View with images and charts Incidence of Pathogens and Their Drug Susceptibility Pattern in Blood and Urine Culture INTRODUCTION 1.1 Background Blood and Urine are the most commonly received specimen in routine microbiology laboratories, with many thousands of antimicrobial sensitivity results being issued each day (Barrett et al., 1999). The bacteriological examination of the urine is must for the diagnosis of urinary tract infection. Bacterial bloodstream infections are a leading cause of morbidity and mortality worldwide. In the United States approximately 200,000 patients develop bacteramia of fungaemia annually with estimated associated mortality ranges of 20 of 50% and septicaemia is the 13th leading cause of death. In addition, antimicrobial resistance in some of the most frequent bacterial species isolated from blood such as Staphylococcus aureus or Streptococcus pneumoniae has reached worrying levels (Decousser W.J, 2003). The urine and blood specimen is easy to obtain and can be collected in several different ways. A quantitative culture result can help diagnose significant bacteriuria and is performed by most laboratories.
Microorganisms are causative agents of many diseases. The identification of the bacteria causing the disease is often essential for the life and wellbeing of a patient. Blood borne pathogens are bacteria, viruses and parasites found in human blood and other body fluids. They can infect and cause disease in humans (Fatima Hamadi1 et at., 2008). Two pathogens recently receiving the greatest attention are the Hepatitis B virus (HBV) and Human Immunodeficiency Virus (HIV). Other pathogens which can also be of concern are Herpes, Meningitis, Tuberculosis, Epstein-Barr Virus, Lyme Disease, Malaria, and Syphilis, to name a few (Roy K.R., 2001). In healthy persons, properly obtained blood specimens are sterile. Although microorganisms from the normal respiratory and gastrointestinal flora occasionally enter the blood, they are rapidly removed by the reticuloendothetial system. Blood culture is the single most important procedure to detect systemic infection due to bacteria. If a blood culture yields microorganisms, this fact is of great clinical significance provided the contamination should be excluded. Urine secreted in the kidney is sterile unless
the kidney is infected. Uncontaminated bladder urine is also normally sterile. The urethra, however, contains a normal flora, so that normal voided urine contains small numbers of bacteria. Because it is necessary to distinguish contamination organisms form etiologically important organisms, only quantitative urine examination can yield meaningful results (Jawetz, 2001). The wide variability of clinical symptoms and the ongoing difficulties concerning the rapid and specific laboratory diagnosis, contribute to the fact the sepsis primarily remains a clinical diagnosis. To contribute to a more tailored antibiotic coverage of the patient early in the course of the disease, modern diagnostic concepts favour the qualitative and quantitative molecular biological detection of blood stream pathogens directly from whole blood. This offers a very attractive alternative to the currently applied less sensitive and much more timeconsuming blood culture-base laboratory methods. Moreover, recent study results suggest an increasing impact of molecular detection methods with short turn-around times for more effective treatment and better outcomes of patients with sepsis and septic shock. In the short term, such tests will not substitute conventional blood culture despite their superior rapidity and sensitivity, mainly because of higher cost. The amazing speed of ongoing scientific developments means, however, that techniques that might appear complicated, labour intensive and costly today, will develop to become the future standards in the microbiological diagnosis of patients with sepsis and septic shock. (Barrett p., et. Al., 1999). In this context, the current study was designed to isolate and identify the etiological agents of blood and urine infections. Besides these, antimicrobial resistance and sensitivity pattern of the isolate was also determined to find out suitable prophylactic agent to treat the above mentioned infections. 1.2 Literature review 1.2.1 Urinary tract infections
The anatomical structure of the mammalian urinary system is such that the external genitalia and the lower aspects of the urethra are normally contaminated with a diverse population of microorganisms. The tissues and organs that compose the remainder of the urinary system, the bladder, ureters, and kidneys are sterile and therefore urine that passes through these structures is also sterile. When pathogens gain access to this system, they can establish
infection (Cappuccino, 1996). Urinary tract infections (UTIs) are some of the most common infections experienced by humans, exceeded in frequency among ambulatory patients only by respiratory and gastrointestinal infections. It is also the most common cause of nosocomial infections in adults. E.coli is the leading pathogen and it was significantly more predominant in bacteremia Urinary tract infection (UTI) than non bacteremia UTI. Escherichia coli is the commonest cause of community and nosocomial urinary tract infection (UTI). Urinary tract infection (UTI) is a broad term that encompasses both asymptomtic microbial colonization of the urine and symtomic infection with microbial invasion and inflammation of urinary tract structures. Apart from the outer one-third of the female urethra, the urinary tract is normally sterile. From a microbiologic perspective, urinary tract infection exists when pathogenic microorganisms are detected in the urine, urethra,, bladder, kidney, or prostate. In most instances, growth of more than 105 organisms per milliliter from a properly collected midstream “clean-catch” urine sample indicates infection. The kids of infections that ESBLproducing E.coli can cause range from urinary tract infection, to – at the more serious end of the spectrum-case where they enter the bloodstream and cause blood poisoning. Infections with ESBL- producing E.coli are most common amongst the elderly, or those who have recently been in hospital or received antibiotic treatment. ESBL-producing E.coli are extremely rare in simple cystitis (Kenechukwu et al., 2000). 1.2.2 Circulatory system and blood infection The main components of the human circulatory system are the heart, the blood, and the blood vessels. An average adult contains five to six quarts (roughly 4.7 to 5.7 liters) of blood, which consists of plasma, red blood cells, white blood cells, and platelets. Normally blood is sterile, bacteria occur transiently in the blood steam. Bacteremia may be a phase in the natural course of some infections such as typhoid fever and brucellosis and meningococcal infection, it also occurs as a spill-over effect in a serious infection when the patient’s defenses become inadequate, as in severe pneumonia or an extended soft tissue infection. Bacterial bloodstream infections are a leading cause of morbidity and mortality worldwide. (Alferd et al., 2005). Blood culturing is the “gold standard” for diagnosis of bloodstream infections (BSI). The identification of bacteremia and fungemia by culturing blood remains one of the most important roles of the microbiology laboratory. Most infections are caused by many different microbes, so it’s important to figure out which of them is causing the infection. A blood culture can be performed to determine whether the infectious agent is in bloodstream. Symptoms of an infection in bloodstream might include a high fever or chills. Blood culture procedure must design to overcome the intermittency and low order of magnitude bacteremias and fungemias and inhibit any antimicrobial properties or components of the blood. Among the seven variables affecting yields, the volume of blood cultured appears to be most important (Washington et al., 2000). Septicaemia denotes as overwhelming invasion of the blood steam from a focus of infection. The distinction between bactaemia and septicaemia is essentially clinical but there is a quantitative implication. Thus septicaemia is thought of as a life-threatening emergency that must be dealt with urgently (Andrew et al.,1989). 1.2.3 Microorganisms associated with blood and urine
Human microflora has been shown to contain microorganisms which refer to bacteria, microscopic fungi and protozoa. Some of them are intracellular parasites. Unfortunately, currently available methods are not sufficiently rapid and universal for slow growing bacteria, anaerobes, nonfermenters and other extraordinary microbes. Bacteria, the most familia of infectious agents, cause 90 percent of hospitalized infections in developing countries, although they compete with viruses for being the most diversified. For the most part, bacteria are the smallest free-living organisms in nature, having all the genetic material necessary to live independently. This is not to say that many of them don’t enjoy off other organisms, but as a class they are complete life forms unto themselves. Bacteria cause most of the serious, short-term infections we get and can be stopped with medications called antibiotics (Cappuccino, 1996). A sample of urine from a patient with a suspected urinary treat infection is the most common type of specimen received by most clinical microbiological laboratories. The commonest condition of UTI is cystitis, due to infection of the bladder with a uropathogenic bacterium, which most frequently is Escherichia coli but sometimes Staphylococcus saprophyticus or especially in hospital-acquired infection, Klebsiella spp, Proteus mirabilis, other coliforms Pseudomonus aeruginosa or Enterococcus faecalls. Candida infection may occur in diabetic and immunocompromised patients. Rare infection organisms including Sreptococcus agalactiae, Steprococcus milleri, other, Streptococci, anaerobic Streptococci and Gardnerella vaginalis (Collius et al., 1986). More serious bacterial infections are acute phelitis and pyelonephuritis, in which the symptoms usually include join pain and fever and accompanied by a bactaeremia detectable by blood culture. The causative organism may be any of those that cause cystitis but, Staphylococcus aureus is responsible for some the cases (Stamm et al., 1980). Some bacterial and fungal agents of urinary tract diseases are illustrated bellow. Organisms other than bacteria may also act as etiological agents of urogenital infection. Trichomonas vaginalis, a pathogenic flagellated protazoa is coomonly found in the vagina and under appropriate condition it is responsible for a severe inflammatory vaginitis. Candida albicans, pathogenic yeast is normally found in low numbers in intestines.
Bacteria
Gram Negative
Gram Positive
Streptococcus aureus
Enterococci
Streptococcus pyotenes Escherichia coli
Streptococcus faecalis
Psendomonas aerugnosa
Streptococcus faecium
Proteus uvlgsris Klebisella pneumoniae Figure 1.1: Bacterial agents of urinary tract diseases
Fungi
Candida albicans
Blastomyces dermatitidis
Coccidioides immitis
Figure 1.2: Fungal agents of urinary tract disease (Cappuccino, 1996).
1.2.3.1 Escherichia coli Escherichia coli (E.coli) is a bacterium that commonly lives in the intestines of people and animals. Most of the E.coli are normal inhabitants of the small intestine and colon and are non-pathogenic, meaning they do not cause disease in the intestines. Nevertheless, these nonpathogenic E.coli can cause disease if they spread outside of the intestines, for example, into the urinary tract (Where they cause bladder or kidney infections) or into the blood stream (sepsis) (Mitchell E., et al 2000).
1.2.3.2 Acinetobacter baumannii
Acinetobacter baumannii is a nonfermentive, aerobic, opportunistic, gram-negative coccobacillary rod. Morphological findings vary according to the phase of cell growth and exposure to antimicrobial agents. Acinetobacter baumannii commonly isolated from the hospital environment and hospitalized patients. This organism is often cultured from hospitalized patients sputum or respiratory secretions, wounds, and urine. In a hospital setting, Acinetobacter commonly colonizes irrigating solutions and intravenous solutions. Acinetobacter species have low virulence but are capable of causing infection. Most Acinetobacter isolates recovered from hospitalized patients, particularly those recovered from respiratory secretions and urine, represent colonization rather than infection (Shih1 M J 2007). 1.2.3.3 Staphylococci Staphylococci are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes. Staphylococcus aureus form a fairly large yellow colony on rich medium; S.epidermidis has a relatively small white colony. S. aureus is often hemolytic on blood agar; S. epidermidis is non hemolytic. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. The bacteria are catalasepositive and oxidase-negative. S.aureus can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations as high as 15 percent. Nearly all Strains of S.aureus produce the enzyme coagulate; nearly all strains of . lack this enzyme. S.aureus should always be considered a potential pathogen; most strains of S.epidermidis are nonpathogenic and may even play a protective role in their host as normal flora. Staphylococcus epidermidis may be a pathogen in the hospital environment (Bae. T. 2004). 1.2.3.4 Candida Candida is yeast and the most common cause of opportunistic mycoses worldwide. It is also a frequent colonizer of human skin and mucous membranes. Candida is a member of normal flora of skin, mouth, vagina, and stool. As well as being a pathogen and a colonizer, it is found in the environment, particularly on leaves, flowers, water, and soil. Infections caused by Candida spp. Are in general referred to as candidiasis. The clinical spectrum of candidiasis is extremely divers. (Jo-Anne H et al., 2001). Candidacies may be superficial and local or deep-seated and disseminated. Disseminated infections arise from hematogenous spread from the primarily infected locus. Candida albicans is the most pathogenic and most commonly encountered species among all (Abi-Sai et al 1997). 1.2.3.5 Salmonella typhi Salmonella typhi is part of the Enterobacteriaceae family; it is a Gram-negative motile, aerobic rod which is faculttively anaerobic and there is serological identification of somatic and flagellar antigen (Reveendran R et al. 2007).
1.2.4 Antibiotics Antibiotics are used for treatment or prevention of bacterial infection. Antibiotics may be informally defined as the subgroup of anti-infective that are derived from bacterial sources and are used to tract bacterial infections (M.K et al 1898). Some antibiotics are bactericidal (kill bacteria), others are bacteriostatic (arrest bacterial growth). Some antibiotics have a broad spectrum of activity, being active against a wide range of pathogens; others have a narrow spectrum of action. Streptomycin was the first major antibiotic to be discovered after penicillin. Many of the newer penicillins, such as ampicillin, such as ampicillin, possess a much broader spectrum of activity, some of them being active against Gram negative bacteria. Despite some adverse reactions in the human, effective antibiotics have been developed that have one or more of these modes of action on the bacterial cell: A. Inhibition of cell wall synthesis B. Alternation of cell membranes C. Inhibition of protein synthesis D. Inhibition of nucleic synthesis E. Antimetabolic activity or competitive antagonism (Warren J.W, 1999). 1.2.4.1 Treatment of urine infection: The major organisms causing UTIs are the Coliforms, Staphylococcus aureus and Proteus spp. Antibiotics which have been recommended to treat UTIs includes Ampicillin, Trimethoprim-Sulfamethoxazole, Flouroquinolones and Nitrofurantion. However due to incessant abuse and misuse of these antibiotics, extensive resistance of micro-organisms to these antibiotics has developed. The usual treatment for both simple and complicated urinary tract infections is antibiotics. The type of antibiotic and duration of treatment depend on the circumstances (Mezue Kenechulwu et al, 2004). For patients with troublesome dysuria, phenazopyridine may help control symptoms until the antibiotics do (usually within 48 h). Ceftriaxone 125 mg IM plus either azithromycin or a fluoroquinolone is given for 10 to 14 day. For non-STD urethritis in men, trimethoprimsulfamethoxazole or a fluoroquinolone is given for 10 to 14 days; women are treated with a regimen for cystitis asymptomatic bacteriuria in pregnant women is actively sought and treated as a symptomatic UTI, although many antibiotics cannot be safely used. Orar β-
lactams, sulfonamides, and nitrofurantoin are considered safe in early pregnancy, but sulfonamides should be avoided near parturition because of a possible role in the development of kernicterus. Common regimens include ampicillin plus, gentamicin and a Fluoroquinolone, and broad-spectrum cephalosporins (eg, ceftriaxone, Aztreonam, β lactam/β-lactam inhibitor, combinations (ampicillin-sulbactam) and imipenem-cilastatin clavulanate, piperacillin-tazobactam ticarcilli are generally reserved for patients with more complicated pyelonephritis (Mayo Clinic, 2007). 1.2.4.2 Treatment of blood infections Identifying the specific causative agent ultimately determines how sepsis is treated. However, time is of the essence, so a broad-spectrum antibiotic or multiple antibiotics will be administered until blood cultures reveal the cause and treatment can be made specific to the organism. Intravenous antibiotic therapy is usually necessary and is administered in the hospital. A number of different types of medications are used in treating sepsis. They include: Antibiotics, vasopressors, activated protein and others. Treatment with antibiotics begins immediately – even before the infectious agent is identified. The antibiotics are administered intravenously (IV). People with severe sepsis usually receive supportive care including intravenous fluids and oxygen (Mayo Clinic, 2007). 1.2.4.3 Trends of antibiotic resistance in Bangladesh All antibiotics cause risk of overgrowth by non-susceptible bacteria. Excessive or inappropriate use may promote growth of resistant pathogens. Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome and a rate of about 1 in 10 8 per chromosomal replication. Some common pathogens are now resistant to antibiotics previously used frequently for treatment. Some strains of Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa are resistant to almost every antibiotic available. Uropathogens are resistance to commonly used antibiotics is causing concern in several other countries. Although rates of resistance to antibiotic in Bangladesh is about 60%. In Bangladesh, a resistance rate 18% to ciprofloxacin has also been reported and in Spain the resistance rate to norfloxacin is 13%. 1.3 Aims and Objectives Probably 10% or fewer of asymptomatic bacteriuria patients develop renal failure attributable to the infection; hypertension is even rare. Chronic urinary tract infection is eradicate by short-term therapy (2-6 weeks) in about 25-35% of patients. Some of the others have relapses caused by the same organism; some have re-infection caused by other organisms. Again, infection in blood is very much fatal and can cause meningitis and other serious complications. So, antibiotic should be advised more carefully by keeping in mind the after effect of antibiotic resistance. The present study had following aims and objectives: a) b) c) d)
To isolate and identify the causative agents of urine infections. To isolate and identify the causative agents of blood infections. To determine the antibiotic sensitivity and resistance pattern of isolated pathogens. To find out the most suitable antimicrobial agents for treatment of urine and blood infections.
MATERIALS AND METHODS 2.1 Collection site and Number of specimens The blood and urine samples were collected from the out-patients and admitted patients of United Hospital Limited, Gulshan, Dhaka from 10 October 2011 to 31 December 2011. A total 250 clinical specimens of blood and urine were cultured for isolation and identification of aerobic bacteria and antimicrobial susceptibility testing was also carried out. 2.2 Specimen collection and handling 2.2.1 Collection of blood samples Venous blood samples were aseptically collected into blood culture bottles using sterile needles and syringes. The blood culture bottles utilized in this study were the BD BACTED PLUS Aerobic/ F and Anaerobic/ F (Becton Dickinson and Company). 10 ml of venous blood were collected from adult patients 50 ml blood culture bottles, while 5 ml were taken in the case of children.
Fig 2.1: Blood culture vial. 2.2.2 Collection of urine samples In case of urine sample collection, patients were usually asked to submit mid-stream urine samples about 10-20 ml for analysis and culture, preferable the first urine excreted by the patient early in the morning in the commercial urine bottles. 2.3 Inoculation of the specimens The bottles with specimens were immediately placed in the automated BACTEC 9120 incubator system. This system incubates specimens at 350 C with continuous agitation and uses a fluorescent technology to detect the quantity and rate of CO 2 production (indicative of microbial growth) in every 10 min. Blood culture bottles were removed from the incubator after the automated system after determining that they were positive. After the incubation period, they were inoculated onto MacConkey agar and blood agar and incubated at 37 0 for 24.48 hours. The urine samples were immediately inoculated onto MacConkey agar, blood agar, and then the plates were incubated at 37 0 C for 24.48 hours. Each sample was plated onto 5% sheep blood agar and MacConkey agar using a calibrated 100p, delivering 0.01 ml of the sample. This was incubated at 370 C overnight and the observation was made the next day.
For the culture of blood and urine, 4 types of media were used: 1. 2. 3. 4.
Blood agar for isolation of pathogenic bacteria, MacConkey agar for isolation of Enterobacterioceae, SDA agar for culture the fungal strains, and Mueller-Hinton agar medium for antibiotic susceptibility test.
2.4 Microscopic examination of urine The microscopic examination of urine sample was performed as a wet film of uncentrifuged urine to determine whether polymorphs (pus cells) are present in numbers which indicated the infection in the urinary tract. 2.5 Identification of isolates Following the incubation, colonies from the different media were characterized and identified using standard microbiological and biochemical scheme. The tests included gram and spore test, catalase test and oxidase test. Coagulase test was done for conformation of Staphylococcus spp in the catalase test positive. 2.5.1 Catalase test The demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide. A small amount of the culture to be tested was picked from the agar plate with a clean sterile platinum loop and mixed with 3% hydrogen peroxide solution. The production of bubbles from the surface of the culture indicated the positive reaction. 2.5.2 Tube test/ coagulase test To peform the test one coloni was emulsified with a small amount of citrate-treated plasma, and incubate at 370 C. The reaction is slow and usually requires overnight incubation. Lessvirulent Staphylococci do not produce coagulase and are often collectively referred to as ‘coagulase-negative’. If the test is positive the plasma solidifies into a solid plug. To visualize this, tip the tube and watch the meniscus – if negative, it will remain horizontal; if positive it will rotate with the tube. 2.5.3 Oxidase test The Oxidase test depends on the presence in bacteria of certain oxidases the transport of electron between electron donor in the bacteria and a redox dye – tetramethyle-p-phenylenediamine. 2.5.4 MICROGEN TEST (A biochemical identification system for the common Enterobacteriaceae) The Microgen GN – ID system employs 12 (GN A) standardized biochemical substance in micro wells to identify the family. Enterobacteriacease and other non-fastidious negative bacteria (oxidase negative and positive). The kit is intended laboratory use only. 2.5.4.1 Principle of the test: The Microgen GN – ID system comprises to separate micro well test strips GN A and GN B. Each micro well test strips contains 12 standardize biochemical substrates which have been selected to the basis of extensive computer analysis. Dehydrated substrates in each well are
reconstituted with a saline suspension of the organism to be identified. If the individual substrates are metabolized by the organisms, a colour change occurs during incubation or after addition of specific reagents. The permutation of metabolized substrates can be interred interoperated using the microgen identification system software (MID-60) identify the test organism. The GN A micro well test strip is intended for the identification of oxidase negative, nitrate positive glucose fementers comprising the most commonly occurring genera of the family Enterobacteriaceae. The GNA and GNB micro well test strips are used together to produced a 24 substrate system to identify non-fastidious gram negative bacilli (oxidase negative and positive) in addition to all currently recognized species of the family Enterobacteriaceae.
Figure 2.2: Inoculation of culture in micro wells of microgen test 2.5.4.2 Identification of isolates: On the Microgen GN-ID A+B report from, the substrates have been organized into triples with each substrate assigned a numerical value (1, 2, 3 or 4). The sum of the positive reactions for each triplet forms a single digit or the Microgen Identification System Software, which generates a report of the five most likely organisms in the selected database. Table 2.1: List of microorganisms that could be identified by microgen test. Acinetobacter baumannii
Salmonella typhi
Enterobacter aerogenes
Shigella (GroupC)
boydii
Morganella morganii
Enterobacter agglomerans
Salmonella IV
Acinetobacter lwoffii
Salmonella cholerae-suis
Salmonella Group V Yersinia enterocolitica
Proteus mirabilis
Enterobacter gergoviae
Salmonella Group I
Acinetobacter haemolyticus
Salmonella Paratyphi A
Salmonella Illa
Proteus vulgaris
Enterobacter sakazakii
Salmonella Group II
Serratia liquefaciens
Citrobacter freundii
Salmonella
Enterobacter
Klebsiella oxytoca
Group Serratia rubidaea
Klebsiella pneumoniae
Group Serratia Marcescens
gallinarum
cloacae
Providencia rettgeri
Escherichia coli
Shigella (Group D)
Citrobacter diversus
Salmonella pullorum
Hafnia alvei
Providencia stuartii
Escherichia Inactive
coli- Providencia alcalifaciens
Edwardsiella tarda
Shigella dysenteriae
sonnei Klebsiella ozaenae Klebsiella rhinoscleromatis Shigella (Group B)
flexneri
Salmonella Group VI
2.6 Antibiotics susceptibility test by Disk Diffusion method: (the Kirby Bauer technique) Mueller-Hinton agar was considered to be the best for routine susceptibility testing which was done by Kirby-Bauer technique. 2.6.1 Inoculation of the Mueller-Hinton agar plate with test organism: The isolated colony from the various media was inoculated on the Mueller-Hinton agar plate by the spreading technique. 2.6.2 Application of Discs to Inoculated Agar Plates: 1. The antimicrobial discs were dispensed onto the surface of the inoculated agar plate. Each disc must be pressed down to ensure complete contact with the agar surface. Whether the discs were placed individually or with a dispensing apparatus, they must be distributed evenly so that they are no closer than 24 mm plate or more than 5 discs on a 100 mm plate. Because some of the drug diffuses almost instantaneously, a disc should not be relocated once it has come into contact with the agar surface. 2. The plates were inverted and placed in an incubator set to 350C for over night. The antibiotic disk potency and the specific organism for the specific antibiotic are: Table 2.2 Antibiotic potency of the various antibiotics used in the study Antibiotic
Antibiotic potency (Îźg)
Organism
Ampicillin
10
Gram positive
Azithromycin
15
Gram positive
Amoxyclve
30
Gram positive
Vancomycin
30
Gram positive
Penicillin G
10 Units
Gram positive
Doxycylin
30
Gram positive
Gentamincin
10
Gram positive
Imipenem
10
Gram positive
Amikacin
30
Gram positive
Amoxycilin
10
Gram positive
Azithronam
30
Gram positive
Ceftazidime
30
Gram positive
Ceftriaxone
30
Gram positive
Ciprofloxacin
5
Gram positive
Morepenem
10
Gram positive
Cotrimoxazole
25
Gram positive
Imipenem
10
Gram positive
Nalidixic Acid
30
Gram positive
Nitrofurantion
300
Gram positive
Zone sizes were measured from the edge of disc to the zone which is given in the following table. Table 2.3 Interpretation of antibiotic susceptibility
Zone size
Interpretation
1. Equal to wider than or not more than 3 Susceptible mm smaller than the control 2. Zone size greater than 3 mm, but smaller Intermediate than the control by more than 3 mm 3. Zone sizes 3 mm or less
Resistant
RESULTS 3.1 The number of patients, age and sex distribution The total numbers of 250 patients were studied in this research work, and among them 111 were female and 139 were male. 115 blood and 135 urine samples were collected from the patients and the age range was 0 to 90 years. Table 3.1 the age and sex distributions of the patients
Age range-0 to 90 years Sex Male
Female
Type of Specimen
No. of Sample
No. of Positive Culture
Blood
67
18(26.86%)
Urine
72
30(41.66%)
Blood
48
12(25%)
Urine
63
17(26.98%)
Table 3.1 shows that out of 115 patients blood culture were positive in 30(26.08%) study cases, out of 67 male patients blood culture were positive in 18(26.86%) and out of 48 female patients blood culture were positive in 12(25%) patients. No significant difference was observed in between sex groups. A total 30 strains of bacteria were isolated. Among them, A.baumannii 7(23.33%) most common predominantly islolated bacteria followed by S.paratyphi A 6(20.00%), S.typhi 5 (16.67%), E.coli and Pseudomonas sp. there were same 3(10.00%) in each bactria. Furthermore, Klebsiella sp. 2(6.67%), Candida sp. 2(6.67%), Enterobacter sp. 1(3.33%) and S.epidermidis 1(3.33%) were isolated. Table 3.2: Prevalence of microorganism in specimen collected from male / female patients in blood culture Organisms
Numbers identified organisms
of Percentage
Acinetobacter baumannii
7
23.33%
Pseudomons sp
3
10%
S.epidermidis
1
3.33%
Salmonella typhi
5
16.67%
Salnonella Paratyphi “A�
6
20.00%
Klebsiella sp
2
6.67%
Candida sp
2
6.67%
Enterobacter sp
1
3.33%
E.coli
3
10%
Total number
30
Table (3.1) shows that out of 135 patients urine culture were positive in 47 (34.81%) study cases. Out of 72 male patients urine culture were positive in 30(41.66%) and out of 63 female patients urine culture were positive in 17(26.98%) patients. No significant difference was observed in between sex groups.
Table 3.3: Prevalence of microorganism in specimen collected from male / female patients in urine culture Organisms
Numbers of Percentage identified organisms
E.coli
26
55.32%
Candida
4
8.51%
Acinetobacter baumunnii
3
6.38%
Pseudomonas sp.
4
8.51%
Beta haemolytic Streptococcus
2
4.26%
Non haemolytic Streptococcus
3
6.38%
Klebsiella sp
4
8.51%
Serratia sp
1
2.13%
Total number
47
A total 47 strains of bacteria were isolated. Among them E.coli 26 (55.32%) was the most common predominantly isolated bacteria followed by klebsiella sp., Pseudomonas sp., Furthermore, Acinetobacter baumannii, Non heamolytic Streptococcus 3(6.38%) each were isolated. 3.2 Pus cell count in urine sample Counts of the pus cells were given in table 3.4 Table 3.4: Enumeration of pus cells in urine sample Sample (n=22)
number Pus cell count/H.P.F Age (years) (high power field)
Sex
Sample 1
50-60
55Y
Male
Sample 2
30-40
69Y
Female
Sample 3
30-40
66Y
Female
Sample 4
20-30
58Y
Male
Sample 5
10-15
69Y
Female
Sample 6
8-10
40Y
Female
Sample 7
8-10
37Y
Female
Sample 8
5-8
42Y
Female
Sample 9
5-7
65Y
Female
Sample 10
3-5
26Y
Male
Sample 11
2-4
65Y
Male
Sample 12
2-4
45Y
Male
Sample 13
2-3
39Y
Female
Sample 14
2-3
53Y
Male
Sample 15
2-3
65Y
Female
Sample 16
2-3
47Y
Female
Sample 17
1-2
85Y
Male
Sample 18
1-2
20Y
Female
Sample 19
1-2
15Y
Female
Sample 20
Uncountable
33Y
Female
Sample 21
Uncountable
27Y
Female
Sample 22
Uncountable
31Y
Female
3.3 Identification of isolates 3.3.1 Culture characteristics of different microbes Cultural characteristics of isolated microbes are given in the table 3.3 and following figures. Table 3.5: Colony morphology of different types of microorganisms on different media Microorganisms
Media
Colony characteristics
E.coli
MacConkey agar
Small, pink colony
Acinetobacter spp
Blood agar
Non-Lactose fermenting round shape cology
K. pneumonia
MacConkey agar
Mucoid, large pink colonies
Candida sp.
Saboroud dextrose agar
Mucoid, colonies
Enterobacter sp
Blood agar
Gray white small colony
Salmonella paratyphi
Blood agar
Gray white small colony
Pseudomonas sp.
MacConkey agar
Small transparent, colonies
Staphylococcus epidermidis
Blood agar
Large white colonies with zones of B heamolysis
wet
convex
Figure 3.1 Culture of E.coli in MacConkey agar plate form urine sample.
Figure 3.2 Culture of Enterobacter in MacConkey agar plate from blood sample
Figure 3.3 Culture of Pseudomonas sp. in MacConkey agar plate 3.2.2 Gram staining
All the microorganisms were negative except S. epidermidis and S. aureus. Results of Gram staining of the isolates are given below. Table 3.6: Results of Gram staining Microorganisms
Gram reaction
Pseudomonas sp.
Gram Negative
E.coli
Gram Negative
Enterobecter sp.
Gram Negative
Staphylococcus epidermidis
Gram Positive
Staphylococcus aureus
Gram Negative
Klebsiella sp.
Gram Negative
Acinetobacter baumannii
Gram Negative
Salmonella paratyphi paratyphi “A�
and
salmonella
Gram Negative
Table 3.7 Results of oxidase tests, Catalase tests and coagulage tests
Sample
Colony characteristic Oxidase test Catalase and media test
Urine
Small pink colony on MacConkey agar
-
E.coli
Blood
Non-Lactose fermenting round shape colony
+
Acinetobacter baumonnii
Blood
Non-Lactose fermenting transparent colony of MacConkey agar
+
Salmonella paratyphi A
Blood
Non-Lactose fermenting transparent colony of MacConkey agar
+
Salmonella paratyphi B
Blood and Large white colonies + Urine with zones of a heamolysis
+
Blood and Non-Lactose
+
+
Coagulage test
-
Presumptive result
S.epidermidis
Pseudomonas
Urine
fermenting grape like colony
Blood
Mucoid, colonies
pink -
+
Klebsiella pneumoneae
Urine
Pin point colony on blood agar
-
Enterobacter sp.
large
sp.
3.3 Results of Biochemical test (Microgen test) After reading the result of Microgen GN ID A and B considering the microgen idenfication system software, the following organisms were identified (Table 3.6 and 3.7). The distribution of the isolates and their sources are presented in table. The microorganisms identified in the blood were candida sp, E.coli, S.epidermidis, salmonella para typhi “A�. Enterobacter sp, Acinetobacter baumannii, salmonella typhi. Pseudomonous, Klebsiella sp. The most frequently occurring organism in blood samples was Acinetobacter baumannii (23.33%) followed by S. Paratyphi A (20.00%)
Figure 3.4: Interpretation and result of microgen tests Out of the 47 positive cultures of urine, E.coli was most frequent (55.32%) and candida sp. (8.51%), Klebsiella sp. (8.51%), Pseudomonas sp. (8.51%) were the second highest in occurrence. And other organisms that were isolated from the urine specimens were beta haemolytic Streptococcus, Serratia sp, Acinetobacter sp, Non-haemolytic Streptococcus sp. 3.4 Antibiogram of isolates The antibiogram of bacteria associated with the blood and urine of the patients in the hospital were reported here.
Figure 3.5: Drug sensitivity pattern of Pseudomonas spp. on Mueller-Hinton agar plate.
3.4.1 Sensitivity / Resistance of Antibiotics against Microorganisms of urine sample The sensitivity patterns of organisms from urine sample were shown in the table 3.8 for E.coli, table 3.9 for Klebsiella sp. against the following antibiotics: AMC=Amoxyclavonic acid, AK=Amikacin, CAZ=Ceftazidime, CRO=Ceftriaxone, CIP=Ciprofloxacin, MEM=Meropenem, IPM=Imipenem, CN=Gentamycin, CFM= Cefixime, FEP=Cefepime, NET= Netilmycin, SXT= Cotrimoxazole, F= Nitrofurantoin, NA= Nalidixic acid, PB= Polymyxin B, TZP= Tazobactam/Piperacillin
\ Figure-3.6: Sensitivity pattern of E. coli against different types of antibiotics. E.coli were sensitive (100%) to imipenem, Amikacin, Morepenem, Nitrofuration were 84.62% each sensitive. On the other hand, E.coli showed 96.15% resistance against tazobactam and followed by nalidixic acid (88.46%).
Figure--3.7: Sensitivity pattern of Klebsiella sp. against different types of antibiotics. Klebsiella sp. Isolates in this study were found 75% sensitive to amiacin, imipenem, morepenem, tazobactum and 100% resistance to amoxyclave, ciprofloxacin, ceftraxone, ceftazidime, cefepime and gentamycin. Non haemolytic Streptococcus showed 100% sensitivity to amoxyclave, nitrofuration, linezolid, vancomycin and 100% resistance to cefepime, cotrimoxazole, cefixime, nalidixc acid. Sensitivity / Resistance of antibiotics against Microorganisms of blood sample For the blood isolated organisms, the sensitivity and resistant patterns were shown in the table 3.10 for Acinetobacter baumannii, the table 3.11for E. coli, table 3.12 for S. paratyphi A, table 3.13 for Klebsiella sp. and table 3.14 for Pseudomonas sp. against the following antibiotics: AMC=Amoxyclavonic acid, AK=Amikacin, ATM=Aztreonam, AZM= Azithromycin, CAZ=Ceftazidime, CRO=Ceftriaxone, CIP=Ciprofloxacin, MEM=Meropenem, IPM=Imipenem, CN=Gentamycin, Do=Doxycycline, OX=Oxacillin, TZP=Piperacilline/Tazobactam, CFM= Cefixime, FEP=Cefepime, NET= Netilmycin, C=Chloramphenicol, SXT= Cotrimoxazole,
Figure-3.8: Sensitivity pattern of Acinetobacter baumannii against different types of antibiotics. A.baumannii were 100% sensitive to cotrimozazole and 85.71% each sensitive to ciprofloxacin and imipenem. Resistances were found 100% against ceftazidime, gentamicin and netelmicin and 85.71% to amikacin, amoxyclavonic, cefepime and ceftriaxone.
Figure -3.9: Sensitivity pattern of S. paratyphi A against different types of antibiotics. Bacterial isolates of S.paratyphi A showed 100% sensitivity to ceftriaxone and cefepime each. Sensitivity to ciprofloxacin, cefixime, cotrimoxazole, chloramphenicol and Aztreonam were also 100% and resistance to ampicillin was 16.67%. Finally, 100% highly resistivity was found against Azithromycin.
S.typhi were 100% sensitive to ceftriaxone, ciprofloxacin, cefepime again, cefixime and Aztreonam were also showed 100% sensitivity followed by Azithromycin 80%. On the other hand, resistance were observed to cotrimoxazole, chloramphenicol and ampicillin 60% each.
Figure-3.10: Sensitivity pattern of E. coli against different types of antibiotics. E.coli were sensitive (100%) to amikacin, amoxyclavonic acid, cefepime, ceftriaxone, ceftazidime, cefixime, imipenem, netilmycin wrer also showed 100% sensitivity. But were 66.7% resistant to cotrimoxazole followed by ciprofloxacin, gentamycin, Morepenem, Tazobactum 33.33% each..
Figure-3.11: Sensitivity pattern of Pseudomonas sp. against different types of antibiotics. Isolates of Pseudomonas sp. were 100% sensitive to imipenem, morepenem and tazobactum but 100% each resistant to contrimoxazole and cefixime. Amikacin, cefepime, ciprofloxacin, ceftazidime, gentamycin and netelmycin showed 66.7% sensitivity each. However, 66.7% resistances were found against amoxyclave and ceftriaxone.
Figure -3.12: Sensitivity pattern of Klebsiella sp. against different types of antibiotics. Klebsiella sp isolates in this study were found 100% sensitive to imipenem and meropenem, 50% to Amikacin, cefepime and ciprofloxacin. On the other hand, 100% resistance were observed against ceftriaxone, ceftazidime and cefixime, Klebsiella sp showed 50% resistance against contrimoxazole, netelmycin and tazobactum, 50% resistivity was also found against amoxyclavonic acid. 3.4.3 Identification of ESBL and MRSE group There were eleven ESBL producing E.coli and two Klebsiella sp. identified from the urine. One was resistance to 10 antibiotics and another was resistance to 13 antibiotics. The name of the antibiotics against which these bacteria were resistant were presented table 3.8
Table 3.8: ESBL producing microorganisms and name of the antibiotics Number of resistant Name of resistance antibiotics isolates (n=13)
Name of antibiotics
Ecoli (11)
AMC, FEP, CEP, CIP, SXT, 11 CN, NET, CRO, CAZ, NA, F
Klebsiella sp. (2)
AMC, FEP, CEP, CIP, CN, 9 NET, CRO, CAZ, NA,
From the antibiotic sensitivity tests from blood samples one S. epidermidis was isolated that was MRSE. It showed resistance to 7 antibiotics. The name of the resistance antibiotics were given in the following table Table: 3.9 MRSE group and the antibiotics
Number of resistant Name of resistance antibiotics isolates (n=1) 1
Name of antibiotics
AMP, DO, OX, CIP, SXT, CFM, 7 E
DISCUSSION Blood and urine infection is very severe and immediate diagnosis is necessary to find out the causative agents, because both infections may become fatal if untreated. Sometimes patients use antimicrobial agents before diagnosis. But antimicrobial resistance is an issue of great significance for public health at the global level. Considered as wonder drugs, antibiotics are after prescribed inappropriately and inadequately and have thus become one of the highly abused agents. Bacterial pathogens causing acute infections are increasingly exhibiting resistance to the commonly used antibiotics and have become a great threat to public health. The increasing antibiotic resistance problems, largely due to widespread and irrational use of antimicrobial agents in hospitals and the community, is a cause of great concern, especially in developing countries (Lakshmi V., 2008). For this reason, this project work was regarding to the collection of two types of samples e.g. blood and urine and the microbiological study of the pathogens isolated from those samples. A total 250 specimens were collected from the indoor and outdoor patients of United Hospital Ltd. Dhaka. Among the total number of patients, blood samples were collected from 67 male and 48 female. Urine samples were collected from 72 male and 63 from female patients. Firstly, urine was assessed under a high power field (HPF) for the presence of pus cells before microbiological analysis. Two urine samples collected from two female patients had uncountable pus cells which indicated severe urine infections. Some of the urine samples had 30-40, 50-60 pus cells in microscopic examination. That means pathogens caused severe damage in urinary tract so pus cells were released in urine enormously. Normally, there should be only an occasional red blood cell in the urine (2-3 per high power field). Hematuria, the presence of abnormal numbers of red blood cells in the urine may be due to: Glomerular disease, tumors that erode any part of the urinary tract, kidney trauma, renal infarcts, acute tubular necrosis, upper and lower urinary tract infections, traumatic catheterization, passage of renal stones. In male the normal range is 5-8 and in females it is up to 10 per high power field (HPF). All investigations are to be interpreted in the background of patient’s symptoms. Pus cells don’t always mean infection (Shigemura K. et. al., 2005). Some of the urine samples that were containing pus cells showed no growth. The more interesting aspect of this study was use of automatic system named Microgen test by which pathogenic microbes specially members of Enterobacteriocae family were identified my microngen identification system software. The automated system’s ability to enumerate the bacterial populations in the original clinical specimen attained a high degree of accuracy (Isenberg et al., 1979). 47 positive cultures were found from all the urine samples and it was found that Escherichia coli were the most frequent causative agents. E. coli is the most common gram-negative bacterium isolated in clinical laboratories and is the organism responsible for up to 70 to 95% of urinary tract infections (Edberg et al., 1983), Candida (8.51%) was also identified mainly from female patients. Beside these, Beta haemolytic
Streptococcus. Pseudomonas, Klebsiella sp, Serratia sp, Acinetobacter sp etc were found after culturing all the positive samples. Antibiotic sensitivity pattern of E.coli, Pseudomons and was determined by Kirby – Bauer method. All of them sensitivity to imipenem. Because these antibiotics were not widely used in Bangladesh and administered by injection. In the urine culture, E.coli from 18 male patient and 8 female patient, showed resistance to most of the antibiotics which were Extended spectrum Beta Lactamase (ESBL) producing microbes. Few studies have compared the abilities of different laboratory methods to detect ESBL producing organisms among members of the family Enterobacteriaceae (Coudron et al., 1997). ESBLs are capable of hydrolyzing penicillins, many marrow spectrum cephalosporins, many extended-spectrum cephalosporins, oxyimino-cephalosporins (cefotaxime, ceftazidime), and monobactams (aztreonam). Beta-lactamase inhibitors (e.g. clavulanic acid) generally inhibit ESBL producing strains (Anaissie et al., 1997) ESBL producing isolates are most commonly Klebsiella sp, Predominantly Klebsiella pneumoniea, and E.coli, but they have been found throughout the Enterobacteriaeae. In case of blood samples, 30 showed positive results out of 115 samples. Whereas 47 culture of urine showed positive result out of 135 samples. The cause of rest of the samples showed no growth may be the patients were having log time antibiotic treatments. The results revealed the presence of E.coli in 26 (55.32%), candida in 4 (8.51%), Pseudomonas in 4 (8.51%), beta hemolytic streptococcus in 2 (4.26%). Non hemolytic streptococcus in 3(6.38%), Klebsilla sp in 4 (8.51%), Serratia1 (2.13%) in 47 positive urine samples. On the other hand, out of 30 positive blood samples Acenetobacter baumannii was found in 7(23.33%), Pseudomonas in 3(10.00%), Staph epidermidis in 1(3.33%), Salmonella typhi in 5 (16.67%), Salmonella paratyphi A in 6 (20%), Klebsiella sp. in 2 (6.67%), Candida in 2(6.67%), Enteobacter spp in 1(3.33%), E.coli in 3(10.00%). After antibiotic suspectibility test, Staphylococcus Epidermidis from 1 male showed resistance to most to the antibiotics which suggested that it falls under MRSE. MRSE (Methicillin-resistant staphylococcus Epidermidis) is a bacterium responsible for difficult-totreat infections in humans. Like MRSA which is a resistant variation of the common bacterium Staphylococcus Epidermidis. It has evolved an ability to survive treatment with beta-lactam antibiotics. It can cause infection in hospital including central venous catheters associated infections (Graham 2008). Like MRSA there are few treatment options available to treat MRSE. Vancomycin is often the last choice. The Control of emergence and spread of antimicrobial resistance among the most common human bacterial pathogens is probably one of the most important challenges for the scientific and medical community. It is essential to evaluate prospectively the distribution of bacterial species isolated from blood and their susceptibility to the major antimicrobial agents and alternative drugs to adapt antibiotic therapy strategies. The risk of antibiotic therapy strategies. The Risk of antibiotic resistance in bloodstream isolates, particularly Gram-positive cocci, emphasize the importance of hospital control measures, rational prescribing policies and new vaccine strategies (Decousser et al., 2003). Multi drug resistant organisms (MDROs), for e.g. E.coli and Pseudomonas spp were found in this study which was alarming. Resistance factors, particularly those carried on mobile elements, can spread rapidly within human and animal populations. Multidrugresistant pathogens travel not only locally but also globally, with newly introduced pathogens spreading rapidly in susceptible hosts. Antibiotic resistance patterns may very locally and regionally, so surveillance data needs to be collected from selected sentinel sources. Patterns
can change rapidly and they need to be monitored closely because of their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by physicians in that area (Lalitha. M.K.2004). Conclusion: E.coli, candida sp. Klebsiella sp., and Pseudomonas sp. in urine and Acinetobacter baumannii, E. coli, Salmonella paratyhi A and salmonella typhi in blood were highly prevalent of this investigation. The organisms which were isolated from urine culture, were not different from other studies normally which are found from the urine culture, and this sensitivity patterns were also very much same to the other studies. The organisms isolated from blood culture make a big difference from the other studies. In the particular state the majority of Acinetobacter baumannii. Most of the blood cultures were collected from intensive care patients; these patients had coronary heart by pass surgery and they developed complications after surgery. Acinetobacter baumannii is a low grade pathogen and is term as hospital resident pathogen, which are causes hospital infections. Organisms which are resident in hospital such as S.aureus, E. coli, Pseudomonas sp., Acinetobacter baumannii, are very much resistance to all kinds of antibiotics. With the isolation of ESBL, these organisms are resistance to most of the antibiotics except Imipenem and Meropenem. Extensive and indiscriminate use of antibiotics can also rise to resistance to many kinds of antibiotics. It should also be realized that the prevalence of drug resistance in developing nations is rather unique, as the poor environmental sanitation, nutritional deficiencies in the host and certain endemic infections further enhance the risk of dissemination of resistant strains both in the community and in healthcare areas. Monitoring of antimicrobial resistance is the single most important recommendation of many professional societies and national agencies that critically address the growing problem of antibiotic resistance, Sharing of expertise, cooperation, and collaboration between the clinicians using antibiotic therapy and the clinical microbiologists at the regional levels may be the simplest and the most useful public health measure to optimize the use of antibiotics and manage infectious diseases. REFERENCES
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