Microbiology World Issue 12

Microbiology World

Issue 12

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July – Aug 2015

ISSN 2350 - 8774

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Microbiology World

Issue 12

July – Aug 2015

ISSN 2350 - 8774

Chief Editor Mr. Sagar Aryal (Founder) M.Sc. Medical Microbiology, St. Xavier’s College, Nepal

Editor Mr. Sunil Pandey ELSEVIER Student Ambassador South Asia 2014 B.Sc. Medical Microbiology, Nobel College, Kathmandu, Nepal Mr. Sushan Dhakal Medical Microbiology, St. Xavier’s College, Nepal

International Country Editors Mr. Saumyadip Sarkar ELSEVIER Student Ambassador South Asia 2013 Ph.D Scholar (Human Genetics), India Mr. Hasnain Nangyal M.Phil., Department of Botany, Hazara University, Pakistan

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Microbiology World

Issue 12

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Table of Content Title

Page No.

A Review of Prevention and Control Dynamics of Methicillin Resistant Staphylococcus aureus in Tertiary Care Hospitals

4-16

Antimicrobial activity of the plant extractions against Methicillin Resistant Staphylococcus aureus (MRSA)

17-25

Preventing Antimicrobial Drug Resistances

26-29

Antibiotic- An Introduction

31-38

Characterization of Salmonella Serotypes from Enteric Fever Suspected Patients

39-46

Designing and Development of Bioassay

47-54

IL-17 and its role in autoimmune disease modulation

55-61

Single cell oil (SCO): A product of oleaginous Microorganisms

62-67

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Microbiology World

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A Review of Prevention and Control Dynamics of Methicillin Resistant Staphylococcus aureus in Tertiary Care Hospitals Abid Jan1 1

Institute of Basic Medical Sciences, Khyber Medical University Peshawar, Pakistan Corresponding Email: abidmicrobiologist@live.com

Abstract Excessive use of antibiotics and long duration of treatment in infections can lead to bacterial resistance. Methicillin and Vancomycin resistance in Staphylococcus aureus is a new global problem in hospitals due to lack of hygiene practice and proper management of infectious diseases. Countries like Pakistan are facing this problem in its tertiary care hospital due to lack of infections control facilities and trained nursing staff. In order to stop this problem and to eradicate MRSA form our hospitals we need to use antibiotics in proper dosage and for proper duration. Establishment of Infection Control Committee is the need of the day. Intradepartmental communication and in time response must be insured to tackle the problems. The developed countries has taken a step and developed guidelines to control the spread of MRSA in community hospitals. The development of new drugs and research on controlling bacterial resistance is required on priority basis.

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) are bacteria which are resistant to methicillin antibiotic and can colonize on the skin or in the nose of healthy persons. The bacteria are normally non-pathogenic unless it reaches to deep tissues inside wounds and in surgical incisions. Inside the bloodstream it causes bacteraemia while in the lungs, it causes pneumonia.

Flucloxacillin were used as a drug of choice since 1960 and was found effective in the treatment.

Later on most of the strains became resistant. Therefore methicillin was the first drug used against staphylococcal strains to which these strains become resistant and were called as MRSA. www.microbiologyworld.com

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Today it is the most challenging issue to stop the spread of MRSA inside community hospitals (1).

MRSA is a consistent threat to health in hospitals from the early 70s and the work done so for on highlighting the importance and clinical manifestations of MRSA is a great achievement in the field of science (2, 3). At the inception it was diagnosed from surgical incisions, wounds, burns sites and other skin cuts (4). The in time identification of MRSA colonization by isolating the organism, its proper diagnosis and controlling the carriers in hospitals are helpful in the spread of MRSA (6). MRSA affecting individuals in a Health care facility is known as Healthcareassociated MRSA or HA-MRSA. These individuals usually get infection of MRSA due to frequent exposure to the bacteria (7, 8).

In order to eliminate the health care associated MRSA, the Veterans Affairs Pittsburg has taken the initiative in 2001 with the cooperation of Pittsburg Regional Affairs and Center for Diseases Control (CDC). They used the word “MRSA Bundle” which was based on all the published guidelines consisted of surveillance of MRSA colonization, personal hygiene and precautions which should be taken during attending a patient.

(9). With the implementation of those

guidelines within the community hospitals, the rate of MRSA infection was reduced to 60% in surgical units and 75% in ICU(10,11), those guidelines helped in implementing the rule that MRSA infection will be diagnose through a laboratory test rather than the sign and symptoms found by the physician. If a patient gives a positive MRSA result within 48 hours of admission, it will be considered as community acquired MRSA (12).

The aim of this review is to highlight the strategies and methods used for controlling spread of MRSA inside the hospitals. The reason for controlling MRSA is to insure patient care, minimize the rate of mortality and morbidity due to nosocomial infection of MRSA.

Prevention and Control UK (16, 17), USA (18), New Zealand (5), and Ireland (1) have been taken steps and developed guidelines for the prevention and control of MRSA inside hospitals. These counties have reviewed the literature and give their decision on the basis of the outcome. SARI (A Strategy to www.microbiologyworld.com

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Control Antibacterial Resistance in Ireland) had done a great job by reviewing that part of literature which was totally related to control and prevention of MRSA while the earlier guidelines were based on the expert opinion rather that randomized control trials. The SARI subcommittee has divided their strategy into four (4) grades. Grade “A” was based on randomized control trails , Grade “B” was based on Quasi-experimental study, Grade “C” was related to correlation studies and Case control studies and Grade “D” was the outcome of the recommendations given by the expert committee. The prevention and control measures have been divided into different parts in order to segregate one source of infection from other.

Decolonisation of MRSA Decolonization of MRSA can be done through various chemical agents, such as topical agents like nasal ointment and shampoo for body washing. The antibiotics are used for continuous presence of the organism in patient. However the efficiency of these antibiotics depends on the burden of organisms and the sites of colonization (1). The use of five (5) days nasal mupirocin is effective about 18% along with body washing through chlorhexidine (13). The excessive use of mupirocin must be avoided in the MRSA endemic areas (14). These agents can also be used in decolonization of skin with the combination of 3% hexachlorophene and oral anti MRSA. This regimen reduces the chances of infection from 0.84 to 0.03 per month. Longer duration of treatment with this regimen will have longer effectiveness (13, 15). Transmission through hospital staff is also a reason for the spread of MRSA but it can be stopped through adaptation of good hygiene practice, such as wearing protective clothes and washing hands before and after handling an MRSA patient (1). For decolonization of throat carriage of MRSA, the role of systemic treatment on exceptional bases may be recommended. The use of systemic antibacterial agents should be first consulted with the patient and the hospital staff.

Personal Hygiene Before and after handling the patients, hands must be washed with soap or any other disinfectants. Sterilization of equipments and devices must be carried out properly. Self www.microbiologyworld.com

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protection before entering to intensive care unit (ICU) or any segregated area used for the treatment of MRSA patients. Proper dressing of wounds, cuts or breaks in the skin is important.

Wearing of masks and sterilized or washed uniform on daily basis should be used, so that cross contamination and infection should be avoided. Change of previous gloves when checking another patient of MRSA or of any other infectious disease.

Hospital Hygiene Cleaning of hospital from dirt and any other unpleasant waste is necessary so that the environment is become pleasant to patients and working staff. The floors of hospital must be washed with detol or any other detergent on regular intervals. The walls and ceilings must be clean with 70% v/v ethanol/IPA once in a week. For the prevention of air borne infection, the air circulation system must be modified to the infections control standard. Every tertiary care hospital has a central air circulating system which must be set with the air pressure of 90 ft/mint.

Both the “IN” and “OUT” chambers of the air system must be balanced in such a way that some of the “IN” air pressure is more than the air going through “OUT” chamber. This will allow fresh air to maintain its pressure on the doors of the hospitals also (this pressure is called positive air pressure). Positive air pressure will allow the air borne pathogens to go outside the hospital through doors or “OUT” chamber. The filters of the air chamber should be checked at every 6 months intervals and a DOP test of the HEPA filters should be performed.

The hospitals should establish an infection control committee, which will help in developing strategies for prevention and control of infection inside the hospital. The incharge of the committee must be a microbiologist who will supervise the other nursing and health providing staff. The chief executive officer must take the responsibility for providing all necessary equipments and devices to the infection control committee. The nursing staff must be trained and well educated regarding hygiene practice and safe handling of the patient. They should be aware of precautions during insertion of intravenous and urinary catheters and should wear clean and neat dress washed on the daily bases. www.microbiologyworld.com

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Proper use of Antibiotics The excessive use of antibiotics and prophylaxis must be strictly avoided. The patient should be first diagnosed and then proper antibiotic in proper dosage should be given. Long duration of antibiotic therapy particularly glycopeptides antibiotics should be stopped. The drugs that are creating resistance should be stopped of being prescribed. Broad spectrum antibiotic such as third generation cephalosporin and floroquinolones must be stopped.

All the hospitals should plot a proper way for the antibiotics stewardship. Every patient should be first screened and then the sample taken from that patient should be sent for culture and sensitivity test in order to find out the organisms and proper antibiotic to which the organism is sensitive.

Screening of Patients A good diagnostic and screening system can help in early detection and management of MRSA. Detection of the patients having MRSA is depending on laboratory methods, sample collection and processing that are performed for diagnosis. Surveillance cultures should be performed with regular intervals in order to keep the record updated. The patients, who had three negative tests consecutively during screening after every 72 hours after giving decolonization agents, should be transferred from the isolation chamber. Those patients who have MRSA with wounds and ulcers should be kept in isolation. If a patient is readmitted to the hospital who had MRSA previously should be kept in isolation until the results of the diagnostic tests confirmed.

Screening of Hospital Staff Each and every staff member of the hospital should be screened regularly. In the past, only those staff members were screened who was working in the specific units where patients of MRSA were kept. But today as MRSA is becoming a global problem and gone worst. Therefore it has been decided to screen all the working staff. For this purpose nasal and body swabs are sufficient. www.microbiologyworld.com

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Treatment of MRSA Carrying Patients Patients having colonized MRSA and who is going to be pass through surgery or if they are in ICU or any other clinical area having high chances of transferring infection to others. Therefore nasal decolonization should be done but not excessively as it is creating resistance.

Mupirocin 2% within base (paraffin) with the help of cotton swab should be applied to the inner surface of the nose 3 to 4 times daily for 4 to 5 days. This treatment is almost enough for decolonization of MRSA. After completion of the above treatment, at least 48 hours latter a swab should be taken from the internal surface of the nostrils. In case of positive result, repeat the treatment but only once. If the organism was found resistant to mupirocin then use the alternatives such as naseptin which is the combination of 0.5% neomycin and chlorhexidine 0.1%.

Daily baths with body wash and if the patient’s body is not allergic to chemicals like chlorhexidine 4% and povidone-iodine 7.5% can also be used. If the results are satisfactory after treatment then all the bed sheets and clothing should be washed with detergents.

Control of Glycopeptide Resistant Staphylococcus aureus (GRSA) Personal hygiene must be practiced first because it is the initial step for eradication of MRSA and GRSA from the hospital environment. If resistances to Vancomycin or any other glycoprotein are found in the laboratory, so it must be consulted with the infection control committee. The infection control committee will be responsible for the detection of source of infection.

All the units in hospital should be alerted and necessary precaution should be taken in case of suspected case. It should be determined that wither the resistance was already there or it was developed due to excessive use of antibiotics. The infection control committee must take the responsibility to ward off the organism as soon as possible.

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Conclusion MRSA has become a global problem due to excessive use of antibiotics in infections. Today MRSA is the main nosocomial problem in most of the tertiary care hospital. In developing countries like Pakistan people can buy unprescribed antibiotics from the corner shops which are creating bacterial resistance in most of the patients. Inside the public sector hospitals the hygiene practice is almost nil and none of the precautionary measures are being taken for eradication of hazards. One bed used by one patient is giving to the next one without changing bed sheet or pillow. Health education and hygiene should be implemented in hospitals so that to control infections transfer from one patient to the other.

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8. Otter J. A., French G. L. (2011). Community-associated meticillin-resistant Staphylococcus aureus strains as a cause of healthcare-associated infection. J Hosp Infect,79, 189–193.

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Infection Society and the Infection Control Nurses Association.( 1998). J Hosp Infect, 39(4), 253-290.

17. Guidelines on the control of methicillin-resistant Staphylococcus aureus in the community. Report of a combined Working Party of the British Society for Antimicrobial Chemotherapy and the Hospital Infection Society.( 1995). J Hosp Infect, 31(1),1-12.

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25. Abramson M. A., Sexton D. J.(1999). Nosocomial methicillin-resistant and methicillinsusceptible Staphylococcus aureus primary bacteremia: at what costs?Infect Control Hosp Epidemiol, 20(6), 408-411.

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33. Fridkin S. K., Lawton R., Edwards J. R., Tenover F. C., McGowan J. E., Jr., Gaynes R. P.(2002). Monitoring antimicrobial use and resistance: comparison with a national benchmark on reducing vancomycin use and vancomycin-resistant enterococci. Emerg Infect Dis, 8(7), 702-707. www.microbiologyworld.com

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34. Centers for Disease Control and Prevention. Recommendations for preventing the spread of vancomycin resistance: recommendations of the Hospital Infection Control Advisory Committee (HICPAC). MMWR 44[(No. RR-12)], 1-33. 1995.

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45. O’Connell N. H., Humphreys H.(2000). Intensive care unit design and environmental factors in the acquisition of infection. J Hosp Infect, 45(4), 255-262.

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48. Vasquez J. E., Walker E. S., Franzus B. W., Overbay B. K., Reagan D. R., Sarubbi F. A.(2000). The epidemiology of mupirocin resistance among methicillin-resistant Staphylococcus aureus at a Veterans’ Affairs hospital. Infect Control Hosp Epidemiol, 21(7), 459-464.

49. Guidelines on the control of methicillin-resistant Staphylococcus aureus in the community. Report of a combined Working Party of the British Society for Antimicrobial Chemotherapy and the Hospital Infection Society.(1995). J Hosp Infect, 31(1), 1-12.

50. Burd M., Humphreys H., Glynn G., Mitchell E., McDonald P., Johnson H., et al.(2003).

Control and the prevention of methicillin-resistant Staphylococcus aureus in hospitals in Ireland: North/South Study of MRSA in Ireland 1999. J Hosp Infect, 53(4), 297-303.

51. European Antimicrobial Resistance Surveillance System. EARSS Annual Report 2002.

2003. Netherlands, RIVM. 52. Department of Health and Children. Guidelines for the control of methicillin-resistant Staphylococcus aureus (MRSA) in acute hospital wards, including specialist units. 1995.

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53. Kim T., Oh P. I., Simor A. E.(2001). The economic impact of methicillin-resistant Staphylococcus aureus in Canadian hospitals. Infect Control Hosp Epidemiol, 22(2), 99104.

54. Hiramatsu K.(2001). Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis, 1(3), 147-155.

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55. Enright M. C., Robinson D. A., Randle G., Feil E. J., Grundmann H., Spratt B. G.(2002).

The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA, 99(11), 7687-7692.

56. Safdar N., Maki D. G.(2002). The commonality of risk factors for nosocomial colonization

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Antimicrobial activity of the plant extractions against Methicillin Resistant Staphylococcus aureus (MRSA) Sathiswary Selvaraj1, Ranjutha Valiappan1 1

Faculty of Biomedical and Health Sciences, University Selangor (Unisel), Shah Alam Campus, Jalan Zircon A7/A, Section 7, Shah Alam, Selangor Darul Ehsan, Malaysia.

Abstract The objective of this study was to investigate the traditional medicinal plants includes Mangrove, Melastoma Malabathricum Leaves , Neem and Cinnamon that used to treat infectious disease, were used to look for potential antimicrobial activity against multiresistant bacteria of medical importance especially Methicillin Resistant Staphylococcus aureus (MRSA). S.aureus strains were susceptible to extracts of selected plants. The Minimum Inhibitory Concentrations (MICs) of the total and of additional fractions of these plants were determined by employing strains of Methicillin Resistant S.aureus (MRSA), and Methicillin Sensitive S.aureus (MSSA). The occurrence of Vancomycin-resistant S.aureus(VRSA) and multidrug resistant(MDR) strains of this organism necessitate the discovery of new classes of anti-staphylococcus drug leads. At present there are no single chemical entity plant derived antibacterial used clinically, and this biologically diverse group deserves consideration as a source of chemical diversity for two important reasons. Firstly, plants have exceptional ability to produce cytotoxic agents and, secondly, there is an etiologic rationale that antibacterial natural products should be present or synthesized de novo following microbial attack to protect plants from invasive and pathogenic microbes in their environment. This review cites plants natural products that either modify resistance S.aureus or are antibacterial through bacteriostatic or bactericidal properties. Present study reveals significantly Cinnamon extract is the best used for antibacterial activity against MRSA.

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Introduction The developing countries are now having a greater burden of infection diseases caused by susceptible pathogens. The S.aureus is a pathogen of major concern due to its ability to cause diverse array of diseases ranging from minor infection to life threatening diseases. It has the ability to adapt various conditions of environment. To treat MRSA, medicinal plants are significant developing aspects which become a safer antibacterial principle [1]. Several plant extractions are used by some scientist to study their antimicrobial activity against MRSA.

Resistance towards Antimicrobials S.aureus is commonly known for its resistance towards methicillin like antimicrobials. Where, these antimicrobials typically prevent the bacterial cell wall synthesis. The resistance of S.aureus towards methicillin is mediated by mecA gene which encodes for a novel penicillin-binding protein (PBP), PBP-2a. The mecA resistance gene in bacterial DNA keeps the β-lactam (betalactam) antibiotic from inhibiting the bacterial transpetidase enzymes which are critical for cell wall biosynthesis. β-lactam ring is a part of core structure in several antibiotic families, which are responsible in inhibiting the bacterial cell wall production. The mecA gene is carried on a mobile Staphylococcal Cassette chromosome (SCC) [figure.1]. It contains a variety of genes which leads them to antibiotics resistance. Instead of producing normal PBP, the bacteria start to produce PBP-2a which is influenced by action of other gene. The produce PBP-2a does not have affinity towards β-lactam ring. Therefore, PBP-2a is not inhibited by antibiotics such as methicillin and penicillin [22].

Essential Plant Extraction Mangroves like R. mucronata, R. lamarkii and Bruguiera cylindrica had antibacterial activity against MRSA. The bioactives such as, alkaloid, tannin, flavonoid, triterpenoid, glycosida and saponin from plants that have biological activities or chemical constituents is important mainly for the discovery of new therapeutic agents [17]. The leaf of mangrove E. agallocha is one of the best resources for bioactive properties to cure MRSA [17]. www.microbiologyworld.com

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Figure 1. Structure of the staphylococcal cassette chromosome mec, with the recombinase genes complex upstream of the mec complex. The mec complex contains the mecA gene responsible for b-lactam resistance in S.aureus. IS1272 = insertion sequence–like element; ccrA and ccrB = cassette chromosome recombinase genes A and B that mobilize the mec element; mecR1 = mec sensor transducer and repressor genes that regulate production of PBP-2A, which is responsible for b-lactam resistance; IS431 = integrated plasmid that encodes tetracycline resistance; and orfx = open reading frame in which the mobile elements (staphylococcal cassette chromosome) are located. [23]

On the other hand Rhizophora leaf is the best source to combat MRSA [18]. This difference was probably because of the habitat of the mangroves. The production of bioactive substances by plant is related to several factors, such as independent evolution, genetic, and climate [19] [20].

Based on the result of antibacterial susceptibility testing against MRSA, mangrove plant leaves could be considered to be rich sources of novel anti-MRSA [17]. It was clear that all mangroves (Sonneratia caseolaris, Acanthus ilicifolius, Rhizophora mucronata and Excoecaria agallocha) methanolic extracts indicated anti-MRSA. However, the most potential anti MRSA compound was found on E. agallocha leaf. Hence, further research needs to be done to purify and to characterize anti-MRSA compound from E. agalloca. www.microbiologyworld.com

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Melastoma malabathricum Linn (Melastomataceae) is one of the most important herbs or shrubs found in Malaysia and known to Malays as “senduduk”. The chromatographic separation of Melastoma malabathricum Leaves (MMML), active fraction of M. malabathricum leaves (ML5) by VLC afforded six M. malabathricum Leaves fractions (ML1- ML6). Bioassay-guided fractionation, direct TLC bio-autography revealed that the ML5 fraction showed the highest numbers of antibacterial components with sufficient amount (2.5 g per 1 kg of MMML) [21].

The bioactive constituents, such as kaempferol, responsible for antibacterial activity against S. aureus and MRSA were detected [21]. The MMML displayed potent antibacterial activity with larger zones of inhibition diameter equal to 19±0 mm for S. aureus between 18±0 to 20.33±0.58 mm for MRSA. Kaempferol-3-O-(2",6"-di-O-ptrans-coumaroyl)-β-glucopyranoside,Kf, at concentration ranging from 0.125±0 to 0.25±0 mg mL-1 inhibit the growth of S. aureus and MRSA. Treatment of S. aureus with MMML at concentrations 1×, 2× and 4× MIC was successful in killing bacterial cells within 8 hours. However, the treatment of MRSA with MMML at concentrations 2× and 4× MIC showed lethal effect after 8 h [21]. (×: fold)

Azadirachta indica, commonly known as Neem, with the active compound naming azadirachtin, is a multipurpose tree with variety of health benefits. The leaves, barks and seeds work efficiently against human pathogen bacteria S.aureus. In particular, the leaf extract exhibits a stronger antimicrobial activity against S.aureus at 500μg/ml- 2000μg/ml concentration. While, bark extract works moderately on these particular bacteria at the concentrations of 1000μg/ml2000μg/ml. But, seed extract exhibits only minimum antimicrobial activity [2]. The thickness and uniformity of the extract used, size of the inoculum, temperature and pH might affect the accuracy of the test result. On another study conducted, a greater zone of exhibition obtained from neem leaf ethanol extract (100% conc.) when test its in-vitro antimicrobial activity against MRSA [6].

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The antibacterial activity against MRSA by in-vitro method was tested using ethanolic extract of cinnamon, cumin and clove. Where, cinnamon and clove were found to be bactericidal after 6 hours of incubation [9]. All the three extracts show their bactericidal activity against S.aureus after 24 hours of incubation at 200Îźg/ml- 300Îźg/ml concentration. Cinnamon which has the active compound known as cinnamaldehyde was claimed as the most effective spice against microorganism tested and S.aureus is the most susceptible bacterial strain to that spice (Zone Diameter of Inhibition ZDI: 32mm). From this particular experiment, was found that cinnamon exhibits the best anti-S.aureus activity with the ZDI ranging between 22mm to 27mm. Where, cinnamon and clove shows a stronger in-vitro compared to cumin [10].

Action Mechanism of Components of Plant Extract against MRSA The cell wall of gram positive bacteria, consisting of peptidoglycan,composed of teicocic acid and several proteins. The structure of the cell wall of the gram positive bacteria permits hydrophobic molecules to penetrate easily the cell wall and act on both the cell wall and cytoplasm [10]. The active compounds of the stated plants, alkoids, tennin, quercitrin, kaempferol, azadirachtin and cinnamaldehyde, affect both the cell wall and cytoplasm. The hydrophobicity of the component of extract causes an increasing permeability of the cell membrane of the bacterium due to the inability of the separation of extract active component from cell membrane. Therefore, the damaged cytoplasmic membrane will allow the active compounds to enter in and coagulate the cytoplasm [11] [12] [13]. Yet, the active compounds of the natural products will damage the membrane protein, decrease the ATP production of the bacteria, reduce the proton motive force [14], further increase the permeability of the cell membrane [15], which lead to the leakage of cytoplasmic contents. Since the plant extracts consist of numerous of molecules and active compounds, their antimicrobial activity cannot be attributed to a single mechanism. The active compounds of plant extracts chemically modify the cell membrane, cytoplasm, proteins and enzymes, causes a complete change in the bacteria.

Thus, a continuous loss of ions, ATP and metabolites due to the exposure to active compound of plant extract; it leads to cell death [16] [Figure 2.] www.microbiologyworld.com

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Figure 2: Mechanism of action and target sites of the plant extract active compounds on microbial cell [16]

Conclusion Plants are rich in variety of secondary metabolites such as tannins, terpenoids, alkaloids, flavonoid, etc., which have been fond upon in-vitro to have medicinal characters. The antibacterial activity in essential plant extracts are significantly higher compared to antibiotics such as ampicillin and vancomycin used [7]. The hydrophobicity of the essential plant oils enables them to permeate the lipid of bacterial cell membrane and mitochondria, disturb the structure and rendering them more permeable [2]. From the review here, it can be concluded that, Cinnamon extract and its active compound, cinnamaldehyde, is the best used for antibacterial activity against MRSA. Further research is needed to identify the plant extract’s toxicity and the dosage to be used for clinical purposes.

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References 1. S.Chanda, B.R.M. Vyas, Y. Vaghasiya, H. Patel. Globalresistance trends and the potential impact of Methicillin Resistant Staphylococcus Aureus (MRSA) and its solution.2010.

2. Raja Ratna Reddy Y, Krishna Kumari C, Lokanatha O, Mamatha S, Damodar Reddy C. Antimicrobial activity of Azadirachta indica (neem) leaf, bark and seed extracts.

2013;2231-010X. 3. Shuchi Kaushik, Rajesh Singh Tomar, Vikas Shrivastava, Archana Shrivastava, Sudhir Kumar Jain. Antimicrobial efficacy of essential oils of selected plants and vaccine design against mecA protein of MRSA. 2014;vol7,suppl 1, 0974-2441.

4. Chaied K, Hajlaoui H, Zmantar T, Ben Kahla-Nakbi A, Rouabhia M, Mahdouani K et al, The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata: a short review phytotheraphy research 2007;21(6);501-506.

5. Bensky D, Clavey S, Stoger E, Gamble A, Lai Bensky L; Chinese herbal medicine: MateriaMedica, (III Ed.) Eastlend Press. Beuteilung. DtschZahnarztl Z 2004;3:189-191.

6. Wendy C.Sarmiento, Cecilia C. Maramba, Ma. Liza M. Gonzales. An in-vitro study on the antibacterial effect of neem leaf extract on Methicillin-Sensitive and MethicillinResistant Staphylococcus aureus.PIDSP Journal 2011,vol 12 No1.

7. Shilpa M Shenoy, A.Veena Shetty, Vinaya Bhat. Comparative evaluation of antimicrobial activity of the essential plant oils on multidrug resistant nosocomial pathogens.IJPCBS 2014, 4(2), 372-377.

8. Aditi Grover, B.S. Bhandari, Nishant Rai. Antimicrobial activity of medicinal plantsAzdirachta indica A.Juss, Allium cepa L and Aloe vera L. international Journal of Pharmtech Research. 2011;3(2):1059-1065.

9. Shyamapada Mandal, Manisha DebMandal, Krishnendu Saha, Nishith Kumar Pal. Invitro antibacterial activity of three Indian spices against MRSA. Oman Medical Journal 2011;Vol 26,No5; 319-323. www.microbiologyworld.com

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10. Filomena Nazzaro, Florinda Fratianni, Laura De Martino, Raffaele Coppola, Vincenzo De Feo. Effect of Essential Oils on Pathogenic Bacteria. Pharmaceuticals 2013, 6, 14511474; doi:10.3390/ph6121451.

11. Ultee, A.; Bennik, M.H.; Moezelaar, R. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ.

Microbiol. 2002, 68,1561–1568.

12. Ultee, A.; Kets, E.P.W.; Alberda, M.; Hoekstra, F.A.; Smid, E.J. Adaptation of the foodborne pathogen Bacillus cereus to carvacrol. Arch. Microbiol. 2000, 174, 233–238.

13. Gustafson, J.E.; Liew, Y.C.; Chew, S.; Markham, J.L.; Bell, H.C.; Wyllie, S.G.; Warmington, J.R. Effects of tea tree oil on Escherichia coli. Lett. Appl. Microbiol. 1998, 26, 194–198.

14. Ultee, A.; Smid, E.J. Influence of carvacrol on growth and toxin production by Bacillus cereus. Int. J. Food Microbiol. 2001, 64, 373–378.

15. Burt, S. Essential oils: their antibacterial properties and potential applications in foods— A review. Int. J. Food Microbiol. 2004, 94, 223–253.

16. Burt, S.A.; Reinders, R.D. Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Lett. Appl. Microbiol. 2003, 36,162–167.

17. Asep Awaludin Prihanto, Muhamad Firdaus, Rahmi Nurdiani. Anti-Methicillin Resistant Staphylococcus aureus (MRSA) of Methanol Extract of Mangrove Plants Leaf: Preliminary Report. Drug Invention Today ISSN: 0975-7619 Short Communication www.ditonline.info.

18. Chandrasekaran M, Venkatesalu V, Sivasankari S, Kannathasan K, Sajit-Khan AK,, Prabhakar K, et al. Antibacterial activity of certain mangroves against methicillin resistant Staphylococcus aureus. Seaweed Res Utiln, 28, 2006, 165–170.

19. Ibrahim T, Ajala L, Adetuyi FO, Ojei BJ, Assessment Of The Antibacterial Activity Of Vernonia amygdalina and Occimum gratissimum Leaves On Selected Food Borne Pathogens. The Internet Journal of Third World Medicine, 8 (2), 2009 from: www.microbiologyworld.com

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http://www.ispub.com/journal/the-internet-journal-of-thirdworld-

ISSN 2350 - 8774 medicine/volume-8-

number-2.html 20. Pimentel MR, Molina G, Dionísio AP, Junior MRM, Pastore GM. The Use of Endophytes to Obtain Bioactive Compounds and Their Application in Biotransformation Process. Biotechnology Research International 2011, 2011, 1-11.

21. Mourouge Saadi Alwash, Nazlina Ibrahim and Wan Yaacob Ahmad. Identification and Mode of Action of Antibacterial Components from Melastoma Malabathricum Linn Leaves. American Journal of Infectious Diseases 9 (2): 46-58, 2013 ISSN: 1553-6203 ©2013 Science Publication doi:10.3844/ajidsp.2013.46.58 Published Online 9 (2) 2013 (http://www.thescipub.com/ajid.toc).

22. Michael J. Rybak, Pharm.D., and Kerry L. LaPlante, Pharm.D. Community-Associated Methicillin-Resistant Staphylococcus aureus: A Review. PHARMACOTHERAPY Volume 25, Number 1, 2005.

23. Mongkolrattanothai K, Boyle S, Kahana MD, Daum RS. Severe Staphylococcus aureus infections caused by clonally related community-acquired methicillin-susceptible and methicillinresistant isolates. Clin Infect Dis 2003;37:1050–8.

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Preventing Antimicrobial Drug Resistances Mr. Binod Rayamajhee1 1

M. Sc. in Medical Microbiology, Teaching fellow of Kathmandu University High School Corresponding Email: binod@kuhs.edu.np

Antibiotics are the gift of science to human life. Use of the antibiotics has undoubtedly gained access in almost all aspects of biological applications ranging from food preservation to medical purpose. In medical aspects antibiotics have been used either for prophylaxis, empiric therapy or pathogen directed therapy which contributes the central role in control and management of infectious diseases. The use of antibiotics shortly after its discovery has widened to such an extent that the overuse of then resulted into development of resistance by various bacteria. The trend of the development of resistance by bacteria against antibiotics is too high with respect to the discovery of new antibiotic that we can sorrowfully assume a time is close to us when no antibiotic available will be effective against infectious bacteria.

The morbidity and mortality of infectious disease have increased in parallel with the greater acquisition of antibiotic resistance by organisms, especially in regards to isolates that are completely resistant to antibiotic. Without gathering the information about the existing MDR (multi drug resistant) isolates, we cannot reduce the morbidity and mortality due to infectious caused by MDR pathogens; neither can we reduce the rate of emergence and spread of antimicrobial resistance. Appropriate use of antibiotics is central to limiting the development and the spread of resistant bacteria in hospital and communities. Use of broad-spectrum antibiotics, in particular the third generation cephalosporin in nosocomial infections have been linked to the emergence of antibiotic resistance and increase in treatment costs.

The hospital setting is particularly conducive to the development of antibiotic resistance as patients who are severely ill, immune-compromised or have devices and /or implants in then are likely to receive frequent courses of empirical or prophylactic antibiotic therapy. Easier access to antibiotics leads to the in appropriate use of antibiotic and often the high cost of antibiotic results in an incomplete course being purchased, sufficient only to alleviate symptoms. Developing www.microbiologyworld.com

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countries are often unable to afford costly second line antibiotics to treat infectious due to resistant organisms, resulting in prolonged illness with longer periods of infectivity and further spread of resistant isolates. These factors contribute to emergence of antibiotic resistance worldwide; however condition is even worst in developing countries.

The major trouble causing MDR isolates that have been widely observed and studied include Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin Resistant coagulase negative Staphylococci, Glycopeptide intermediate sensitivity S. aureus (GISA), and vancomycin resistant Enterococus (VER). In later years however, extended spectrum β-lactamase (ESBL), Metallo betalactamase (MBL) and AmpC β-lactamase encoding organisms have been observed which not only resist β-lactam antibiotics but also non β-lactam antibiotics. These later organisms may even exhibit resistivity towards these antibiotics in vivo which they are susceptible in vitro.

According to the centers of Disease control and prevention (CDC) in Atlanta, Georgia (USA), widespread antimicrobial resistance is highly prevalent. Nearly 2 million patients in the USA develop hospital acquired (nosocomial) infections each year. Nosocomial infections are difficult to treat because up to 70% of the infecting microorganisms are resistant to antimicrobial drugs.

Staphylococcus aureus is one of the most problematic human pathogens causing dangerous and costly infections worldwide. It is associated with a variety of clinical infections including septicemia, pneumonia, wound sepsis, septic arthritis, osteomyelitis, and post-surgical toxic shock syndrome with substantial rates of morbidity and mortality. Staphylococcus aureus, which causes over 10% of these infections in intensive care units, research shows a two fold increase in drug resistance to methicillin and oxacillin over a 13-year period. Methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) rank as the second most common causes of hospital-associated blood stream infections and associated with increased mortality and longer hospital stay. Ever since its isolation, MRSA has emerged as one of the most common cause of hospital acquired infection and continues to remain as an important factor contributing to failure of management. MRSA is frequently resistant to most of the commonly used antimicrobial agents including the aminoglycosides, macrolides, chloramphenicol, tetracycline, www.microbiologyworld.com

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and fluoroquinolones. Resistance in Mycobacterium tuberculosis, Enterococcus faecium and Candida albicans are also of major concern.

Antimicrobial resistance has been a widely discussed topic in the public health world over the past few years. Antimicrobials antibiotics being the most common have greatly reduced infections disease incidence and mortality since they were first used in the 1940s. However, their widespread over use and misuse in both food and animal production and human medicine have led to the adaptation of infections microorganisms that are resistant to the very drugs trying to kill them. These "superbugs", which include bacteria, fungi, parasites and viruses, help to spread infections rapidly and make antimicrobials ineffective. Experts fear that this increasingly dangerous problem could have dire consequences on worldwide health if it is not quickly and adequately addressed.

The major health institution and professional should espouse a ten steps program to prevent resistance to antimicrobial agents and the program is summarized infra. The program stresses the importance of preventing infection rapidly and positively diagnosing and treating infections, using antimicrobial agents wisely and preventing pathogen transmission.

1. Immunize to prevent common disease: Keep immunizations up to date, especially for likely diseases, exposures, etc. in addition to required vaccinations, this should include yearly influenza vaccination for nearly everyone, meningitis vaccines and pneumococcal immunizations for health care providers or those exposed to large number of people, as in school, colleges and the military.

2. Avoid unnecessary introduction of parenteral devices, such as catheters: All present a risk of introducing infections agents into the body. If such devices are necessary, remove them as soon as possible.

3. Target the pathogen: Attempt to culture the infections agent while targeting antimicrobial drug treatment for the most likely pathogens. After positive culture results, adjust the therapy to target the known pathogen & its antibiotic susceptibility.

4. Access the experts: For serious infections, follow up with an infectious disease expert. Get a second opinion if conditions do not rapidly improve after treatment has begun. www.microbiologyworld.com

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5. Practice antimicrobial control: Be aware and current in knowledge of appropriate antimicrobial drugs and their use. Be sure the treatment offered is current and recommended for the pathogen.

6. Use local data: Obtain and understand the antibiotic susceptibility pattern or profile for the infections agent from local health care sources.

7. Treat infection, not contamination: Aseptic techniques must be followed to obtain appropriate samples from infected tissues. Contaminating organisms may be present on skin, catheters, or intravenous lines. Obtain cultures only from the site of infection.

8. Treat infection, not colonization: Treat the pathogen and no other colonizing microorganisms that are not causing disease. For example, cultures from normal skin and throat are often colonized with potential pathogens such as Staphylococcus spp. These may have nothing to do with the current infection.

9. Treat with the least exotic antimicrobial agent that will eliminate the pathogen: Treatment with the latest broad - spectrum antibiotic, while efficacious, may not be warranted if other drugs are still effective. The more a drug is used, the greater the chance that resistant organisms will develop. For example some Enterococcus isolates are already resistant to Vancomycin, a relatively new broad - spectrum antibiotic, largely because the drug was overprescribed to treat MRSA infections when it was first introduced.

10. Monitor antimicrobial use: Antimicrobial use should be discontinued as soon as the prescribed course of treatment is completed. If an infection cannot be diagnosed, treatment should be discontinued. For example, in the case of pharyngitis (sore throat), antibiotic treatment for Streptococcus pyogenes is often started before throat culture results are confirmed. If throat cultures are negative for S. pyogenes before throat culture results are confirmed. If throat cultures are negative for S. pyogenes treatment with antibiotics should be stopped. Antibiotics are ineffective for treatment of the viruses that are most probable causes of pharyngitis.

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Antibiotic- An Introduction Shubha Ratna Shakya1 1

NAST Ph.D. Fellow, Associate Professor of Zoology, Amrit Campus, Tribhuvan University, Kathmandu, Nepal Corresponding Email: shubharatnashakya@gmail.com

Introduction Antibiotics are used to kill or inhibit the growth of microbes. The term “antibiotic” was first used

by Selman Waksman and his collaborators in 1942 to describe “any chemical substance produced by or derived from a microorganism such as bacteria or fungi and is capable of inhibiting the growth of or even destroying other microorganisms in high dilution i.e., small concentration”. This definition does not include substances such as gastric juices, hydrogen peroxide and synthetic antibacterial compounds such as the sulfonamides that have an inhibitory effect on bacteria only. These substances are not of microbial origin. Today with the advent of synthetic and semi-synthetic compounds modifies the definition and an antibiotic now refers to a substance of microbial origin, or to a similar substance (produced wholly or partly by chemical

synthesis),

which

in

low

concentrations inhibits

the

growth

of

other

microorganisms. Antimicrobial agents such as sulphonamides and the 4-quinolones, produced solely by synthetic means, are often referred to as antibiotics.

The first antibiotic to receive widespread attention was penicillin. Penicillin is the product of the molds (fungi), Penicillium notatum and Penicillium chrysgnum. Penicillium was discovered by Scottish Physician Sir Alexander Fleming at St. Mary’s Hospital in London in 1928. Since then, many new antibiotics have been developed and are being used to treat variety of infections caused by bacteria. In other words, antibiotics are among the most frequently prescribed medications in modern medicine.

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Antibiotics Versus Antimicrobials The word antibiotic, derived from the Greek word anti (against) and biotikos (concerning life) refers to substances produced by microorganisms that act against another microorganism. Thus, antibiotics do not include antimicrobial substances that are synthetic (sulfonamides and quinolones), or semisynthetic (methicillin and amoxicillin), or those that come from plants (quercetin and alkaloids) or animals (lysozyme). An antibiotic is a low molecular substance produced by a microorganism that inhibits or kills other microorganisms in high dilution.

The word antimicrobial, derived from the Greek words anti (against), mikros (little) and bios (life) refers to all agents such as natural, semisynthetic or synthetic origin that kills or inhibits the growth of all types of microorganisms: bacteria (antibacterial), viruses (antiviral), fungi (antifungal) and protozoa (antiprotozoal). All antibiotics are antimicrobials, but not all antimicrobials are antibiotics.

General Features of Antibiotics 1. Antibiotics are biologically active against a large number of pathogens even when present in extremely low concentration.

2. They kill or inhibit the growth of pathogens.

3. They should be safe (non-toxic) to the host.

4. They should not cause any allergic reactions to the host.

5. They remain in specific tissues in the body, long enough to be effective.

6. They kill pathogens before pathogens mutate and become resistant to it.

Types of Antibiotics On the basis of action against microorganisms, antibiotics are of two types: bacteriostatic and bactericidial.

1. Bacteriostatic antibiotics inhibit growth and multiplication of susceptible bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism under special pathological conditions. Chloramphenicol, www.microbiologyworld.com

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erythromycin, tetracyclines, sulphonamides are bacteriostatic antibiotics. The bacteria can continue to grow and multiply if the antibiotics are removed.

2. Bactericidial antibiotics generally kill bacteria by interfering with either the formation of the bacterium's cell wall or its cellular contents. Streptomycin, penicillin, cephalosporin, polymyxin, colistinfluoroquinolones, and metronidazole are some of the bactericidial antibiotics. Bactericidial antibiotics may become bacteriostatic antibiotic at lower concentrations.

The range of antibiotics effectiveness varies. On the basis of application, antibiotics may be of the following two types:Narrow-spectrum and broad-spectrum antibiotics.

1. Narrow spectrum antibiotics are effective against a limited variety of bacteria.

Penicillin and erythromycin are primarily effective against gram positive bacteria (e.g. streptococci) and streptomycin acts against gram-negative bacteria (e.g. E.coli) 2. Broad spectrum antibiotics act effectively against large number of related and unrelated bacteria including non-pathogenic forms. Chloramphenical and tetracycline are effective against both gram positive and gram negative bacteria.

Side Effects of Antibiotics Antibiotics can save lives and are effective in treating illnesses caused by bacterial infections.

However, like any other drugs, they may cause unwanted side effects and allergic reactions.

When antibiotics are ingested to control disease-causing bacteria, they also affect the normal bacteria flora. Although many of these side effects are not dangerous, they can make life miserable while the drug is being taken.

In general, antibiotics rarely cause serious side effects. The most common side effects from antibiotics are diarrhea, nausea, constipation, and vomiting. Fungal infection of mouth, digestive tract and vagina can also occur with antibiotics because they destroy the protective 'good' bacteria in the body along with the 'bad' ones that cause diseases.Some people are allergic to www.microbiologyworld.com

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antibiotics, particularly penicillin. Allergic reactions cause swelling of the face, itching and a skin rash and, in severe cases, breathing difficulties.

Antibiotics can also interfere with action of other drugs.These unintended reactions to antibiotics are called adverse drug events.

Mechanism of action of Antibiotics Antibiotics are effective for a variety of reasons. The antibiotics interfere with some aspect of metabolism of the bacteria, which is not found in its host. Thus, the bacteria suffer, but the host does not. The antibiotics during the course of action disrupt a vital link in the metabolism of the pathogen such as synthesis of enzymes, conversion of glucose into energy, destroy cell wall, inhibit synthesis of cell, inhibit protein synthesis, inhibit production of nucleic acids etc. For instance, bacterium exposed to the action of penicillium swells and stops dividing. Such bacteria are easily destroyed by the body’s white blood cells.

Antibiotic Resistance A major problem of widespread use of antibiotics is the emergence of resistant bacteria.

Antibiotic-resistant bacteria are not killed by commonly used antibiotics. When bacteria are exposed to the same antibiotics over and over, the bacteria can change and are no longer affected by the drug. In other words, the antibiotic doesn’t actually cause the resistance. It allows resistance to happen by creating an environment in which groups of naturally resistant bacteria can flourish.

Because antibiotic resistance occurs as part of a natural evolution process, it can be significantly slowed but not stopped. Therefore, new antibiotics will always be needed to keep up with resistant bacteria as well as new diagnostic tests to track the development of resistance.

Any use of antibiotics can increase selective pressure in a population of bacteria that promotes resistant bacteria to survive and the susceptible bacteria to die off. As resistance to antibiotics becomes more common, a greater need for alternative treatments arises. www.microbiologyworld.com

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Antibiotics may also be destroyed by enzymes present inside the cell. Beta-lactamases (also called penicillinases) are enzymes that deactivate penicillins. Beta-lactamases allow bacteria to be resistant to penicillin.

Bacteria may acquire resistance to antibiotic in one of the following ways.

1. Mutation: Mutation may occur at any time. Natural mutations may create gene alterations producing structural or biochemical changes that lead to resistance. Bacteria are present in such a large numbers that there is high chance of development of resistant bacteria in a population. As soon as they develop resistant, use of the antibiotic to which they are resistant will give them a selective advantage over non-resistant types and they will multiply to became the dominant type.

2. Transfer of resistance: Resistance can spread from one bacterium to another by transfer of the resistant genes. Conjugation, a simple form of sexual reproduction, is the most common mechanism to transfer resistance.

Bacterium resistance to two or more than two antibiotics is called multi-antibiotic resistant or superbug. It can also be transferred from one bacterial species to another.

The antibiotic resistant bacteria in animals can be transmitted to humans via three pathways; through consumption of meat, from close or direct contact with animals and through the environment. Antibiotic resistance is a serious and growing phenomenon and has emerged as one of the biggest public health concerns of the 21st century.

Applications 1. Antibiotics in human health: Antibiotics are among the most commonly prescribed drugs used to fight bacterial infections in humans. Different types of antibiotics are available for treatment of different types of diseases. Antibiotics should be used properly to save lives. They have greatly contributed to the maintenance of human health. www.microbiologyworld.com

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However, up to 50% of all the antibiotics prescribed for people are not needed or are not optimally effective as prescribed.

2. Antibiotics in agriculture: Antibiotics are commonly used in food for animals such as cows, buffaloes, pigs, chickens etc. to prevent, control and treat diseases. They are also used to promote the growth of food-producing animals and poultry. Antibiotics are also used to control diseases in plants. These antibiotics can affect safety of meat, milk, and eggs produced from those animals and can be the source of superbugs. Farm animals particularly pigs, are able to infect humans with methicillin-resistant Staphylococcus aureus (MRSA).

3. Antibiotics in aquaculture: Infectious diseases are a major problem in aquaculture causing heavy loss to the fish farmers. Antibiotics are used for controlling diseases in fishes. In aquaculture, antibiotics at therapeutic levels are frequently administered for short periods of time via oral route to groups of fish that share tanks or cages. The most common route for the delivery of antibiotics to fish occurs through mixing the antibiotic with specially formulated feed.

As a consequence, these antibiotics occur as traces in human diets and then result in hazards to health. The hazards are direct toxic effects, hypersensitive reactions and the production of antibiotic reactions to pathogenic bacteria transmissible to humans. Bacterial resistance to antibiotics is increasing due to misuse or overuse of antibiotic in animals, humans and vegetables. These bacteria are difficult to treat. The rational use of antibiotics may reduce the chances of development of antibiotic-resistant bacteria.

Conclusion Antibiotics are both beneficial as well as harmful to human beings and other living beings. If antibiotics are taken properly and the drug dose is completed, it cures diseases. If the antibiotic selection is wrong or the dose is incomplete, then drug resistant bacteria develop. Drug resistant bacteria are difficult to treat. www.microbiologyworld.com

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Public health hazards related to antibiotics use in aquaculture include the development and spread of antibiotic-resistant bacteria and resistance genes, and the presence of antibiotics residues in aquaculture products and in the environment.

Careless and injudicious use of antibiotics has been the major contributing factor in the development of multiple drug resistant bacteria. Antibiotic resistance is an emerging problem in the world. From a public health perspective, poorly supervised or incomplete treatment of infectious diseases is worse than no treatment at all.

References 1. Arias CA, Murray BE; Murray, BE (2009). "Antibiotic-Resistant Bugs in the 21st Century — A Clinical Super-Challenge". New England Journal of Medicine 360 (5): 439–443.

2. Bengtsson B, Wierup M; Wierup, M. (2006). "Antimicrobial resistance in Scandinavia after ban of antimicrobial growth promoters". Anim. Biotechnol. 17 (2): 147–56.

3. Cabello, F.C. (2006) Heavy use of prophylactic antibiotics in aquaculture: A growing problem for human and animal health and for the environment. Environmental Microbiology, 8, 1137-1144.

4. Castanon JI (2007). "History of the use of antibiotic as growth promoters in European poultry feeds". Poult. Sci. 86 (11): 2466–71.

5. Clardy, J., Fischbach, M. and Currie, C. (2009). The natural history of antibiotics.

Current Biology, 19, R437-R441.

6. CVM-MSU (2011) The golden age of antibacterials: Antimicrobial resistance learning site. Michigan State University, East Lansing.

7. Davies, J. (2013) Antibiotic discovery: Then and now. Issue: Antimicrobials.

Microbiology-today.

8. Fischbach, M.A. and Walsh, C.T. (2009) Antibiotics for Emerging Pathogens. Science, 325, 1089-1093. www.microbiologyworld.com

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9. Gardiner Harris ( 2012). "U.S. Tightens Rules on Antibiotics Use for Livestock". The New York Times.

10. Glew, R. (2010) Bacterial resistance to antimicrobials: From the Golden Age to the Bronze Age of antibiotic use. Medical Center.

11. Mathew AG, Cissell R, Liamthong S; Cissell, R; Liamthong, S (2007). "Antibiotic resistance in bacteria associated with food animals: a United States perspective of livestock production". Foodborne Pathog. Dis. 4 (2): 115–33.

12. Torrence, M.E., et al. (2008) Antibiotics: Mode of action, mechanisms of resistance, and transfer. Microbial food safety in animal agriculture: Current topics. John Wiley & Sons, Inc.

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Characterization of Salmonella Serotypes from Enteric Fever Suspected Patients Adhikari R1, Manandhar S1, Acharya J2 1

Department of Microbiology, Tri-Chandra Multiple Campus, Ghantaghar, Kathmandu, Nepal 2

Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal Corresponding Email: rajib.adhikari11@gmail.com

Abstract Enteric fever is still an important public health problem in developing countries like Nepal. A changing antibiotic susceptibility pattern of Salmonella Typhi and S. Paratyphi A has increased to a great concern. A cross-sectional study was carried out at Sukraraj Tropical and Infectious Disease Hospital from mid of Septemberr 2012 to mid of June 2013. Blood culture samples were collected from all suspected enteric fever patients visiting the hospital and tested microbiologically by standard procedure. AST was based on Kirby disc diffusion method. The results were interpreted according to CLSI (Clinical Laboratory Standards Institute) guidelines.

Of total 56 (5.5%) Salmonella serotype isolated from 1,011 blood culture samples, 29 (51.8%) were S. Paratyphi A and 27 (48.2%) were S. Typhi. Among the culture positive cases, the incidence rate was high in male 69.6% and the age group of 16-30 years showed maximum number of growth 47 (75%). Most of the serotypes were isolated between the period of the mid of May to the mid of June 15(26.8%). Among the tested antibiotics S. Typhi was fully susceptible towards chloramphenicol and 92.6 of the isolated S. Typhi were susceptible towards ceftriaxone and cotrimoxazole. In case of S. Paratyphi A chloramphenicol and ceftriaxone showed 96.7% susceptibility followed by Cotrimoxazole (92.6), whereas 93.1% were resistant to nalidixic Acid. Enteric fever still poses the challenge to the public health in Nepal as 16.1% of patients were still admitted in the hospital for the treatment. 87.5% of the isolates were resistant to nalidixic acid leading to the reduced susceptibility and resistance towards ciprofloxacin.

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ciprofloxacin susceptibility or ciprofloxacin resistant cases. The reemergence of the susceptibility of the first line drugs was seen which should be studied in the large samples.

Keywords: Enteric fever, S. Typhi, S. Paratyphi A, Ceftriaxone, Cotrimoxazole, Reemergence.

Introduction Enteric fever is a systemic infection caused by the human adapted pathogens Salmonella enterica serotype Typhi (S. Typhi) and S. enterica serotype Paratyphi (S. Paratyphi) A, B, and C1. Enteric fever remains one of the major public health issues globally, especially in Asia. According to recently revised global estimate, above 22 million cases of typhoid fever occur each year round the world while 90% of the sufferers are from the South East Asia.2 Its incidence is highest in children and young adults between 5 and 19 years old3. In context of Nepal, enteric fever caused

by S. Typhi and S. Paratyphi A is the most common clinical diagnosis among febrile patients.4 A changing antibiotic susceptibility pattern of Salmonella isolates and emergence of S. Paratyphi A as a cause of enteric fever has increased to a great concern. So, this study was undertaken to characterize the Salmonella isolates, determine the enteric fever burden, to study the effects of first line drugs on the isolates and Fluoroquinolone susceptibility characters of the isolates.

Materials And Methods The study was carried out at Sukraraj Tropical and Infectious Disease Hospital from mid of September 2012 to mid of June 2013. Blood culture samples were collected from all 1,011 enteric fever suspected patients visiting the hospital. Brain-heart infusion (BHI) broth was used as enrichment media which supports the growth of all common pathogens causing enteric fever.

Collection of blood, incubation, and subcultures onto blood agar and Mac-Conkey agar were done as per the standard methods. Suspected colonies were further processed and identified by biochemical reactions and confirmed by group and type specific Salmonella antisera (Denka Seiken, Japan). Antimicrobial susceptibility was determined by the Kirby-Bauer disc-diffusion method. The antibiotic discs used were Ampicillin (30µg), Nalidixic acid (30µg), Ofloxacin www.microbiologyworld.com

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(5µg), Ciprofloxacin (5µg), Cotrimoxazole (1.25/23.75µg), Chloramphenicol (30 µg), Ceftriaxone (30µg), Azithromycin (15µg) and results were interpreted according to CLSI (Clinical Laboratory Standards Institute) guidelines. Data were entered in Microsoft Excel and analyzed by SPSS version 16.0

Results A total of 1,011 patients suspected of enteric fever were studied, among them 5.5% were culture positive. Out of 56 Salmonella isolates, 27 (48.2%) were S. Typhi and 29 (51.8%) were S. Paratyphi A. Among the culture positive cases, the incidence rate was high in male 69.6% than female 30.4% and the age group of 15-30 years showed maximum number of growth i.e. 42 (75%). However, the association of organism isolated with respect to different gender was found to be insignificant (P>0.05) but the association between the isolated organism with respect to age groups was found statistically significant (P<0.001). Most of the isolates (26.8%) were isolated from the mid of May to mid of June and the association was statistically significant (P=0.012).

Chloramphenicol was found to be 100% effective in all the S. Typhi isolates. It was followed by Cotrimoxazole and Cotrimoxazole with 92.6% susceptibility. In case of S. Paratyphi A 96.6% of the isolates were sensitive to Ceftriaxone and Chloramphenicol followed by Cotrimoxazole (93.1%). Out of 56 Salmonella isolates 87.5% were resistant to Nalidixic acid.

Discussion In this study, out of 1,011 blood culture samples, 56 (5.5%) were culture positive. Similar results have been reported by Acharya et al5 and Adhikari et al,6 who found 7.6% and 8.99% culture positive result respectively. The low isolation of the pathogen in blood culture may be due to the partial treatment received elsewhere before coming to the hospital Shakva et al,7 and our reliance upon a single blood culture for diagnosis.

Out of 56 isolates 29 (51.8%) were S. Paratyphi A and 27 (48.2%) were S. Typhi. Increasing numbers of S. Paratyphi A infection have been noted in Nepal by Acharya et al, 5 and Versa et www.microbiologyworld.com

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al.8 A possible reason proposed for the higher incidence for paratyphoid is more likely the involvement of food from street vendors.9

Though 39 (69.6%) of the positive samples were from the male patients and rest from the female patient, the relation between gender and growth positivity was not found to be statistically significant (P>0.05). On the other hand, the association of organism isolated with respect to different age group was found to be statistically significant (P <0.001) with highest no of isolates www.microbiologyworld.com

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i,e. 42(75%) from the age group of 15-30 years. This may be due to high mobility and exposure of this age group.

The most diagnosed enteric fever cases were from the mid of May to the mid of June. The association was statically significant (P<0.05). This may be possibly due to the sewage-mediated contamination of water sample during the rainy seasons.10 Chloramphenicol was 100% susceptible towards all S. Typhi isolates and 96.55% to S. Paratyphi A isolates. Moreover, reemergence of first line drug (chloramphenicol, cotrimoxazole and ampicillin) was observed in the study where 58.9% isolates were susceptible. A number of studies in recent years have shown an increase in sensitivity to the first line antibiotics in case of S. Typhi and Paratyphi A Neupane et al11, Acharya et al12 and Bhandari13 This study showed 92.6% susceptibility of ceftriaxone for S. Typhi and 96.55% for S. Paratyphi A strains and the remaining isolates were also intermediately susceptible to it. Khatiwada,14 and Raza et al,10 also revealed that ceftriaxone is found to be one the most effective (100%) antibiotics against Salmonella Paratyphi A ceftriaxone remains the last line drug against infections with ciprofloxacin resistant Salmonella when it is resistant to other first line drug.

Azithromycin was found to be the least effective antibiotic with high degree of resistance, 40.7% in S. Typhi and 27.6% in S. Paratyphi A. Similar finding was reported by Rai15 where 63.9% of S. Typhi and 55% of S. Paratyphi A. The emerging resistant of azithromycin has also been reported in India by Chaudary et al.16 since only 46% isolates were susceptible. This may be the result of misuse of azithromycin that has been propelled due to its oral route of administration, as well as broad-spectrum antimicrobial activity with minimal side effects and interactions.17 Out of 56 isolates, 49 (87.5%) were nalidixic acid resistant. S. Paratyphi A strains showed higher rate of resistance (93.1%) towards nalidixic acid than S. Typhi (81.48%). These trends have also been noted by Shirakawa et al.18 in which the resistance to nalidixic acid in S. Typhi and S. Paratyphi A was 73.3% and 94.9% respectively.

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Acknowledgements Respectfully, I would like to express my sincere gratitude to Mrs. Sarita Manandhar, Lecturer, Mrs. Manju Shree Hada, HOD, Asso. Prof. Shova Shrestha, former HOD, Asso. Prof. Dr. Bidya Shrestha, Program coordinator, Asso. Prof. Pradeep Sah, former Program Coordinator M.Sc. Microbioogy all the respected teachers and staffs of Department of Microbiology, TriChandra Multiple Campus, Kathmandu and Mrs. Jyoti Acharya, Senior Medical technologist, Sukraraj Tropical and Infectious Disease hospital, Kathmandu for their continuous guidance, all the Patients for being a part of my study and Dr. Prakash Ghimire for moral support and encouragement. I am very thankful to all the laboratory team of hospital and to the microbiology department of NPHL for their support, valuable advices, and cooperation. I also would like to thank Dr. Sabita Dongol. Finally, I express my deepest gratitude to my family members and my friends Microbiologist Mrs. Apsara Bhandari, Mr. Kamal Rai, Mr. Santosh Thapa, Mrs. Pooja Poudel, Mr. Sailesh Raj Dhungana and Mr. Bibek Limbu for their various supports.

References 1. Crump JA and Mintz ED (2010). Global trends in typhoid and paratyphoid fever. Clin Infect Dis. 50(2): 241â&#x20AC;&#x201C;246.

2. Rahman AKMM, Ahmad M, Begum RS, Hossain MZ, Hoque SA, Matin A, Yeasmin L and Mamun MGS (2011). Prevalence of typhoid fever among the children in a semi urban area of Bangladesh. J Dhaka Med Coll. 20(1): 37-43.

3. "Typhoid Fever". World Health Organization. Retrieved 2007-08-28.

4. Maskey AP, Basnyat B, Thwaites GE, Campbell JI, Farrar JJ and Zimmerman MD (2008). Emerging trends in enteric fever in Nepal: 9124 cases confirmed by blood culture 1993-2003.Royal Society of Trop Med and Hyg. 102: 91-95.

5. Acharya

D,

Trakulsomboon

S,

Madhup

SK

and

Korbsrisate

S

(2012).

Antibioticsusceptibility pattern and the indicator of decreased Ciprofloxacin susceptibility of Salmonella enterica serovar Typhi isolated from Dhulikhel Hospital, Nepal. Jpn J Infec Dis. 65(3): 264-267. www.microbiologyworld.com

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6. Adhikari D, Acharya D, Shrestha P and Amatya R (2012). Ciprofloxacin susceptibility of Salmonella enterica serovar Typhi and Paratyphi A from blood samples of suspected enteric fever patients. Int J Infect Microbiol 1 (1): 9-13.

7. Shakva KN, Baral MR and Shrestha R (2008). A study of atypical manifestations of entericfever in children, J Nepal Health Res Counc 6(12): 1-4.

8. Versa G, Jaspal K and Chander J (2009). An increase in enteric fever cases due to Salmonella Paratyphi A in and around Chandigarh. Indian J Med Res.129: 95- 98.

9. World Health Organization (2003). Background document: The diagnosis, treatment and prevention of typhoid fever. WHO/V&B/03.07. Geneva: World

Health

Organization.

10.

Raza S, Tamrakar R, Bhatt CP and Joshi SK (2012). Antimicrobial Susceptibility

Pattern of Salmonella Typhi and Salmonella Paratyphi A in a Tertiary Care Hospital J Nepal Health Res Counc. 22: 214-7.

11.

Neupane A, Singh SB, Bhatta R, Dhital B and Karki DB (2008). Changing

spectrumof antibiotic sensitivity in enteric fever. KUM J. 6(21): 12-15.

12.

Acharya D, Malla S, Bhatta DR, Adhikari N and Dumre SP (2012). Current

Fluoroquinolone

Susceptibility Criteria for Salmonella Needs Re-evaluation.

Kathmandu Univ Med J. 37(1):24-9.

13.

Bhandari A(2013). Reduced Ciprofloxacin susceptibility pattern among the

Salmonella isolates from the enteric fever suspected patients visiting Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu. M.Sc. Dissertation submitted to Central Department of Microbiology, St. Savier College, Tribhuwan University pp 2850.

14.

Khatiwada S (2006). Study of Prevalence of Enteric Fever and Assessment of

Widal Test in the Diagnosis of typhoid Fever. A M.Sc. Dissertation

Submitted to

The Central Department of Microbiology, Tribhuvan University.

15.

Rai K (2013). Salmonella serotypes and their antibiogram in enteric fever patients

visiting Nepal Police Hospital, Kathmandu. M.Sc. Dissertation Submitted to www.microbiologyworld.com

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Department of Microbiology, Tri-Chandra Multiple Campus, Tribhuwan University, pp 29-47.

16.

Chaudary A, Gopalkrishanan R, Senthur NP Ramasubraminian V, Ghafur KA and

Thirunarayan MA (2013). Antimicrobial Susceptibility Testing of Salmonella enterica serovars in Tertiary Care hospital in South India. Indian J Med Res.137 (4): 800-802.

17.

Capoor MR and Nair D (2010). Quinolone and Cephalosporin Resistance in

Enteric Fever. J Glob Infect Dis. 2(3): 258â&#x20AC;&#x201C;262.

18.

Shirakawa T, Acharya B, Kinoshita S, Kumagai S,Gotoh A and Kawabata M

(2006). Decreased susceptibility to fluoroquinolones and gyrA gene mutation in the Salmonella enterica serovar Typhi and Paratyphi A isolated in Katmandu, Nepal, in 2003. DiagnMicrobiol Infect Dis. 54: 299-303.

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Designing and Development of Bioassay Faiza Ahad Khan1, Hasnain Nangyal2 1 2

Department of Microbiology Jinnah University for Women, Karachi

Department of Botany, Faculty of Sciences, Hazara University, Mansehra Khyber Pakhtoonkhwa Corresponding Email: hasnain308@gmail.com

Abstract Bioassay or biological assay/screening is any qualitative or quantitative analysis of substances that uses a living system, such as an intact cell, as a component. Bioassays are typically conducted to measure the effects of a substance on a living organism or other living samples.

Essential components of bioassays/assays are Stimulus (Test sample, drug candidate, potential agrochemical, etc), Subject (Animal, Tissues, Cells, Sub-cellular organelles, Biochemicalâ&#x20AC;&#x2122;s, etc., Response (Response of the subject to various doses of stimulus). It is not only used to standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptic, but it also helps to avoid the use of whole organism for testing the pharmacokinetics properties of newly synthesized drugs and related leading compounds on human. Certain complex compounds like Vitamin B-12 which cannot be analyzed by simple assay techniques, but it can be effectively estimated by bioassays.

Introduction It is advancement in biological experiment, which is used to compare biological effect produced by test substance, with that of standard preparation and analyses how much test substance is required to produce some biological effects as produced by the stander (MjaW 20th Sept 2013 ).Between potency of influenced factors of environment judge the behavior and survival of organisms (pest, fungi, and bacteria) in this environmental analyses the structure activity relationship (SAR) and the qualitative (statistical problem include) and quantitative (statistical problems not include) ( MjaW 20th Sept 2013), most of the experimental techniques may be www.microbiologyworld.com

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same but the difference created in statistical analysis, is used to measure concentration of experimental objects (pesticides, fungicides, drug, vitamins and plant extract etc.) in comparison between particular tested substances and concentration with known amount of same substances(R.Srivastava et all).. For this purpose scientists use different biological activities such as animal-based biological assays (analysis for organismsâ&#x20AC;&#x2122; biological response towards end product), cell culture-based biological assays (at cellular level, analyze biochemical and physiological response) and biochemical assays (analysis of biological activities in a living cell).

In developmental years of bioassay, it was used to standardize many drugs, specifically extraction from organic sources with a complex chemistry. (Heparin, hormones, insulin etc.) And some newly discovered autacoids rely heavily on bioassay (amines, prostanoids, no etc). It is also helpful in increasing the precision and accuracy of all physiochemical methods, which are used to analyze the presence in concentrations of particular objects and absence of appropriate chemical or biochemical method. (Fent, 1998).

As we discussed above typical bioassay involves stimulus, applied to subject. Varieties of dose are used for the intensity of different stimulus in experiments. Application of stimulus is followed by a change in measurement of its characteristics such as weight of the whole subject or some particular organ, analyzed value of blood sugar content or bone ash percentage, occurrence or non-occurrence of a certain muscular contractions, symptoms of dietary deficiency or death is the response of subject. ( Parascandola et al,1981 & Gohlke et al, 2002)

Principle of bioassay The bioassay compares the test sample with a same internationally applicable standard substance. It determines the quantity of test sample required to produce an equivalent biological response to that of standard substance. Standard samples are accepted by expert committee at international level and they represent fixed units of activity(self written)

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Types Of Bioassay

Direct Assay: first we perform this type of bioassay. For this assay test, standard preparations of doses are enough to manufacture a specified response. This assay is directly measure. Example of direct assay is cat.

Indirect Assay: Secondly we perform this type of assay. In this we firstly ascertain the relationship between the dose and response of each preparation, then the dose related to a particular response is acquired from the relation for each preparation separately.

The bioassay systems vary based on the biological system used like animals (mouse, rat, guinea pig, rabbits etc), plant bioassay (using plant constituents to evaluate a sample like(hemolytic activity) microbiological or cell based assay (using microbes like bacteria, fungi or cultured cells for anti biotic compound screening etc). Based on techniques they can be differentiated into two broad types like, two types of technique are including in bioassay (1978, statistical methods of biological assay, 3rd addition)

Vitro technique: Bioassays perform in glass ware (outside the organism) Examples of In vitro bioassay: Activity Assay (DPPH assay, Xanthenes oxidize inhibition assay), Bioassay (DNA level, protein level, RNA level) cell based.

Vivo technique: Bioassay performs in living organism such as anti-diabetic assay, CNS assay, and antihypertensive assay. Example of in vivo bioassay: Animal Toxicity (acute toxicity, chronic toxicity), Animal study (animal model with induced disease, animal model with induced injury). Preclinical trials (clinical trials)

Fields of Application In different fields of application bioassay is applied. Mainly five fields of application of bioassays can be distinguished: regulatory purposes, effect screening, biomonitoring and early www.microbiologyworld.com

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warning systems, research and teaching, and hazard/risk assessment. Regulatory purposes: internationally there are variable structure of environmental laws and regulations; one may summarize that the aim and level of environmental protection is defined in National environmental laws, by referring to environmental standards. Environmental standards can be different in standards, Bioassays are used to define and control the application of both standards emission and emission, especially emissions standards (Peters, 1999).

Effect screening: Sometimes bioassays are applicable in order to get an overview of toxic potentials (effect screening) in ecotoxicological studies which cover huge number of samples. It also gives a rough classification of toxic and non-toxic samples (ecotoxicological methods as well as chemical analysis) in a cost-effective manner.

Biomonitoring and early warning systems: Bioindicator: organisms, which accumulate substances and respond sensitively when any change in environmental conditions takes place, are use in biomonitoring to record and evaluate the state of the environment. (Plachter, 1991; Klein & Debus, 1994).

Research and teaching: The kind of application of bioassays in research and teaching varies, but it can be summarized, that the general aim of their application is to gain knowledge about the effect of substances on ecosystems.

Hazard/risk assessment: Hazard risk assessment of a substance or sample is another important application of bioassay. While for hazard assessment the toxic potential of a substance or sample is evaluated, it should predict the harmful effects of the substance or sample in an ecosystem (Peters, 1999).

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Bioassay and Pharmaceutical Companies In the past, most drugs were either discovered by trial or error (traditional remedies) or by serendipitous discoveries. After suffering 20 years of decline, natural product field shows new distinction. Many objects like plants, animals, and micro-organisms have always been playing a role in production of contrasting compounds. Modern techniques either analytical or genetic make us able to isolate and extract the leading compounds with its defined biological activity which can be used as antibiotics and pesticides. Among these, the ones that are most rapid and specific take as effective bioassays. Micro-organisms (bacteria, fungi, actinomycetes) use as assay organism specifically when work to isolate new antibiotics. The bioassay of Antibiotics is done by using a genetic technique; used widely all over the world, which is disc diffusion technique (susceptibility test). In initial developmental years small glass capillary tubes are used for susceptibility test for Antibiotics, the compound (leading material that needs to be checked) is filled in these tubes and placed in the center of plates, which contain the object on the surface of solid agar or gel. Then this modified and well diffusion technique was introduced in which compounds are filled on wells make on solid media or gel surface. But now a dayâ&#x20AC;&#x2122;s 2 techniques are widely used for this purpose of automated assays disc diffusion method (discs containing the compound) and micro titer plates. Micro titer plates allow the testing of large numbers of samples in a short time. Once a target compound has been successively identified, the validity test will perform on it (J.-P. Salvador et al.).

Hallmarks of a Good Assay:

Precision: Is a measure of reproducibility and can be thought of as the closeness to each other of individual estimates when they are repeated. The index of precision of a good bio-assay should be of the order of 5% (Standard Deviation/Mean x 100%). Precision will obviously depend on the type of bio assay used.

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Accuracy: Accuracy is how close the answer is to the actual. In simple bioassays in vitro this implies actions on the same receptor but since the same response can also be obtained from different drugs acting on the same receptor, or via different receptors, an inaccurate answer might be obtained.

Speed: It helps if an answer can be obtained quickly, but this is generally at the expense of precision (and often, but not necessarily, accuracy) ( MjaW 20th Sept 2013)

Conclusion Bioassay is any qualitative or quantitative analysis of substance that uses a living system.

Typical bioassay involves stimulus, applied to subject. Application of stimulus is followed by a change in measurement of its characteristics which is the response of subject. The sample of biological assay is compared with a same internationally applicable standard substance to determine the ratio of required sample and of standard substance. For direct assay, preparations of doses are enough for a specified response whereas in indirect assay the link between the dose and response of each preparation is needed. Indirect assay then requires the relation for each preparation individually. Bio assay is used in regulating drugs, vaccines, toxins or antiseptic and in testing the pharmacokinetics of newly synthesized drugs and leading compounds. Application of bioassay involves effective screening to overview toxic potentials, biomonitering for evaluation of environment, Researching about positives responses to ecosystem as well as the hazardous effects of sample on the ecosystems. Modern techniques make us able to isolate and extract the foremost compound with its defined biological commotion, which can be used as antibiotics or pesticides. Bioassays have appeared as a great milestone in the success rate of pharmaceutical companies through different modified techniques. The traits of appropriate bioassays are measures of reproducibility, accuracy and speed which depend upon precision.

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Refernces 1. http://www.math.iitb.ac.in/~ashish/workshop/gmsahaw3.pdf 2. http://apt.med.ubc.ca/files/2014/09/404_Bioasays_2014_walker_notes.pdf 3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444097/ 4. http://www.mdpi.com/1424-8220/12/7/9181 5. http://www.sednet.org/download/Part_E_Bioassays_as_tool_for_dredged_material_quality_ assessment_(POR_II).pdf 6. file:///C:/Users/Farah%20Maalik/Downloads/Salvador%202007_Comp.%20Anal.%20Chem _Ch2.8.pdf 7. https://kb.osu.edu/dspace/bitstream/1811/3443/1/V44N06_278.pdf 8. http://cw.prenhall.com/bookbind/pubbooks/brock/chapter11/deluxe.html 9. http://ei.cornell.edu/toxicology/bioassays/daphnia/culture.asp 10. http://ei.cornell.edu/toxicology/bioassays/lettuce/RefTest.asp 11. http://ei.cornell.edu/toxicology/bioassays/lettuce/ 12. http://ei.cornell.edu/toxicology/bioassays/lettuce/RefTest.asp 13. http://www.enviroliteracy.org/article.php/1231.php 14. http://ei.cornell.edu/toxicology/bioassays/lettuce/ 15. http://www.apsnet.org/education/labexercises/fungistaticagent/default.html 16. http://www.scielo.br/scielo.php?pid=S1984-82502013000400015&script=sci_arttext 17. http://www.annualreviews.org/doi/abs/10.1146/annurev.en.07.010162.002253?journalCo de=ento 18. http://www.iqac.csic.es/index.php?option=com_content&view=article&id=750%3Aappli cation-of-bioassaysbiosensors-for-the-analysis-of-pharmaceuticals-in-environmentalsamples&catid=136%3Apublications&Itemid=98&lang=en 19. http://www.bsac.org.uk/susceptibility_testing/guide_to_antimicrobial_susceptibility_testi ng.cfm

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20. http://www.microbiologyonline.org.uk/misac.htm 21. http://www.chemicool.com/definition/bioassay.html 22. . http://www.answers.com/topic/bioassay?cat=health 23. Fent, K. (1998): Ökotoxikologie. 1. Edition., Georg Thieme Verlag, Stuttgart 24. Parascandola, J. The theoretical basis of Paul Ehrlich’s chemotherapy. J. Hist. Med. All. Sci.1981, 36, 19–43.

25. Gohlke, H.; Klebe, G. Approaches to the description and prediction of the binding affinity of small-molecule ligands to macromolecular receptors. Angew. Chem. Int. Ed. 2002, 41, 2645–2676.

26. Peters, C. (1999): Die "Belastbarkeit" von Ökotoxizitätstests aus naturwissenschaftlicher und umweltrechtlicher Sicht aufgezeigt an Beispielen des Gewässerschutzes. Diplomarbeit an der Universität Hamburg, Institut für Allgemeine Botanik und Botanischer Garten.

27. Plachter, H. (1991): Naturschutz. UTB Gustav Fischer, Stuttgart.

28. J.-P. Salvador et al.(2007)

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IL-17 and its role in autoimmune disease modulation Rakesh Kumar1, Mukesh Bhatt2 1 2

Division of Pathology, ICAR-IVRI, Izatnagar, Bareilly, U.P. 243122 Division of Virology, ICAR-IVRI, Izatnagar, Bareilly, U.P. 243122 Corresponding Email: rakudoc@gmail.com

Introduction IL-17 first cloned in 1993 from a murine cDNA library and known originally as CTLA-8 (Rouvier et al., 1993; Yao et al., 1996) and human IL-17A shows 63% similarity with murine IL17A. .Based on the structure similarity, IL-17 family comprises six members -IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F (Xinyang Song, Youcun Qian). IL-17F -most similar to IL17A. IL-17E -least similar to IL-17A (Iwakura Y et al). Uncommitted (naive) Th cells differentiate to particular lineages Th1, Th2, Th17 and Treg depending upon mode of stimulation, antigen concentration, co-stimulation, and cytokine and differentiation factors.Th17 or Th1 are pleiotropic cytokine producing cells with multiple: 1. Pro-inflammatory functions (Singh et al, 2013) 2. Progression of autoimmune diseases (Weaver et al., 2006) Treg. Cells are anti-inflammatory lineage- cause quiescence of Ads. Cellular sources of IL-17: Th17, γδT, NK cells, monocytes, neutrophils, epithelial cells, paneth cells, local tissue cells-like epithelial cells, keratinocytes, macrophages, T cells and B cells are main sources of IL-17 (Korn et al., 2009). If IL-17 cytokine is over-expressed, which up-regulate and synergize with local inflammatory mediators such as IL-6 and TNF-a, nitric oxide etc. leads to conditions like, SLE (Wong et al., CK, 2000), Psoriasis (Arican et al., 2005; Homey et al., 2000), Multiple sclerosis (Matusevicius et al., 1999), Rheumatoid arthritis, Systemic sclerosis (Kurasawa et al., 2000), Chronic inflammatory bowel disease (Fujino et al., 2003).This can be antagonized by treatment with IL-4, IL-13or anti-IL-17 blocking antibody. Uncommitted Th cells differentiate to: Th1, Th2, Th17 and Treg depending upon differentiation factor involved. www.microbiologyworld.com

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The pathways of differentiation (Nelms et al., 1999) 1. IL-12 [signalling through (STAT)-4] towards Th1.

2. IL-4 (signalling through STAT-6) towards Th2.

3. TGF-beta towards Treg Once differentiated the each committed lineage is characterized by expression of specific transcription factors and cytokines (Szabo et al., 2000; Szabo et al., 2002.).

Fig.1. Origin of IL-17 www.microbiologyworld.com

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Plasticity of Th17 cells: TGF-β has dualistic role in development of Th17 and Treg cells. TGF-β 1. At low conc. Synergizes with IL-6 and IL-21 to promote IL-23 favoring Th17 differentiation.

2. At high conc. suppress IL-23R expression and favor Foxp3+ Treg cells. TGF-β promotes the production of IL-10 & suppresses its production as it supports Th17eff. Production.

What Leads To Autoimmunity

1. IL-23 increase (key player).

2. Increase GM-CSF (by IL-23 from Th17) 3. IFN-gamma and IL-4 reduction.

4. IL-6 &TGF-β reduction.

5. IL-23 can also inhibit Foxp3 gene expression while it synergizes with TGF-β to elevate the level of IL-17.

6. Th17 cells that lack the ability to produce GM-CSF do not transfer autoimmune disease.

7. GM-CSF is important for the pathogenicity of Th17 cells.

8. TGF-β suppresses its production.

9. IFN-gamma and IL-4 inhibit naïve not committed 10. IL-2, IL-15, IL-18 and IL-21 stimulate IL-17 production

Cross Talk: Th1 has reciprocal relationship with Th17 &Th17 inversely related with Treg.

To summarize, in modulation of autoimmunity three main player are involved while others mediators helps them in this regulation. These are, IFN-gamma (Th1), IL-10 (Treg.). IL-23 involved in IL-17 production (Hoeve et al., 2006)

Microbial agents used as modulator: 1. CFA or BCG leads to IL-17, IL-22, IL- 10, and IFN-γ production. www.microbiologyworld.com

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2. Zymosan and CFA stimulate TGF-beta production from DCs. 3. Zymosan induces DCs to secrete IL-10 and IL-12.

4. BCG vaccination is not effective in humans so far 5. Treg cells grown with IL-23 – Pathogenic.

6. DCs require IL-27R on T cells for IL-10 production.

Conclusion Role of Th17 cells in autoimmune diseases is pro-inflammatory as well as immunoregulatory.

Th1 has reciprocal relationship with Th17 & Th17 inversely related with Treg. IL-23 is key player in differentiation and generation of pathogenic Th17 cells. Th1 and Th17 cells are able to cross regulate each other and lead to autoimmunity. Dendritic cell keep balances between Th1, Th17 and regulatory T cells. The plasticity of Th17 cells can give rise to Th1 cells. BCG or CFA induce Th17 response are protective in autoimmune diseases.

References 1. Singh, B, Schwartz, J.A.et.al. Modulation of autoimmune diseases by interleukin (IL)-17 producing regulatory T helper (Th17) cells. Indian J Med Res. November 2013; 8:591594.

2. Afzali,B,Lombardi,G.et.al.The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. 2007 British Society for Immunology, Clinical and Experimental Immunology, 148: 32–46.

3. Song,X ,QianY. IL-17 family cytokines mediated signaling in the pathogenesis of inflammatory diseases. Cellular Signaling 25 (2013) 2335–2347.

4. Nikoopour§, E,Jordan A.S.et.al. Therapeutic Benefits of Regulating Inflammation in Autoimmunity. Inflammation & Allergy - Drug Targets, 2008;7, 203-210 .

5. Lee,Y.K.Ryuta Robin,M.et.al. Developmental plasticity of Th17 and Treg cells.Opinion in Immunology 2009; 21:274–280. www.microbiologyworld.com

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6. Hawiger,D & Flavell,R.A. Regulatory T cells that become autoaggressive . Nature immunology.september 2009; 10:9 7. Korn T et al. IL-17 and Th17 Cells. Annu Rev Immunol 2009; 27:485–517.

8. Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol 2010; 10(7):479–89.

9. Iwakura Y et al. Functional specialization of interleukin-17 family members. Immunity 2011; 34(2):149–62.

10. Nakae S et al. Suppression of immune induction of collagen-induced arthritis in IL-17deficient mice. J Immunol 2003; 171(11):6173–7.

11. [17] Yang XO et al. Regulation of inflammatory responses by IL-17F. J Exp Med 2008; 205(5):1063–75.

12. [18] Hu Y et al. IL-17RC is required for IL-17A- and IL-17F-dependent signaling and the pathogenesis of experimental autoimmune encephalomyelitis. J Immunol 2010; 184(8):4307–16.

13. Wong CK et al. Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 2000; 9(8):589–93.

14. Wong CK et al. Hyperproduction of IL-23 and IL-17 in patients with systemic lupus erythematosus: implications for Th17-mediated inflammation in auto- immunity. Clin Immunol 2008; 127(3):385–93.

15. Rizzo HL et al. IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL17A. J Immunol 2010; 186(3):1495–502.

16. Ito R et al. Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice.

Biochem Biophys Res Commun 2008; 377(1):12–6.

17. Emamaullee JA et al. Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 2009; 58(6):1302–11.

18. Miossec P. Interleukin-17 in rheumatoid arthritis: if T cells were to contribute to inflammation and destruction through synergy. Arthritis Rheum 2003; 48:594–601. www.microbiologyworld.com

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19. Wong CK, Ho CY, Li EK, Lam CW. Elevation of proinflammatory cytokine (IL-18, IL17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 2000; 9:589–93.

20. Arican O, Aral M, Sasmaz S, Ciragil P. Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity. Mediat Inflammation 2005; 2005:273–9.

21. Homey B, eu-Nosjean MC, Wiesenborn A et al. Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis. J Immunol 2000; 164:6621–32.

22. Hoeve MA, de Savage ND, BT et al. Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells. Eur J Immunol.2006; 36:661–70.

23. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. The IL-4 receptor: signaling mechanisms and biologic functions. Annu Rev Immunol 1999; 17:701–38.

24. SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000; 100:655–69.

25. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 2002; 295:338–42.

26. Rouvier E, LucianiMF,Mattei MG, Denizot F, Golstein P. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene. J Immunol 1993; 150:5445–56.

27. Yao Z, Timour M, Painter S, Fanslow W, Spriggs M. Complete nucleotide sequence of the mouse CTLA8 gene. Gene 1996; 168:223–5.

28. Wong CK, Ho CY, Li EK, Lam CW. Elevation of proinflammatory cytokine (IL-18, IL17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 2000; 9:589–93.

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29. Arican O, Aral M, Sasmaz S, Ciragil P. Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity. Mediat Inflammation 2005; 2005:273–9.

30. Homey B, eu-Nosjean MC, Wiesenborn A et al. Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis. J Immunol 2000; 164:6621–32.

31. Matusevicius D, Kivisakk P, He B et al. Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Mult Scler 1999; 5:101–4.

32. Kurasawa K, Hirose K, Sano H et al. Increased interleukin-17 production in patients with systemic sclerosis. Arthritis Rheum 2000; 43:2455–63.

33. Fujino S, Andoh A, Bamba S et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003; 52:65–70.

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Single cell oil (SCO): A product of oleaginous microorganisms Mr. Shaikh Rajesh Ali 1 1

Assistant Professor, Dept. Of Microbiology, Acharya Prafulla Chandra College, New Barrackpore, Kolkata -700131, West Bengal, India.

Email: raaz_boseinst@yahoo.co.in

Introduction Microbial lipids, which are also known as single cell oils (SCO), are produced by oleaginous microorganisms including oleaginous bacteria, yeast, fungus and algae through converting carbohydrates into lipids under certain conditions.

Single cell oil might be defined as the edible oils (ARA, DHA etc.) obtainable from microorganisms being similar in type and composition to those oils and fats from plants or animals.

Single cell oils (SCO) are the edible oils extracted from microorganisms the singlecelled entities that are at the bottom of the food chain. The best producers with the highest oil contents are various species of yeasts and fungi with several key algae that are also able to produce higher level of nutritionally important PUFA. Their potential to produce PUFA has now galvanized the current interest in these SCO as oils rich in highly desirable fatty acids essentially for our well-being and not readily available either from plants or animals.

They are usually used to be added in infant food for required nutrition.

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Composition of Single Cell Oils SCO encompass the oils containing high amounts of polyunsaturated fatty acids (PUFAs), particularly PUFAs having long carbon chains, such as Omega-6 fatty acid arachidonic acid (ARA) and Omega-3 fatty acids docosahexanoic acids (DHA).

These acids are found in human milk and proved indispensable for the development of infants.

Since there are no alternative plant sources of ARA and DHA, various researchers sought to develop SCOs that were rich in ARA and DHA. Indeed, many industrial processes for SCO production are currently on the run.

In addition to the products currently on the market, there are a number of cross-over technologies taken from the growing bioenergy industry where organisms grown to produce algal oil for biofuels were found to produce oils relatively rich in the desired Omega-3 fatty acids.

Microorganism used in SCO production Although all microbes have the ability to produce some amount of lipids for their structural components such as membranes, only a few of them are able to accumulate lipid in amounts which are of commercial importance. Those microorganisms which have the ability to amass lipids more than 20% of their dry cell weight are characterized as oleaginous microorganisms.

Oleaginous microorganisms fall within the groups of bacteria, algae, yeasts and fungi, the properties and the compositions of the oils varying on the species. ď&#x192;&#x2DC; Among these, yeasts and fungi are the most efficient producers of oils. They produce a variety of oils that are structurally similar to plant oils. Moreover, they produce larger amounts of oils than other microbes and their oils can be easily extracted. These characteristics are considered important in order permit an industrially feasible production. ď&#x192;&#x2DC; Micro algae also produce up to 20-40% (w/w dry weight) lipid content and their lipids include several polyunsaturated fatty acids of dietary significance. However, they require specific growth conditions such as warm temperature, sunshine and clean water making them a not so suitable choice for industrial applications. Furthermore, special recovery www.microbiologyworld.com

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techniques are needed for the extraction of algal oils, thus further restricting their use.

However, micro algae are an excellent choice to be used in waste or sewage treatment plants thereby permitting the production of oils simultaneously with the bioremediation process. ď&#x192;&#x2DC; Bacteria, however, are not extensively used as oil producers. Though many species of bacteria do produce lipids, only a few of them accumulate high enough amounts of extractable lipids. Some species of bacteria such as Mycobacterium, Corynebacterium and Rhodococcus accumulate up to 30-40% of lipids but these lipids are hard to extract and are associated with toxic or allergic factors, making them undesirable as edible oil producers.

Following are microorganisms used in SCO production- Botryococcus braunii, Chlorella sp., Crypthecodinium cohinii, Cylindrotheca sp., Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Nannochloropsis sp., Neochloris oleoabundans, Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp., Tetraselmis sueica, Ulkenia sp., Euglena, Yarrowia lipolytica GM.

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Production way These organisms can be grown in fermentors or in outside ponds (where the climate permits).

Regardless of the method employed, the subsequent processing would be the same; namely separation of the biomass from the liquor, drying of the biomass, recovery of the microbial or single cell oil and the subsequent refining of the oil. The processing steps for making single cell oils are shown in Figure:

Industrial microbial oil refining technology Microbial oil (single cell oil) is obtained from microbes with high dry cell-based oil up to 70% such as bacteria, mold, yeasts and algae, which is a Triglyceride polyunsaturated

(TG) fatty

consists acids

of

multiple

(PUFA).

Microbial

fermentation oil is a great method to develop new oil source and acquires various types of fatty acids. www.microbiologyworld.com

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1. Bacterium selection- The current microbial oil materials mainly include alga, yeasts and molds.

2. Bacteria cultivating expansion- The cultivation medium composition and conditions directly affect microbial oil quality and oil yield.

3. Dry bacterium pretreatment- The bacterium pretreatment by organic solvent extraction is key technology of microbial oil refining.

4. Microbial oil extraction- Organic solvent such as ether, isopropyl ether, chloroform, ether-ethanol, petroleum ether, chloroform-methanol makes effective oil extraction from granulated dry bacterium with high extraction equipment utilization, less powder in mix oil, good quality crude oil, no blocking of extraction pipe system. Keep temperature of no more than 50 degrees during granulation to avoid oil and fat oxidation, and recycle solvent through distillation under reduced pressure.

5. Microbial Oil refinery- High quality microbial oil is available by processing of hydration, degumming, alkali refining, decolorization by active white clay and evaporation.

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The Advantages and Disadvantages of Single Cell Oils The synthesis of lipids by microorganisms has several advantages over the production of plant or animal derived oils. Microbes have shorter life cycles than plants or animals, warranting a rapid production. Unlike other sources, there is no requirement for farm lands and therefore the effect of climatic factors on the production is insignificant. Moreover, microbial fermentation involves less labour and the scaling up the production is easier than in other methods.

On the other hand, the microbial synthesis of lipids is not as economical as the plant oil production due to the high cost of the carbon sources and the requirement of sophisticated extraction techniques. Furthermore, the oil yields which can be obtained by microorganisms are comparatively lower than that with plants or animals.

However, these organisms can be genetically engineered to utilize cheaper substrates such as industrial wastes. Another more economical approach is modifying these organisms into producing more value-added specialty fats and oils or products which canâ&#x20AC;&#x2122;t be extracted through other sources. This will be more profitable than modifying plant oils into products of higher value such as cocoa butter.

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You can also send your articles to

info@microbiologyworld.com Selected ones will be published in our next issue of Sept-Oct 2015.

Thanks, Sagar Aryal Editor-In-Chief Microbiology World

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