Antimicrobials in Agriculture
Edited by Arti Gupta and Ram Prasad
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Chapter 1 Antimicrobial Resistance in Lactic Acid Bacteria 1
Sukanya Phuengjayaem, Jenjuiree Mahittikon, and Somboon Tanasupawat
Chapter 2 Antimicrobial Indexing of Indigenous Pathogens from Sanitary Landfill Leachate ........................................................................................................
Rachna Jain and Pritam Dey Chapter 3 Role of Phytoalexins in Agriculture
Sana Sheikh, Sareen Sheikh, Jyothi Miranda, Vishal Moger, Swaroop Kumar V. S., and Jayalaxmi
Chapter 4 Livestock Manure Application Causes the Spread of Antibiotic-resistant Genes in Agricultural Lands 55
Dileep Kumar, Anam Aslam, Ranjana Gautam, Ankita Gupta, Anurag Yadav, and Kusum Yadav
Chapter 5 Antibiotic Residues in Freshwater: Antimicrobial Resistance Development and Bioremediation 70
Vaishnavi Shedge, Shefali Gupta, Nidhi V. Maheshwari, and Seema Sambrani
Chapter 6 Production of Antimicrobial Substances from Endophytic Microorganisms –Potential Applications as Biocontrol Agents in Agriculture ....................................... 85
Pranay Jain and Ram Kumar Pundir
Chapter 7 Antimicrobial Efficacy of Medicinal Plants: Present Status and Future Prospects 96
Aniket Singh and Neela Singh
Chapter 8 Nanomaterials as Alternative Antimicrobial Agents in Management of Phytopathogens in Agriculture 110
Diksha Tajane, Hariprasad Paikrao, and Anita Patil
Chapter 9 Nanotechnology-based Antimicrobials in Agriculture 133
Thenmozhi Manivel, Sathammaipriya, Steffi P. F., and Thirumalaiyammal B.
Index 150
Preface
Antimicrobials have been used in agriculture for decades, with recent estimates revealing that 73 per cent of all antibiotics worldwide are used in agriculture. Agricultural dependence on antimicrobials suggests habitual communication patterns, which are socially and culturally ingrained. Moreover, several reports indicate that antimicrobials are used imprudently in agriculture. For example, antimicrobials are frequently misused for growth advancement and preventative purposes. Antimicrobial resistance in agriculture is a valuable industrial resource that addresses multifaceted topics regarding farm, wild, friendly animals, and how the environment plays an active role in the amplification and transmission of resistant genes in the human food chain. This book explores the various aspects of microbials in agriculture and their perceived impact on human health including updated information about all the genera of livestock and associated pathogenic microbes. The valuable information may be exploited by policymakers and various groups of experts including academic and research personnel engaged in clinical and medical microbiology, along with all veterinary and medical practitioners. Researchers, scientists, and healthcare and management practitioners will find this an essential resource on the legal use of antibiotics in animals and humans.
The first chapter, by Phuengjayaem et al., reviews the antimicrobial resistance in lactic acid bacteria. In Chapter 2, Jain and Dey describe the antimicrobial indexing of indigenous pathogens from sanitary landfill leachate. Extensive studies and application of phytoalexins in agriculture discussed by Sheikh et al. in Chapter 3 and in Chapter 4, Kumar and his collaborators highlight that livestock manure application causes the spread of antibiotic-resistant genes in agricultural lands. In Chapter 5, Shedge et al. emphasize antibiotic residues in freshwater: antimicrobial resistance development and bioremediation. In Chapter 6, Jain and Pundir describe production of antimicrobial substances from endophytic microorganisms and potential applications as biocontrol agents, and in Chapter 7, Singh and Singh highlight the antimicrobial efficacy of medicinal plants in reference to present status and future research. In Chapter 8, Tajane et al. highlight the nano-materials as alternative antimicrobial agents in the management of phytopathogens in agricultural systems. Finally, nanotechnologybased antimicrobials in agriculture are more prominently explained in Chapter 9 by Thenmozhi Manivel et al.
Arti Gupta and Ram Prasad
Contributors
Anam Aslam
Department of Biochemistry, University of Lucknow, Lucknow, India
Thirumalaiyammal B.
Research Scholar PG and Research Department of Microbiology, Cauvery College for Women (Autonomous), Thiruchirappalli, Tamilnadu, India
Pritam Dey
CSIR-National Environmental Engineering Research Institute (NEERI), Kolkata Zonal Centre, i-8, sector-C, EKDP, E.M. Bypass, Kolkata, India
Ranjana Gautam
Department of Biochemistry, University of Lucknow, Lucknow, India
Ankita Gupta
Department of Biochemistry, University of Lucknow, Lucknow, India
Shefali Gupta
Department of Industrial Microbiology, Sam Higginbottom University of Agriculture, Technology and Sciences, Naini, Prayagraj, Uttar Pradesh, India
Pranay Jain
Department of Biotechnology, University Institute of Engineering and Technology, Kurukshetra University, Kurukshetra, Haryana, India
Rachna Jain
CSIR-National Environmental Engineering Research Institute (NEERI), Kolkata Zonal Centre, i-8, sector-C, EKDP, E.M. Bypass, Kolkata, India
Jayalaxmi
Department of Botany, St Aloysius College (Autonomous) Mangaluru, India
Dileep Kumar
Department of Biotechnology, Khwaja Moinuddin Chishti Language University, Lucknow, India
Ram Kumar Pundir
Department of Biotechnology, University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
Swaroop Kumar V. S. Department of Botany, St Aloysius College (Autonomous) Mangaluru, India
Hariprasad Paikrao
Government Institute of Forensic Science, Nagpur, RT Road, Civil Lines Nagpur, Maharashtra, India
Nidhi V. Maheshwari
Department of Biotechnology, S K Somaiya College, Somaiya Vidyavihar University, Vidyavihar, Mumbai, India
Jenjuiree Mahittikon
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
Thenmozhi Manivel
Department of Microbiology, Indra
Ganesan College of Arts And Science, Thiruchirappalli, Tamilnadu, India
Jyothi Miranda
Department of Botany, St Aloysius College (Autonomous) Mangaluru, India
Vishal Moger
Department of Botany, St Aloysius College (Autonomous) Mangaluru, India
Steffi P. F.
PG and Research Department of Microbiology, Cauvery College for Women (Autonomous), Thiruchirappalli, Tamilnadu, India
Anita Patil
Sant Gadge Baba Amravati University, Mardi Raod, Amravati. Maharashtra, India
Sukanya Phuengjayaem
Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
Seema Sambrani
Department of Biotechnology, S K Somaiya College, Somaiya Vidyavihar University, Vidyavihar, Mumbai, India
Sathammaipriya
PG and Research Department of Microbiology, Cauvery College for Women (Autonomous), Thiruchirappalli, Tamilnadu, India
Vaishnavi Shedge
Department of Biotechnology, S K Somaiya College, Somaiya Vidyavihar University, Vidyavihar, Mumbai, India
Sana Sheikh
Department of Botany, St Aloysius College (Autonomous) Mangaluru, India
Sareen Sheikh
Department of Chemistry, Yenepoya Institute of Arts, Science and Management, Yenepoya deemed to be University, Mangaluru, India
Aniket Singh
Department of Zoology, Syamsundar College, Purba Bardhaman, West Bengal, India
Neela Singh Department of Botany, Vinoba Bhave University, Hazaribag, Jharkhand, India
Diksha Tajane Mustard Agronomist, AICRP on Linseed and Mustard, Dr. Punjabrao Deshmukh Krishi Vidyapeeth Akola, MS, India
Somboon Tanasupawat Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
Anurag Yadav
Department of Microbiology, College of Basic Science and Humanities, S. D. Agricultural University, Banaskantha, Gujarat, India
Kusum Yadav Department of Biochemistry, University of Lucknow, Lucknow, India
1 Antimicrobial Resistance in Lactic Acid Bacteria
Sukanya Phuengjayaem, Jenjuiree Mahittikon, and Somboon Tanasupawat
1.1 INTRODUCTION
Currently, antimicrobials are widely used in household products, agriculture, and medicine. Excessive usage of antimicrobial agents is focused on cost-saving and low toxicity of medication selection in drugs (Griffith et al., 2012). Most of the antimicrobials used in medicine are derived from secondary metabolites of plants, fungi, bacteria, and marine organisms. Microbes can exist in a mutually beneficial relationship. Still, when nutrients are in short supply, they can form an antagonistic connection, resulting in the generation of harmful chemicals and metabolites for other species (Bode, 2009). Natural products have been used as a utilized medicine to treat a variety of diseases such as cancer, hypercholesterolemia, and immune dysfunction (Wang et al., 2018; Gora et al., 2021; Huang, Lu and Ding, 2021).
Antimicrobial usage worldwide between 2000 and 2015, antibiotic consumption climbed by 65%, while the antibiotic use rate increased by 39% (Klein et al., 2018). Furthermore, global antimicrobial usage is expected to rise by 67% from 2010 to 2030, from approximately 63 thousand tons to over 100 thousand tons (Van Boeckel et al., 2015). Changing production techniques in middleincome countries has contributed to a one-third increase in livestock consumption between 2010 and 2030. Large-scale intensive agricultural enterprises that habitually employ antimicrobials at subtherapeutic levels will replace vast farming systems (Van Boeckel et al., 2015). Even though it is arduous to evaluate the actual numbers of antimicrobial agents used because of the distinction in variability and international registration system, the continued increase global sale of antibiotics could indicate the trend of antibiotic accumulation in the environment (Tiseo et al., 2020). This information may be linked to a global assessment on antibiotic resistance, which predicts that by 2050, with a population of 10 million, there will be one fatality per year (Tong, 2021). Besides the utilization of antimicrobials, the dangers of antimicrobial resistance in microorganisms are the global faces. They are the most serious worldwide health issues. Uncontrolled use of antimicrobial reagents and disposal of wastes without waste categorization are causes of antimicrobial contamination and antimicrobial resistance distribution to the environment (Kimera et al., 2020). Therefore, the study of the involvement between antibiotic usage, the generation, the dispersal of antimicrobial resistance, and multi-resistant bacteria should be considered (Busetti et al., 2017).
Lactic acid bacteria (LAB) are microorganisms widely employed in food manufacturing as probiotics. They are living organisms that provide health advantages to the host (Korhonen, 2010). Therefore, they probably account for the most natural gut microbiome of humans and animals (Álvarez-Cisneros and Ponce-Alquicira, 2018). Currently, the demand for probiotic product usage is a rising trend because most people are concerned about improving their quality of life (Colombo et al., 2018). Good nutrition, physical activity, and a healthy lifestyle are connected to good health and well-being (Ouwehand and Röytiö, 2015). Lactobacillus (Lb), Lactococcus (Lc), Pediococcus (P), Streptococcus (S), Enterococcus (E), and Bifidobacterium (B) strains are employed as probiotics. They are usually found in foods and dietary supplements. Lactobacillus bulgaricus
and Streptococcus thermophilus strains are commonly used in yogurt production (Adolfsson et al., 2004). Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus strains are regularly used in dairy products, for example, milk preservation, cheese, kefir, and buttermilk (Clementi and Aquilanti, 2011; Rakhmanova et al., 2018). Additionally, LAB can be used to deliver vaccines and other substances directly into the digestive tract system (Gad et al., 2014).
Although antibiotic-resistant properties of LAB are useful to humans or animals by assisting in maintaining gastrointestinal equilibrium in situations of antibiotic-induced diarrhoea, they may serve as a reservoir of antibiotic resistance genes that could be transferred onto pathogenic bacteria (Erginkaya et al., 2018). LAB antimicrobial resistance in food products has been discovered as either intrinsic or acquired antimicrobial agents (Mathur and Singh, 2005).
Many antibiotics are thought to be inherently resistant to LAB (Preethi et al., 2017). LAB could be resistant to other antimicrobial agents, thereby spreading resistance to diseases found in the gastrointestinal tracts of both animals and humans. Antibiotic-resistant food-borne organisms have largely been reported, for example, antimicrobial resistance in LAB, isolated from Chinese yogurts (Zhou et al., 2017), traditional Turkish fermented dairy (Erginkaya et al., 2018), faeces of broiler chicken in Malaysia (Shazali et al., 2014). In addition, antibiotic resistance in Lactobacillus, Pediococcus, and Enterococcus strains could be isolated from dry-fermented sausages (Fraqueza, 2015). They contributed to the growing wealth of evidence demonstrating the major function of food-borne LAB as a horizontal reservoir of antibiotic-resistant genes that are transferred horizontally through the food chain to pathogens, posing a significant public health concern. Mostly antimicrobial-resistant, vancomycin has been observed in Enterococcus, Lactobacillus, Lactococcus, Pediococcus, and Bifidobacterium species (Temmerman et al., 2003; Jia et al., 2014; Rozman et al., 2020). Furthermore, bacitracin, cefoxitin, ciprofloxacin, fusidic acid, streptomycin, sulphadiazine, teicoplanin, and vancomycin resistance is intense in some lactobacilli (Danielsen and Wind, 2003).
This raises concerns that the biggest threat presented by antibiotic-resistance genes could be transmitted to humans via the food chain (Flórez et al., 2005; Rodriguez-Alonso et al., 2009) Especially, unheated fermented milk products serve as a medium for antibiotic-resistant bacteria. Indigenous bacteria and the human gastrointestinal tract have a direct relationship (Mathur and Singh, 2005). Cheese made from raw milk contains antimicrobial-resistant LAB, which may act as a vector to create resistance to certain antibiotics in bacteria humans infect (Erginkaya et al., 2018).
As a result, the increased use of LAB in food manufacture as probiotics during the previous few decades has raised safety risks worldwide (Ammor et al., 2007). The significant threat to antibiotic usage is establishing and spreading resistance in pathogenic bacteria that have previously failed to respond to previously effective treatments (Erginkaya et al., 2018).
Most of the research has concentrated on historically clinically relevant bacterial strains. However, there has been an increase in interest in isolated strains that have not been clinically proven in recent years, particularly LAB, which accumulate several antibiotic-resistant genes. Because LAB are generally used in the food industry, they may transfer that antibiotic-resistant gene to pathogenic bacteria and humans and animals (Rodriguez-Alonso et al., 2009; Erginkaya et al., 2018). They could make it more difficult to treat a patient with an infection or disease resistant to antibiotics (Korhonen, 2010).
To acquire a perspective of the current situation in LAB regarding antibiotic resistance trends, a wide range of beneficial bacterial strains belonging to Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, and Pediococcus species are summarized in this chapter.
1.2 ANTIMICROBIALS AND THEIR ACTION IN BACTERIAL CELLS
The antimicrobial categories include resisting cell wall synthesis, destroying cytoplasmic membranes, inhibiting protein synthesis, inhibiting nucleic acid replication, and interfering metabolic pathways of bacteria metabolism (Hooper, 2001; Reygaert, 2018; Álvarez-Cisneros and PonceAlquicira, 2018). The resistance cell wall synthesis is subdivided into three functions: preventing the
polymerization and cross-linking process of cell wall peptidoglycan, blocking cell wall precursors from being transported through the cell membrane, and inhibiting peptidoglycan precursors. The case of destroying the cytoplasmic membrane is subdivided into three functions: depolarization of the cytoplasmic membrane, increasing membrane permeability, removing the membrane, and creating pores (Amerikova et al., 2019). Examples of medications and functions from each of these categories are described in Figure 1.1
The mode of action of antimicrobials for clinical usage is divided into five groups according to the mechanism of antimicrobial agents, which is shown in Figure 1.1
Although the various mechanisms are described above for improved management of pathogens, ineffective antimicrobial treatment has contributed to the numerous antimicrobial resistance problems that are now encountered. Excessive usage of antimicrobial agents since medication selection in drugs is focused on cost-saving and low toxicity (Griffith et al., 2012). Increased antimicrobial medication consumption, both by humans and animals, and unsuitably antimicrobial treatment prescribing are important factors contributing to the rising resistance problem (Victor, 2011).
The research of Morar et al. (2015) reported that microbiological and diagnostic evidence seems to indicate that angiotensin receptor blockers (ARBs) and antimicrobial resistance genes (ARGs) are frequently transferred from animals to humans who consume food animals. Ultimately, people are increasingly struggling with more severe diseases that require more extensive treatment and prolonged disease periods that frequently necessitate protracted hospitalization (Amerikova et al., 2019). The expenses of treating these illnesses have risen because of this. Currently, LAB is widely used in the fermented food industry (Amerikova et al., 2019). However, it is important to note that antimicrobial resistance genes are also discovered in the LAB. Therefore, it is extremely important
FIGURE 1.1 Antimicrobial action in bacterial cells.
Source: (Hooper, 2001; Reygaert, 2018; Álvarez-Cisneros and Ponce-Alquicira, 2018).
to pay attention to the dosage, waste disposal methods to the environment, and applications from bacteria that may have drug-resistance genes attached to them. The WHO recommends that LAB used in the food industry be free of resistance (Álvarez-Cisneros and Ponce-Alquicira, 2018).
1.3 SOURCE OF ANTIMICROBIAL CONTAMINATION AND ACCUMULATION IN THE ENVIRONMENT
1.3.1 AntimicrobiAl contAminAtion
Antimicrobials are discarded from the household via the trash bin, clothing, toilets, pharmacies, etc., when unused, expired, or when treatment is finished (Bound and Voulvoulis, 2005). The households release wastewater into suburban canals, city canals, and rivers, respectively. Antimicrobial lists are found in the aquatic environment comprised of sulfamethoxazole, sulfamethazine, sulfapyridine, trimethoprim, erythromycin, clarithromycin, roxithromycin, oleandomycin, oxytetracycline, tetracycline, and lincomycin (Binh et al., 2018). Furthermore, several personal care products containing triclosan, such as handwash soaps, act as a powerful antimicrobial selection source in residential wastewater (Jutkina et al., 2018).
Antibiotics have gradually increased use in the agricultural industries to treat animal infections caused by bacteria as therapeutic usage, to prevent bacterial infections as prophylactic usage, and to reduce management costs by decreasing investment (Kimera et al., 2020). Tetracyclines are used as growth promotors in livestock food additive forms (Rugumisa et al., 2016). Recently, sources of antimicrobial substances have been easily accessed from many resources such as official and unofficial dealers, who provide minimal information to farmers that initiate drug misuse. Human medicines are usually taken for therapeutic and prophylactic treatments. Human drugs (amoxicillin, ampicloxacillin, chloramphenicol, doxycycline, chloramphenicol eye drops, ciprofloxacin, metronidazole, and tetracycline) are used in pig agriculture and poultry (Kimera et al., 2020). Overabundantly using antibiotics is a concern in the public health problem involved with antimicrobial resistance.
The widespread growth of aquaculture is rapidly increasing in the world. Bacterial infection in wild and cultured fish causes huge mortality (Aly and Albutti, 2014). Antibiotics are not only used to enhance growth but also to treat and prevent bacterial infection in aquatic animals (Zhao et al., 2021). After antimicrobial use, over 80% of antimicrobial reagents that were taken in the aquacultural farm were contaminated into the environment, leading to rising antimicrobial resistance via the transition of resistance or multi-resistance genes. Further, the resistance genes such as quinolones, tetracyclines, and β-lactamases, which originated in aquatic bacteria, spread to normalfloras, fish pathogens, and human pathogens (Cabello et al., 2013).
Antimicrobial and pharmaceutical compounds for patient treatment are usually used in the hospital. The wastewater from the hospital is frequently released into natural environments without wastewater management. Pharmaceutical compounds are collected as drug effluents from the hospital, including chlorotetracycline, ciprofloxacin, cyclophosphamide, demeclocycline, paracetamol, metoprolol, diclofenac, ibuprofen, 17β-estradiol, estriol, estrone, doxycycline, oxytetracycline, tetracycline, trimethoprim, sulfamethoxazole, and ifosfamide. These pharmaceutical drugs have analgesic, anti-inflammatory, antirheumatic, hormone contraceptive, and antibiotic activities (Thomas et al., 2007). Over 90% of antimicrobials taken by humans are metabolized and secreted in the faeces forms. Most metabolized antimicrobials in waste products still have antimicrobial functions caused by antimicrobial spreading into environments via wastewater contaminated with resistant bacteria. Additionally, these antimicrobials affect pH, aquatic organic and inorganic compounds, and antimicrobial resistance of organisms in the marine and terrestrial ecosystem. Therefore, the multidrugresistant bacteria from the hospital sewage are isolated and demonstrate the expansion of drug and multidrug resistance into the environment (Serwecińska, 2020). Pharmaceutical industries release a large number of antimicrobial residues and other co-factors, comprising metal and surfactants,
into sewage (Pruden et al., 2013). Ofloxacin, sulfamethoxazole, trimethoprim, azithromycin, clarithromycin, cefotaxime, cephalexin, and cefixime from the pharmaceutical industry are reported to be released in the aquatic environment in Vietnam (Binh et al., 2018).
1.3.2 AntimicrobiAl AccumulAtion in the environment
Eluents from wastewater treatment facilities, the pharmaceutical sector, the agricultural sector, and surface runoff from animal feeding activities are the main sources of antimicrobials in groundwater and surface water (Walters et al., 2010). Nevertheless, due to the high expenses of their usage, these procedures may not be used in all circumstances, and the amount of antimicrobial degradation varies depending on the category of pharmaceuticals and their compounds (Thiele-Brun and Peters, 2007; Mitchell et al., 2015). Furthermore, many traditional wastewater treatments in facilities are not removed and medications and antibiotic residues have also been discovered in the final leaching of wastewater treatment facilities throughout the world (Serwecińska, 2020). Especially hospital wastewater is the source of pathogens and antibiotics that have no normative status. In most countries, hospital wastewater is no separate category of wastewater that is particularly harmful to the environment and enters municipal wastewater treatment. As a result, for these pollutants, a monitoring system is required (Urbaniak et al., 2017). Sewage sludge is regularly distributed over the soil as a feature of agricultural operations because of its high number of organic compounds and nutrients. It is distinguished by high quantities of antibiotic resistance bacteria (ARB), and antibiotic resistance genes (ARG) expelled from entering wastewater. It might be harmful to the environment as well as human health (Calero-Cáceres et al., 2014; Koch et al., 2021).
Antibiotics have been usually found in surface water with concentrations in the range of 0.01 to 1.0 g/L. It indicates that they have migrated into a supply of drinking water sources and that
FIGURE 1.2 Source of antimicrobial release and accumulation in the environment.
traditional drinking water, due to treatments, may not be able to eliminate them. They can also survive for months in the sea. Quinolones, macrolides, sulfonamides, and chloramphenicol are the most commonly discovered antibiotics in drinking water, with ciprofloxacin belonging to the quinolones class (De Kwaadsteniet et al., 2006). According to Vignesh et al. (2016), antibiotic-resistant bacteria were found in water and sediment samples collected in ecoregions along the Chennai coast. Vancomycin and penicillin had the highest antibiotic-resistant bacterial strain frequencies, with 53.6% and 52.6%, respectively. The antibiotics chloramphenicol, ciprofloxacin, gentamicin, and tetracycline were the most efficient against bacteria, with a frequency of less than 7%. Additionally, Harnisz (2013) reported that fluoroquinolone-resistant and tetracycline-resistant bacteria were frequently entered into river water samples downstream (Harnisz, 2013). In surface waters such as lakes, ponds, and rivers, multidrug-resistant opportunistic pathogens with genes resistant to carbapenem, a last-resort antibiotic, pose a severe public health problem (Kittinger et al., 2018). Various methods, such as transport, dilution, degradation, and adsorption, can decrease ARGs and ARBs concentration downstream of the wastewater treatment facilities’ discharges (Yoon et al., 2018).
1.3.2.1 Aquaculture
Antibiotics are also widely used in aquaculture, including fish, shrimp, and shellfish, leading to extraordinarily high levels of antibiotics entering the aquatic environment (Schar et al., 2020). In addition, antimicrobials are ineffective in fish. Approximately 70–80% of their metabolites are active antibiotics floating in fluids and can be diffused across the entire water system. As a result, antibiotic use in fish farming has major environmental implications, as it may result in selection pressure on several ecosystems (Burridge et al., 2010).
Moreover, resistant bacteria in aquaculture have been discovered, and it has expanded to terrestrial animals and humans. They can transfer their resistance genes to opportunistic infections in humans and animals (Serwecińska, 2020). Antimicrobial agents introduced in aquaculture are commonly used as metaphylaxis to whole populations consisting of individuals who are healthy, sick, or carriers, and in quantities that are usually higher than those used in land agriculture (Serwecińska, 2020). Hanna et al. (2018) reported that florfenicol, oxytetracycline, sulfadimethoxine, and ormetoprim have all been approved for aquaculture in the United States. In most European countries, erythromycin, florfenicol, oxytetracycline, ormetoprim, sarafloxacin, sulphonamides, and trimethoprim are permitted (Kümmerer, 2009). Until 2010, China had the largest manufacturing and use of antibiotics (Yang et al., 2010). The country was the top exporter of fish and fisheries products in the world (Serwecińska, 2020).
1.3.2.2
Soil
Bacterial species of clinical relevance are found in soils. Antibiotic pollution’s selective pressure on antimicrobial growth in the soil is of specific importance (Tello et al., 2012). Sludge comprises xenobiotics and resistant bacteria that can affect the metabolic functions and the variety of the soil microbial community, lowering the quality of the soil ecosystem (Knapp et al., 2010). Antibiotic residues, both synthetical material and semi-synthetical material, including fluoroquinolones, tetracyclines, and sulfonamides, attach to soil particles. They have higher chemical stability and are resistant to bacterial digestion, which greatly leads to biodegradation delays. Therefore, they are frequently distributed in soil (Rusch et al., 2019). In addition, ciprofloxacin and oxolinic acid require five months for the removal of only 20% of oxolinic acid (Larsson et al., 2007). Recently, the investigation also necessitates the addition of ARB and ARG to the list of developing pollutants found in sediments and their elimination from sediments and fertilized soils (Wang et al., 2017).
1.3.2.3
Agriculture: Animal Farm, Plant, Vegetable
Antibiotics are used to treat disorders in animals, such as chronic enteritis, which leads to unhealthy food consumption and weight loss (Serwecińska, 2020). Mostly, antibiotics, chlortetracycline,
flavophospholipid, oxytetracycline, penicillin, and bacitracin are widely used to prevent pathogen infections and treat animal diseases (Serwecińska, 2020).
Even though it is arduous to evaluate the actual numbers of antimicrobial agents used in animals that produce food because of the distinction in variability and international registration system, the global sale of medically essential antibiotics for use in food-producing animals reached 93,309 tonnes in 2017. It grew by 3% in 2019 (Tiseo et al., 2020). It is a critical problem since practically all active antimicrobial drugs used in animal agriculture are structurally similar to those used to treat humans, encouraging co-resistance and cross-resistance (EMA, 2014; ECDC et al., 2017).
Besides breeding animals (poultry, cattle, pigs, and rabbits), antibiotics are employed to eliminate pests in agricultural production, as well as beekeeping (McManus et al., 2002; Raymann et al., 2017). Streptomycin is authorized on peppers and tomatoes to control Xanthomonas campestris pv. vesicatoria. However, Erwinia amylovora is resistant to streptomycin (SmR), which produces fire blight and has economic and political ramifications (McManus et al., 2002). On the other hand, tetracycline (tetA, tetB, tetC, and tetR) is also used in animal husbandry and then released into the environment. Tetracycline-resistance genes (TcR) homologous to tetA and tetM have been discovered in a variety of plant-pathogenic bacteria (Sundin and Wang, 2018).
Furthermore, the WHO considers antibiotics such as penicillins, tetracyclines, aminoglycosides, macrolides, quinolones, and sulphonamides, which are extensively used in agriculture, to be particularly significant for human therapy (WHO, 2011). The research of Morar et al. (2015) reported that microbiological and diagnostic evidence seems to indicate that ARBs and ARGs are frequently passed down from animals that produce food for humans. Most of the research has been undertaken on the key parameters that drive the selection and transmission of antimicrobial resistance in food across the food chain.
Lactic acid bacteria (LAB) are utilized in several fields involved in our daily life. They are extensively used in food, chemical, pharmaceutical, and other industries (De Lima et al., 2010). On the contrary, antimicrobial resistance in microorganisms is one of the most serious worldwide health issues. Uncontrolled use of antimicrobial reagents and disposal of wastes without waste categorization from humans, households, hospitals, agriculture, aquaculture, industries, etc. (Figure 1.2), which are released into the environment, are causes of antimicrobial contamination and antimicrobial resistance distribution to the environments (Kimera et al., 2020). Many antibiotics are released into environments because their use enhances the development of a healthy modern society. The selective pressure of agriculture and animal care in the clinical setting increases concern about the human pathogen, which brings about research on the connection between antibiotic usage, the generation and dispersal of antimicrobial resistance, and multi-resistant bacteria (Busetti et al., 2017). Most antimicrobials released into the environment affect the resistance mechanism of lactic acid bacteria, including chloramphenicol, ciprofloxacin, erythromycin, gentamicin, vancomycin, rifampin, and tetracycline. Especially vancomycin resistance gene was found in LAB, including Bifidobacterium, Lactobacillus, Enterococcus, and Streptococcus strains (Erginkaya et al., 2018).
1.4 RESISTANCE OF ANTIMICROBIAL GENES, VIRULENT GENES, AND GENOMIC ANALYSIS IN LAB
1.4.1 BifidoBacterium
Bifidobacteria are the probiotic members involved in the food chain. On the other hand, they are categorized as the antimicrobial resistance strain, one of the global public health concerns. Several genotypic characteristics of Bifidobacterium are observed for data collection and prediction of the antimicrobial resistance genes, mutation, translocation of genetic elements, etc. Flórez et al. (2006) examined antimicrobial susceptibility characteristics of Bifidobacterium strains screened by the minimal inhibitory concentration (MIC) method. The results show antimicrobial resistance phenotypes of this bacteria, including amikacin, ciprofloxacin (CIP), daptomycin (DAP), gentamicin (GEN), kanamycin (KAN), nalidixic acid (NAL), streptomycin (STR), tetracycline (TET)
resistance. Nevertheless, the detection of the resistance gene of the Bifidobacterium strain via the PCR technique has shown only the TET resistance (tetW) gene from healthy human intestinal B. longum H66. Additionally, Ouoba et al. (2008) proposed the resistance gene of lactic acid bacteria, including bifidobacteria. They used the PCR method to amplify and sequence for resistance gene detection. The CIP resistance (gyrA) and STR resistance (aadE) genes represented the positive results in B. longum and B. bifidum. Interestingly, Jung et al. (2018) sequenced and read the wholegenome sequence of B. choerinum FMB-1, using HGAP 3.0, a hierarchical genome-assembly process DNA sequencing for high-quality de novo microbial genome assembly (Chin et al., 2013). This study identifies the antimicrobial resistance gene by MEGARes (Lakin et al., 2016). Three resistance genes BcFMB_02975, BcFMB_04310, and BcFMB_043, express as 3 S rRNA methyltransferases (preventing antibiotic activities), aminoglycoside O-nucleotidyltransferases (aminoglycosides resistant), lincosamide nucleotidyltransferases (lincosamide resistant; LIN), respectively. Esaiassen et al. (2017) analyzed phenotypic and genotypic characteristics from Bifidobacterium spp., including B. longum 11 strains, B. breve 2 strains, and B. animalis strains. They reported that one of B. longum resists LIN by expression of lmrD gene. Resistant of TET in B. animalis and B. breve cause by tetT gene. CIP resistance was found in B. animalis by mfd, gyrA, and gyrB gene expressions. Moreover, Rozman et al. (2020) characterized the antimicrobial resistance genes in bifidobacterial by using in silico detection, which combines several databases, bioinformatic instruments, etc. Antibioticresistant genes of Bifidobacterium strains are represented in Table 1.1.
Mucus display on pathogen adhesive prevention on the intestine. Ruas-Madiedo et al. (2008) reported two genes that involve intestinal mucin degradation. This study amplified two virulence genes from the Bifidobacterium strain by the PCR method as engBF and afcA: engBF of B. longum JCM1217 represent endo-α-N-acetylgalactosaminidase; B. longum JCM1254 encode afcA to produce 1,2-α-l-fucosidases. Esaiassen et al. (2017) expressed twenty virulence genes of bifidobacterial strains, comprised of iron and magnesium ion transportation, inducing bacterial adhesion, stress tolerance protein, immune-activated protein, and toxin secretion. In their results, B. animalis represents gene iap(cwhA), encoded into extracellular protein p60. Moreover, they investigate the virulence gene of B. longum subsp. infantis, which have the ability to encode ureA and ureB to produce urease alpha and beta subunits, respectively. These enzymes hydrolyze urea to produce ammonia and carbamate. In addition, they increase pH to survive and colonize the gastrointestinal tract.
1.4.2 LactoBaciLLus
Normally, antibiotics that impede protein synthesis are toxic to lactobacilli, including clindamycin (CLI), chloramphenicol (CHL), erythromycin (ERY), and tetracycline (TET) (Ammor et al., 2007; Klare et al., 2007). According to Bennedsen et al. (2011), the result from the in silico genome analysis was that Lactobacillus 8 species and 16 strains, including Lb. delbrueckii subsp. bulgaricus strain CHCC8942, CHCC769, CHCC5213; Lb. fermentum strain CHCC10568, CHCC5774, Lb. paracasei strain CHCC3136, CHCC2115, CHCC10665; Lb. acidophilus CHCC3777 Lb. rhamnosus CHCC5366, Lb. rhamnosus CHCC3402, CHCC5150, Lb. reuteri strain CHCC12039, and Lb. plantarum strain CHCC2365, CHCC10668, CHCC10672 had not detected antimicrobial resistance and virulence factor genes.
Similarly, most LAB are isolated from the environment of a bovine feedlot. The prevalence of susceptibility to ampicillin (AMP), CLI, CHL, GEN, KAN, and STR is greater than 80%. However, lactobacilli were detected to be moderately and highly resistant to ERY and TET at 48% and 79%, respectively (Aristimuño Ficoseco et al., 2018).
Additionally, the results of Lactobacillus sp. were isolated from chicken and boar droppings (Klose et al., 2014), including Lb. rhamnosus, Lb. acidophilus, Lb. casei, Lb. plantarum, and Lb. mucosae. They were all sensitive to the β-lactam AMP, and they were commonly utilized as a probiotic (Hummel et al., 2007; Klose et al., 2014). This result was consistent with Cauwerts
TABLE 1.1
Antibiotic Resistance Determinants in Bifidobacterium Species
Strain Antibiotic
B. animalis subsp. animalis ATCC 25527
B. animalis subsp. animalis IM386
LIN lmrD
VAN vanRM
FO murA
MU ileS
FQ acridine dye arlR
TET
tetA(48)
macrolide, LIN RlmA(II)
peptide antibiotic ugd
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
LIN lmrD
VAN vanRM
FQ murA
mupirocin ileS
FQ, acridine dye arlR
TET
B. animalis subsp. lactis Bi-07
Genomic analysis Reference
Whole-genome sequencing and in silico Rozman et al. (2020)
Whole-genome sequencing and in silico Rozman et al. (2020)
tetA(48)
macrolide, LIN RlmA(II) peptide antibiotic ugd
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
LIN lmrD
Whole-genome sequencing and in silico Rozman et al. (2020) tiamulin tvaA
macrolide macB
VAN vanRM
fosfomycin murA
mupirocin ileS
FQ, acridine dye arlR
TET
tetA(48)
TET tetW
macrolide, LIN RlmA(II)
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
B. animalis subsp. lactis BB-12 tiamulin tvaA
VAN vanRM
fosfomycin murA
mupirocin ileS
FQ, acridine dye arlR
TET
tetA(48)
macrolide, LIN RlmA(II) peptide antibiotic ugd
peptide antibiotic ugd
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
Whole-genome sequencing and in silico Rozman et al. (2020)
TABLE 1.1 (Continued)
Antibiotic Resistance Determinants in Bifidobacterium Species
Strain Antibiotic
B. animalis subsp. lactis Bl-04 tiamulin tvaA
Genomic analysis Reference
Whole-genome sequencing and in silico Rozman et al. (2020) VAN vanRM
fosfomycin murA
mupirocin ileS
FQ, acridine dye arlR
TET tetA(48)
TET tetW
macrolide, LIN RlmA(II) peptide antibiotic ugd
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
B. animalis subsp. lactis HN019 tiamulin tvaA
fosfomycin murA
Whole-genome sequencing and in silico Rozman et al. (2020) VAN vanRM
mupirocin ileS
FQ, acridine dye arlR
TET tetA(48)
TET tetW
macrolide, LIN RlmA(II) peptide antibiotic ugd
AMC parY
macrolide, penam mtrA peptide antibiotic, rifamycin rpoB2
B. choerinum FMB-1 LIN BcFMB_04315
B. longum (1 isolate) LIN lmrD
B. animalis (2 isolates)
B. breve (1 isolate) TET tetT
B. bifidum (Europe strain) CIP glyA
B. longum (Europe strain) STR aadE
B. breve strain Yakult STR Mutation of rpsL gene in G residue at 128 positions
B. choerinum FMB-1 23S rRNA methyltransferases BcFMB_02975
Whole-genome sequencing Jung et al. (2018)
Whole-genome sequencing Esaiassen et al. (2017)
Whole-genome sequencing Esaiassen et al. (2017)
PCR Ouoba et al. (2008)
PCR Kiwaki and Sato (2009)
Whole-genome sequencing Jung et al. (2018) Aminoglycoside BcFMB_04310
B. breve UCC2003 Aminoglycoside Bbr_0651
B. breve UCC2003 Aminoglycoside Bbr_1586
B. longum subsp. longum (1 isolate) Penicillin AIE
PCR Fouhy et al. (2013)
PCR Fouhy et al. (2013)
Whole-genome sequencing
Esaiassen et al. (2017)
TABLE 1.1 (Continued)
Antibiotic Resistance Determinants in Bifidobacterium Species
Strain Antibiotic
B. longum subsp. longum (1 isolate)
B. breve (2 isolates) CIP
, gyrA
, gyrA
Genomic analysis Reference
Whole-genome sequencing Esaiassen et al. (2017)
Whole-genome sequencing Esaiassen et al. (2017) B. longum subsp. infantis (4 isolates)
B. longum subsp. longum (7 isolates)
B. animalis (2 isolates)
, gyrA
, gyrA, gyrB
Notes: AMC, aminocoumarin; CIP, ciprofloxacin; FO, Fosfomycin; FQ, Fluoroquinolone; GLY, Glycopeptide; ERY, erythromycin; LIN, lincomycin; MU, Mupirocin; STR, streptomycin; TET, tetracycline; VAN, vancomycin; ND, the result of genes not detected.
et al. (2006), who reported Lb. rhamnosus, Lb. amylovorus, Lb. casei, and Lb. plantarum strains in humans and animals.
On the other hand, some lactobacilli discovered the resistance genes. Cloacal lactobacilli from broilers were found to have a significant prevalence of CLI resistance (Cauwerts et al., 2006). Lb. acidophilus and Lb. fermentum resist ERY while Lb. acidophilus CRL2152, Lb. amylovorus CRL2115, and 4 strains of Lb. mucosae (CRL2101, CRL2112, CRL2114, CRL2154) displayed the highest TET resistance (Aristimuño Ficoseco et al., 2018). Lb. acidophilus, Lb. rhamnosus, and Lb. casei from probiotic products exhibited a high prevalence of KAN resistance (Temmerman et al., 2003). Furthermore, resistance to GEN in Lb. mucosae CRL2158, resistance to KAN in Lb. plantarum CRL2103, resistant to STR in Lb. acidophilus CRL2074, Lb. amylovorus CRL2065, and Lb. mucosae CRL2155 were also discovered (Aristimuño Ficoseco et al., 2018). In agreement, STR resistance in Lb. acidophilus from the human origin (Klare et al., 2007) and Lb. amylovorus from broilers were reported (Cauwerts et al., 2006).
Although most lactobacilli strains were responsive to antibiotics that suppress protein synthesis, research is controversial about the sensitivity and resistance to the antibiotic of Lactobacillus sp. Differences in the examined species, applied methodology, or strain source might cause discrepancies. Because Lactobacillus spp. is widely used as a probiotic in food, pharmaceuticals, and beverages, it is vital to investigate the resistance/sensitive and virulence factor genes.
Aristimuño Ficoseco et al. (2018) , isolated Lactobacillus from feedlot cattle and then determined the virulence gene by using agg , ace , espA , ebp , cylA , hyl , gelE , sprE , fsrA , fsrB , fsrC , atpA as a target gene. However, no feedlot lactobacilli tested positive for virulence factors genes. Contrary, Lb. salivarius strain isolated from a sepsis patient was recently discovered to combine human platelets linked to human fibrinogen via discovering a new fibrinogen- binding protein ( Collins et al., 2012 ). Mostly, Lactobacillus spp. virulence research has focused on the species Lb. rhamnosus and Lb. paracasei are the most commonly utilized Lactobacillus probiotics ( Rossi et al., 2019 ).
Sirichoat et al. (2020), analyzed the comparative genome sequences against the databases CARD, ResFinder, and ARG-ANNOT. They detected antibiotic-resistant genes: Lb. jensenii VA04-2AN
resistant to trimethoprim (TMP), Lb. paracasei VA02-1AN resistant to CHL and VAN, and Lb. reuteri VA24-5 resistant to TMP and VAN. The discovery was the only phenotype and genotype association that was positive. The presence of a conserved phenylalanine residue in the active site of the D-Ala-D-Ala (Ddl) ligase, matching amino acid 261 of the Leuconostoc mesenteroides enzyme, in the inferred sequence of all vancomycin-resistant strains, while the presence of a tyrosine residue at this location was found in the sensitive strains.
The evidence of antimicrobial resistance and virulence genes in Lactobacillus spp. from previous research are summarized in Table 1.2
1.4.3 Lactococcus
Walther et al. (2008) isolated 72 strains of Lactococcus from bovine milk, including 41 strains of Lactococcus lactis and 31 strains of Lc. garvieae, then used a microarray to identify antibioticresistance genes. TET, CLI, ERY, STR, and nitrofurantoin resistances were found. Tetracyclineresistant Lc. garvieae and Lc. lactis carried tet(M) and tet(S), while clindamycin-resistant Lc. lactis contained the erm(B) gene and was also resistant to erythromycin. On the other hand, Lc. lactis subsp. lactis (8 strains) resistance against KAN and TET (Temmerman et al., 2003). In the case of virulence factor genes are not detected in Lc. lactis (Bennedsen et al., 2011) (Table 1.2).
TABLE 1.2
Antibiotic Resistance and Virulence Determinants in Lactobacillus and Lactococcus Strains
Strain Antibiotic Resistance genes
Lb. plantarum KKP 870, KKP 872
CLI lnuA
Lb. plantarum KKP 2021p CLI ermB
Lb. casei CRL 2088 ERY aph(3’’)-III, aadA
Lb. plantarum CRL2126 ERY cat
Lb. rhamnosus CRL2084 ERY ND
Lb. rhamnosus L-015 and L-455 and Lb. paracasei L-005
Lb. acidophilus group
STR ND
Lb. fermentum CRL 2085 STR ermB, ant(6), aadA
Lb. acidophilus CRL2061 and CRL2152 TET ermB
Lb. amylovorus CRL2115 and CRL2116 TET ermB
Lb. mucosae CRL2064, 2069, 2070, 2083, 2111, 2112, 2114, 2154 TET ermB
Lb. fermentum KKP 2020 TET ND
Lb. plantarum (6 strains) VAN ND
Analysis method
Disk diffusion tests, PCR-amplifications
Reference
Stefańska et al. (2021)
Disk diffusion tests, PCR-amplifications Stefańska et al. (2021)
PCR-amplifications
PCR-amplifications
PCR-amplifications
Disk diffusion tests, PCR-amplifications
Aristimuño Ficoseco et al. (2018)
Aristimuño Ficoseco et al. (2018)
Aristimuño Ficoseco et al. (2018)
Klare et al. (2007)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests, PCR-amplifications
Stefańska et al. (2021)
Disk diffusion tests Temmerman et al. (2003)
TABLE 1.2 (Continued)
Antibiotic Resistance and Virulence Determinants in Lactobacillus and Lactococcus Strains
Strain Antibiotic
Lb. acidophilus (13 strains),
Lc. lactis subsp. lactis (8 strains)
Lb. fermentum KKP 811, KKP 830, KKP 843
Lb. buchneri KKP 2047p
Resistance genes
KAN ND
KAN aph(3”)-IIIa
Lb. reuteri L-285 and L-285-2 OXY tet(W), tet(M)
Lb. mucosae CRL2100 and CRL2101 ERY, TET ermB
Lb. mucosae CRL2155 ERY, STR tetS
Lb. plantarum CRL2142 ERY, TET bla, tetS
Lb. acidophilus PCM 2499 STR, TET tetK, strA/strB
Lb. gallinarum LIN, CLI erm(B), erm(C)
Lb. crispatus L-295 and L-296 ERY, CLI erm(B), TET(W)
Lb. amylovorus CRL2044 CLI, TET ermB
Lb. rhamnosus (24 strains),
Lb. casei (29 strains), KAN, VAN ND
Lb. acidophilus CRL2074 CLI, STR, TET ermB
Lb. amylovorus CRL2065 ERY, STR, TET ermB
Lb. diolivorans KKP 2036p KAN, TET, STR aph(3”)-IIIa
Lb. mucosae CRL2158 ERY, GEN, TET tetS
Lb. mucosae CRL2063 CHL, ERY, TET ermB
Lb. plantarum CRL2103 CLI, ERY, KAN bla
Lb. johnsonii KKP 878 CHL, TET, STR ND
Lb. agilis KKP 1834 GEN, ERY, KAN, STR aph(3”)-IIIa, msrC
Lb. mucosae CRL2113 CLI, CHL, ERY, TET ermB
Lb. reuteri (6 strains) KAN, TET, PEN, VAN ND
Analysis method
Reference
Disk diffusion tests Temmerman et al. (2003)
Disk diffusion tests, PCR-amplifications
Stefańska et al. (2021)
Disk diffusion tests, PCR-amplifications Klare et al. (2007)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests, PCR-amplifications Stefańska et al. (2021)
Disk diffusion tests, PCR-amplifications Cauwerts et al. (2006)
Disk diffusion tests, PCR-amplifications Klare Klare et al. (2007)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests Temmerman et al. (2003)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests, PCR-amplifications
Stefańska et al. (2021)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests, PCR-amplifications Stefańska et al. (2021)
Disk diffusion tests, PCR-amplifications
Stefańska et al. (2021)
PCR-amplifications Aristimuño Ficoseco et al. (2018)
Disk diffusion tests Temmerman et al. (2003)
(continued)
TABLE 1.2 (Continued)
Antibiotic Resistance and Virulence Determinants in Lactobacillus and Lactococcus Strains
Strain Antibiotic
Lb. jensenii VA04-2AN TMP
Lb. paracasei VA02-1AN CHL, VAN
Lb. reuteri VA24-5 TMP, VAN
Lc. lactis CHCC6005 TET
Lc. lactis NZ900 TET, ERY
Resistance genes
Mutant gene on active site of Ddl ligases
tet(S)
Tet(S) and erm(B)
Analysis method
Reference
Whole-genome sequencing Sirichoat et al. (2020)
PCR-amplifications Bennedsen et al. (2011)
PCR-amplifications Flórez, et al. (2021)
Notes: AMP, ampicillin; CLI, clindamycin; CHL, chloramphenicol; GEN, gentamycin; ERY, erythromycin; KAN, kanamycin; LIN, lincomycin; OXY, oxytetracycline; STR, streptomycin; TET, tetracycline; TMP, Trimethoprim; VAN, vancomycin; ND, the result of genes not detected. There is no report in virulence genes.
1.4.4 enterococcus
The main point related to antimicrobial resistance is transferring an antimicrobial-resistance gene from bacteria to bacteria, bacteria to an animal, and animal to human via the food chain. Therefore, this problem affects human, animal, medicine, and food production (Dušková and Karpíšková, 2013). Enterococcus spp. is a member of the micro-flora organism, which has colonized the digestive system and plays a role in the immune system modification in humans and animals (Krawczyk et al., 2021). They are generally described as Gram-positive, facultatively anaerobic, ovoid-shape with short chains, pair, single-cell, catalase-negative bacteria (Mohanty and Bhuniya, 2021). They were isolated from many sources, comprising soil, surface water, plants, and animals. In the last decade, this bacterial group has been widely used as probiotics and starter cultures in the food industries. In contrast, they have recently been described as the nosocomial pathogen that causes intensive care infections. Significantly, both Enterococcus faecium and Enterococcus faecalis are important surveillance pathogens because they are able to resist multiple drugs (Mohanty and Bhuniya, 2021). Vancomycin resistance was mediated by nine distinct gene clusters, including vanA, B, C, D, E, G, L, M, and vanN, found (Hollenbeck, and Rice, 2012). De Leener et al. (2004) identified macrolide resistance genes as ermB and TET resistance genes as tetK, tetL, tetM, tetO, and tetS from Enterococcus strains isolated from carcasses of humans, pigs, and pork. The antibiotic resistance of Enterococcus spp. represents the β-lactam resistance gene TEM, aminoglycoside resistance genes aac(6’)-aph(2”), aph(3’)-III, ant(6)-I and ant(2”)-I, the TET resistance gene tetM, erythromycin resistance gene ermB, the vancomycin resistance gene vanA, and the enterococcal multidrug resistance efflux pump gene emeA, which are determined by a PCR (Jia et al., 2014). Raeisi et al. (2017) used disk diffusion and polymerase chain reaction (PCR) to identify the expression of the vancomycin resistance gene as a vanA, gene in Enterococcus faecalis. The DNA microarray is the technique that detects the medium-length oligonucleotides by using the target probe detection (Missoum, 2018). Diarra et al. (2010) examined the antimicrobial resistance genes in enterococci. Moreover, this experiment determined the genes resisting aminoglycoside (aac, aacA-aphD, aadB, aphA, sat4), macrolide (ermA, ermB, ermAM, msrC), TET (tetL, tetM, tetO), streptogramin (satG_ vatE8), bacitracin (bcrR), and LIN (linB) by utilization of DNA microarray. Among enterococci species widely found in nosocomial infections as pathogens, included Enterococcus faecalis and Enterococcus faecium. In addition, Enterococcus spp. contain several virulence genes. The virulence genes determination was investigated using the PCR technique, which uses the specific primers for amplification. They found virulence genes, including the E. faecalis endocarditis antigen (efaA) gene and enterococcal surface protein (esp) gene. In contrast, they did not find the aggregation substance (AS) gene, collagen-binding protein (ace) gene, cytolysin (cylA) gene,
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the Ardo Jídda, to whom belongs the country between Sugúr and Wándalá or Mándará, and the younger of whom was a remarkably handsome man, of slender form, light complexion, and a most agreeable expression of countenance. This, however, is a remark which I have often made on my travels, that the males among the Fúlbe are very handsome till they reach the age of about twenty years, when they gradually assume an apish expression of countenance, which entirely spoils the really Circassian features which they have in early life. As for the females, they preserve their beauty much longer While these young men were giving unrestrained vent to their admiration of my things, the old mʿallem came with a numerous suite of attendants; whereupon they drew shyly back, and sat silently at a distance. In this part of the world there is a great respect for age.
The mʿallem and his companions were not only astonished at my instruments, but manifested much curiosity about the map of Africa, which I unfolded before their eyes, being greatly struck by the extent of the continent towards the south, of which they had previously no idea. I shall show in another part of this work how far the Fúlbe have become acquainted with the regions about the equator, and how a faint rumour of the strong pagan kingdom of Muropúwe has spread over the kingdoms of North Central Africa. Their esteem for me increased when I showed them my little Prayer-Book, which I wore in a red case slung round my shoulders, just as they wear their Kurán; indeed a Christian can never be more sure of acquiring the esteem of a Moslim—at least of a learned one—than when he shows himself impressed with the sentiments of his religion; but he must not be a zealous Roman Catholic, nor broach doctrines which seem to deny the Unity of God. He took great delight in hearing a psalm of the well-known “nebí Dáúd” (David) read in English. He, as well as almost all his companions, spoke Arabic; for, as Saráwu Beréberé is a colony of Bórnu people, Bélem is a pure Arabic colony, that is to say, a colony of the Sálamát, a tribe widely scattered over Bórnu and Wadáy. Mʿallem Óro, or, as he is popularly called, on account of his humility and devotedness, Mʿallem Dalíli, was born in Wadáy, but settled in Bórnu, from whence at the time of the conquest of the country by the Fúlbe or Felláta (in the year 1808) he fled to avoid
famine and oppression, like so many other unfortunate inhabitants of that kingdom, and founded a village in this promising region. This is the country for colonies, and I do not see why a colony of the liberated slaves of Sierra Leone might not be advantageously established here. All these people wear indigo-coloured shirts, and in this manner, even by their dress, are distinguished from the Fúlbe. They are tolerated and protected, although a Púllo head man has his residence here, besides the mʿallem.
We were to start in the afternoon; but my stupid Fezzáni servant, Mohammed ben Habíb, had almost killed himself with eating immoderately of ground-nuts, and was so seriously ill that I was reduced to the alternative either of leaving him behind or waiting for him. Choosing the latter, I made a day of feasting for the whole of my little company, the mʿallem sending me a goat for my people, a couple of fowls for myself, and corn for my horses; besides which, I was so fortunate as to buy a supply of rice. In consideration of his hospitable treatment, I sent the old mʿallem a bit of camphor and a parcel of cloves. Camphor is a most precious thing in these regions, and highly esteemed by the nobler classes, and I cannot too strongly recommend a traveller to provide himself with a supply of it. It is obvious that a small quantity, if well kept, will last him a long time. He may find an opportunity of laying a man of first-rate importance under lasting obligations by a present of a small piece of camphor.
Tuesday, June 17.—We at length set out to continue our journey. The morning was beautifully fresh and cool after the last night’s storm, the sky was clear, and the country open and pleasant. A fine grassy plain, with many patches of cultivated ground, extended on our right to the very foot of Mount Kónkel, which as I now saw is connected by a lower ridge with Mount Holma. We passed the ruins of the village Bíngel, the inhabitants of which had transferred their settlement nearer to the foot of the mountains. Then followed forest, interrupted now and then by cornfields. My friends, the young sons of Ardo Jídda, accompanied me for full two hours on horseback, when they bade me a friendly farewell, receiving each of them, to his great delight, a stone-set ring, which I begged them to present to their ladies as a memorial of the Christian traveller. I now learnt that
the young men were already mixing a good deal in politics; the younger brother, who was much the handsomer, and seemed to be also the more intelligent of the two, had till recently administered the government of his blind father’s province, but had been deposed on account of his friendly disposition towards Wándalá, having married a princess of that country, and the management of affairs had been transferred to his elder brother.
Forest and cultivated ground alternately succeeded each other; a little after nine o’clock we passed on our left a small “rúmde,” or slave-village, with ground-nuts and holcus in the fields, and most luxuriant pasture all around. The country evidently sloped southwards, and at a little distance beyond the village I observed the first watercourse, running decidedly in that direction; on its banks the corn stood already four feet high. The country now became quite open to the east and south, and everything indicated that we were approaching the great artery of the country which I was so anxious to behold. In the distance to the west a range of low hills was still observable, but was gradually receding. About ten o’clock we passed the site of a straggling but deserted village, called Melágo, the inhabitants of which had likewise exchanged their dwelling-place in this low level country for a more healthy one at the foot of the mountains, where there is another village called Kófa, homonymous with that in the Marghí country; for this district belongs to the country of the Bátta, a numerous tribe nearly related, as I have stated above, to the Marghí. All the ruins of the dwellings in Melágo were of clay, and the rumbú or rumbúje—the stacks of corn—were of a peculiar description; fine cornfields spread around and between the huts.
Having rested about noon for a little more than two hours on a rather damp and gloomy spot near a dirty pond, we continued our march, the country now assuming a very pleasant park-like appearance, clothed in the most beautiful green, at times broken by cornfields, where the corn—Pennisetum or géro—stood already five feet high. We soon had to deliberate on the very important question which way to take, as the road divided into two branches, the northern or western one leading by way of Búmánda, while the southern or eastern one went by way of Sulléri. Most of my
companions were for the former road, which they represented as much nearer, and as I afterwards saw, with the very best reason; but fortunately the more gastronomic part of the caravan, headed by Bíllama, who was rather fond of good living, rejected Búmánda, as being inhabited by poor inhospitable pagans, and decided for the promising large dishes of Mohammedan Sulléri. This turned out to be a most fortunate circumstance for me, although the expectations of my friends were most sadly disappointed. For if we had followed the route by Búmánda, we should have crossed the Bénuwé lower down, and I should not have seen the “Tépe,” that most interesting and important locality, where the Bénuwé is joined by the Fáro, and swelled to that majestic river which is at least equal in magnitude to the Kwára. Of this circumstance I was then not aware, else I should have decided from the beginning for the route by Sulléri. Unfortunately, owing to my very short stay in the country, I cannot say exactly where Búmánda lies; but I should suppose that it is situated about ten miles lower down, at a short distance from the river, like the place of the same name near Hamárruwa,[58] and I think it must lie opposite to Yóla, so that a person who crosses the river at that place goes over directly to the capital, without touching either at Ribáwo, or at any of the neighbouring places.
Having, therefore, chosen the eastern road, we soon reached the broad, but at present dry sandy channel of the Máyo Tíyel, which runs in a south-westerly direction to join the Bénuwé; water was to be found close underneath the surface of the sand, and several women heavily laden with sets of calabashes, and belonging to a troop of travellers encamped on the eastern border of the watercourse, were busy in scooping a supply of most excellent water from a shallow hollow or “kénkenu.” The banks of the river, or rather torrent, were lined with luxuriant trees, amongst which I observed the dorówa or meráya (Parkia) in considerable numbers.
Forest and cultivated ground now succeeded alternately, till we reached a beautiful little lake called “gére[59] Páriyá” by the Bátta, and “barre-n-dáke” by the Fúlbe, at present about fourteen hundred yards long, and surrounded by tall grass, everywhere impressed with tracks of the hippopotami or “ngábba,” which emerge during the
night from their watery abode to indulge here quietly in a rich pasturage. This is the usual camping-ground of expeditions which come this way. A little beyond this lake a path branched off from our road to the right, leading to Ródi, a place of the Bátta, whose villages, according to Mohámmedu’s statement, are all fortified with stockades, and situated in strong positions naturally protected by rocky mounts and ridges.
There had been a storm in the afternoon at some distance; but when the sun was setting, and just as we began to wind along a narrow path through a thick forest, a black tempest gathered over our heads. At length we reached the fields of Sulléri, and, having stumbled along them in the deepest darkness, illumined only at intervals by flashes of lightning, we entered the place and pushed our way through the narrow streets, looking round in vain for Íbrahíma, who had gone on to procure quarters.
To our great disappointment we found the house of the governor shut up; and notwithstanding our constant firing and knocking at the door, nobody came to open it, while the heavy clouds began to discharge their watery load over our heads. At length, driven to despair, we turned round, and by force entered his son’s house, which was situated opposite to his own. Here I took possession of one side of the spacious, clean, and cool entrance-hall, which was separated from the thoroughfare by a little balustrade raised above the floor. Spreading my mat and carpet upon the pebbles with which, as is the general custom here, it was strewn, I indulged in comfort and repose after the fatiguing day’s march, while outside the tempest, and inside the landlord, were raging; the latter being extremely angry with Bíllama on account of our forced entry Not the slightest sign of hospitality was shown to us; and instead of regaling themselves with the expected luxurious dishes of Sulléri, my companions had to go supperless to bed, while the poor horses remained without anything to eat, and were drenched with the rain.
CHAPTER XXXV.
THE MEETING OF THE WATERS.—THE BÉNUWÉ AND FÁRO.
Wednesday, June 18.—At an early hour we left the inhospitable place of Sulléri. It was a beautiful fresh morning, all nature being revived and enlivened by the last night’s storm. My companions, sullen and irritated, quarrelled among themselves on account of the selfish behaviour of Íbrahíma. As for me, I was cheerful in the extreme, and borne away by an enthusiastic and triumphant feeling; for to-day I was to see the river.
The neighbourhood of the water was first indicated by numbers of high ant-hills, which, as I shall have occasion to observe more fully in the course of my narrative, abound chiefly in the neighbourhood of rivers: they were here ranged in almost parallel lines, and afforded a very curious spectacle. We had just passed a small village or rúmde, where not a living soul was to be seen, the people having all gone forth to the labours of the field, when the lively Mohámmedu came running up to me, and exclaimed, “Gashí! gashí! dútsi-n-Alantíka kè nan” (“Look! look! that is Mount Alantíka”). I strained my eyes and saw, at a great distance to the south-west, a large but insulated mountain mass, rising abruptly on the east side, and forming a more gradual slope towards the west, while it exhibited a rather smooth and broad top, which certainly must be spacious, as it contains the estates of seven independent pagan chiefs. Judging from the distance, which was pretty well known to me, I estimated the height of the mountain at about eight thousand feet above the plain, or about nine thousand feet of absolute elevation; but it may be somewhat less.
Here there was still cultivated ground, exhibiting at present the finest crop of masr, called “bútalí” by the Fúlbe of Ádamáwa; but a
little further on we entered upon a swampy plain (the savannas of Ádamáwa), overgrown with tall rank grass, and broken by many large hollows full of water, so that we were obliged to proceed with great caution. This whole plain is annually (two months later) entirely under water. However, in the middle of it, on a little rising ground which looks as if it were an artificial mound, lies a small village, the abode of the ferrymen of the Bénuwé, from whence the boys came running after us—slender, well-built lads, accustomed to fatigue and strengthened by daily bathing; the younger ones quite naked, the elder having a leathern apron girt round their loins. A quarter of an hour afterwards we stood on the bank of the Bénuwé.[60]
It happens but rarely that a traveller does not feel disappointed when he first actually beholds the principal features of a new country, of which his imagination has composed a picture from the description of the natives; but although I must admit that the shape and size of the Alantíka, as it rose in rounded lines from the flat level, did not exactly correspond with the idea which I had formed of it, the appearance of the river far exceeded my most lively expectations. None of my informants had promised me that I should just come upon it at that most interesting locality—the Tépe[61]—where the mightier river is joined by another of very considerable size, and that in this place I was to cross it. My arrival at this point, as I have stated before, was a most fortunate circumstance. As I looked from the bank over the scene before me, I was quite enchanted, although the whole country bore the character of a desolate wilderness; but there could scarcely be any great traces of human industry near the river, as, during its floods, it inundates the whole country on both sides. This is the general character of all the great rivers in these regions, except where they are encompassed by very steep banks.
The principal river, the Bénuwé, flowed here from east to west, in a broad and majestic course, through an entirely open country, from which only here and there detached mountains started forth. The banks on our side rose to twenty-five, and in some places to thirty feet, while just opposite to my station, behind a pointed headland of sand, the Fáro rushed forth, appearing from this point not much inferior to the principal river, and coming in a fine sweep from the
south-east, where it disappeared in the plain, but was traced by me, in thought, upwards to the steep eastern foot of the Alantíka. The river, below the junction, keeping the direction of the principal branch, but making a slight bend to the north, ran along the northern foot of Mount Bágelé, and was there lost to the eye, but was followed in thought through the mountainous region of the Báchama and Zína to Hamárruwa, and thence along the industrious country of Korórofa, till it joined the great western river the Kwára or Niger, and, conjointly with it, ran towards the great ocean.
On the northern side of the river another detached mountain, Mount Taife, rose, and behind it the Bengo, with which Mount Fúro seemed connected, stretching out in a long line towards the northwest. The bank upon which we stood was entirely bare of trees, with the exception of a solitary and poor acacia, about one hundred paces further up the river, while on the opposite shore, along the Fáro and below the junction, some fine clusters of trees were faintly seen.
I looked long and silently upon the stream; it was one of the happiest moments in my life. Born on the bank of a large navigable river, in a commercial place of great energy and life, I had from my childhood a great predilection for river-scenery; and although plunged for many years in the too exclusive study of antiquity, I never lost this native instinct. As soon as I left home, and became the independent master of my actions, I began to combine travel with study, and to study while travelling, it being my greatest delight to trace running waters from their sources, and to see them grow into brooks, to follow the brooks, and see them become rivers, till they at last disappeared in the all-devouring ocean. I had wandered all around the Mediterranean, with its many gulfs, its beautiful peninsulas, its fertile islands—not hurried along by steam, but slowly wandering from place to place, following the traces of the settlements of the Greeks and Romans around this beautiful basin, once their terra incognita. And thus, when entering upon the adventurous career in which I subsequently engaged, it had been the object of my most lively desire to throw light upon the natural arteries and hydrographical network of the unknown regions of
Central Africa. The great eastern branch of the Niger was the foremost to occupy my attention; and although for some time uncertain as to the identity of the river of Ádamáwa with that laid down in its lower course by Messrs. W. Allen, Laird, and Oldfield, I had long made up my mind on this point, thanks to the clear information received from my friend Ahmed bel Mejúb. I had now, with my own eyes, clearly established the direction and nature of this mighty river; and to an unprejudiced mind there could no longer be any doubt that this river joins the majestic watercourse explored by the gentlemen just mentioned.[62] Hence I cherish the well-founded conviction, that along this natural highroad European influence and commerce will penetrate into the very heart of the continent and abolish slavery, or rather those infamous slave-hunts and religious wars, destroying the natural germs of human happiness, which are spontaneously developed in the simple life of the pagans, and spreading devastation and desolation all around.
We descended towards the place of embarkation, which at this season of the year changes every week, or even more frequently. At present it was at the mouth of a small, deeply worn channel, or dry watercourse, descending from the swampy meadow-grounds towards the river, and filled with tall reed-grass and bushes. Here was the poor little naval arsenal of the Tépe, consisting of three canoes, two in good repair, and a third one in a state of decay, and unfit for service.
It was now that for the first time I saw these rude little shells, hollowed out of a single trunk—for the boats of the Búdduma are more artificial, being made of a number of boards joined together: and I soon began to eye these frail canoes with rather an anxious feeling, as I was about to trust myself and all my property to what seemed to offer very inadequate means of crossing with safety a large and deep river. They measured from twenty-five to thirty feet in length, and only from a foot to a foot and a half in height, and sixteen inches in width; and one of them was so crooked, that I could scarcely imagine how it could stem the strong current of the river.
On the river itself two canoes were plying; but, notwithstanding our repeated hallooing and firing, the canoemen would not come to our
side of the river; perhaps they were afraid. Roving about along the bushy watercourse, I found an old canoe, which being made of two very large trunks joined together, had been incomparably more comfortable and spacious than the canoes now in use; although the joints being made with cordage just like the stitching of a shirt, and without pitching the holes, which were only stuffed with grass, necessarily allowed the water to penetrate continually into the boat; it, however, had the great advantage of not breaking if it ran upon a rock, being in a certain degree pliable. It was about thirty-five feet long, and twenty-six inches wide in the middle; but it was now out of repair, and was lying upside down. It was from this point, standing upon the bottom of the boat, that I made the sketch of this most interesting locality.
The canoemen still delaying to come, I could not resist the temptation of taking a river bath, a luxury which I had not enjoyed since bathing in the Eurymedon. The river is full of crocodiles; but there could be little danger from these animals after all our firing and the constant noise of so many people. I had not yet arrived at the conviction, that river-bathing is not good for a European in a tropical climate, but this was the first and last time that I bathed voluntarily, with a single exception, for when navigating the river of Logón on a fine day in March 1852, I could not help jumping overboard, and on my return from Bagírmi, in August 1853, I was obliged to do it.
The bed of the river, after the first foot and a half, sloped down very gradually, so that at the distance of thirty yards from the shore I had not more than three feet and a half of water, but then it suddenly became deep. The current was so strong, that I was unable to stem it; but my original strength, I must allow, was at the time already greatly reduced. The only advantage which I derived from this feat was that of learning that the river carries gold with it; for the people, as often as I dipped under water, cried out that I was searching for this metal, and when I came out of the water, were persuaded that I had obtained plenty of it. However, the river was already too full for investigating this matter further.
At length a canoe arrived, the largest of the two that were actually employed, and a long bargaining commenced with the eldest of the
canoemen, a rather short and well-set lad. Of course, as the chief of the caravan, I had to pay for all, and, there being three camels and five horses to be carried over, it was certainly a difficult business. It cannot, therefore, be regarded as a proof of exorbitant demands, that I had to pay five “dóras,” a sum which in Kúkawa would buy two ox-loads of Indian corn. I allowed all the people to go before me, in order to prevent the canoemen from exacting something more from them.
There was considerable difficulty with my large camel-bags, which were far too large for the canoes, and which several times were in danger of being upset; for they were so unsteady that the people were obliged to kneel down on the bottom, and keep their equilibrium by holding with both hands on the sides of the boat. Fortunately I had laid my tent-poles at the bottom of the canoe, so that the water did not reach the luggage; but owing to the carelessness of the Hajji’s companions, all his books were wetted, to his utmost distress, but I saw him afterwards shedding tears while he was drying his deteriorated treasures on the sandy beach of the headland. The horses as they crossed, swimming by the sides of the canoe, had to undergo great fatigue; but desperate was the struggle of the camels, which were too obstinate to be guided by the frail vessels, and had to be pushed through alone, and could only be moved by the most severe beating; the camel of the Hajji was for a while given up in despair by the whole party. At length they were induced to cross the channel, the current carrying them down to a great distance, and our whole party arrived safe on the sandy beach of the headland, where there was not a bit of shade. This whole headland for two or three months every year is covered with water, although its chief part, which was overgrown with tall reed-grass, was at present about fifteen feet above the surface.
The river, where we crossed it, was, at the very least, eight hundred yards broad, and in its channel generally eleven feet deep, and was liable to rise, under ordinary circumstances, at least thirty, or even at times fifty feet higher. Its upper course at that time was known to me as far as the town of Géwe on the road to Logón; but
further on I had only heard from the natives that it came from the south, or rather from the south-south-east.
It was a quarter before one o’clock when we left the beach in order to cross the second river, the Fáro, which is stated to come from Mount Lábul, about seven days’ march to the south. It was at present about six hundred yards broad, but generally not exceeding two feet in depth, although almost all my informants had stated to me that the Fáro was the principal river. The reason of this mistake was, I think, that they had never seen the two rivers at this place, but observed the Fáro near Gúrin, where, a little later in the season, it seems to be of an immense breadth, particularly if they crossed from Bundang; or they were swayed by the great length of the latter river, which they were acquainted with in its whole course, while none of them had followed the upper course of the Bénuwé.
Be this as it may, the current of the Fáro was extremely violent, far more so than of the Bénuwé, approaching, in my estimation, a rate of about five miles, while I would rate the former at about three and a half miles an hour, the current of the Fáro plainly indicating that the mountainous region whence it issued was at no great distance. In order to avoid the strongest part of the current, which swept along the southern shore, we kept close to a small island, which, however, at present could still be reached from this side with dry feet. We then entered upon low meadow-land, overgrown with tall reed-grass, which a month later is entirely inundated to such a depth that only the crowns of the tallest trees are seen rising above the water, of which they bore unmistakable traces, the highest line thus marked being about fifty feet above the present level of the river; for of course the inundation does not always reach the same height, but varies according to the greater or less abundance of the rains. The information of my companions, as well as the evident marks on the ground, left not the least doubt about the immense rise of these rivers.[63]
For a mile and a half from the present margin of the river, near a large and beautiful tamarind-tree, we ascended its outer bank, rising to the height of about thirty feet, the brink of which is not only generally reached by the immense inundation, but even sometimes
overflowed, so that the people who cross it during the height of the inundation, leaving the canoes here, have still to make their way through deep water, covering this highest level.
My companions from Ádamáwa were almost unanimous in spontaneously representing the waters as preserving their highest level for forty days, which, according to their accounts, would extend from about the 20th of August till the end of September. This statement of mine, made not from my own experience, but from the information of the natives, has been slightly, but indeed very slightly, modified by the experience of those eminent men who, upon the reports which I forwarded of my discovery, were sent out by Her Majesty’s Government in the Pleiad and who succeeded in reaching the point down to which I had been able to delineate the course of the river with some degree of certainty. That the fall of the river at this point of the junction begins at the very end of September, has been exactly confirmed by these gentlemen, while with regard to the forty days they have not made any distinct observation, although there is evidence enough that they experienced something confirmatory of it.[64]
On leaving the outer bank of the river, our way led through a fine park-like plain, dotted with a few mimosas of middling size, and clear of underwood. The sides of the path were strewn with skeletons of horses, marking the line followed by the late expedition of the governor of Yóla, on its return from Lére, or the Mbána country. Having then entered upon cultivated ground, we reached the first cluster of huts of the large straggling village Chabajáure, or Chabajáule, situated in a most fertile and slightly undulating tract; and having kept along it for a little less than a mile and a half, we took up our quarters in a solitary and secluded cluster of huts, including a very spacious courtyard.
It was a sign of warm hospitality that, although the whole caravan had fallen to the charge of a single household, sufficient quantities not only of “nyíro,” the common dish of Indian corn, but even of meat, were brought to us in the evening. While passing the village, I had observed that all the corn on the fields was “geróri,” or Pennisetum (millet—dukhn), a kind of grain originally, it would seem,
so strange to the Fúlbe, that they have not even a word of their own for it, having only modified a little the Háusa word “géro”; not a single blade of “baíri,” or sorghum, was to be seen. The scarcity was less felt here than in the northern districts of the country, and we brought some grain for our horses as a supply for the next day.
Thursday, June 19.—We started early in the morning, continuing along the straggling hamlets and rich cornfields of Chabajáule for a mile and a half; when we passed two slave villages, or “rúmde,” belonging to a rich Púllo, of the name of Hanúri. All the meadows were beautifully adorned with white violet-striped lilies. We then entered a wooded tract, ascending at the same time considerably on the hilly ground which juts out from the foot of Mount Bágelé, and which allowed us a clearer view of the geological character of the mountain. Having again emerged from the forest upon an open, cultivated, and populous district, we passed the large village of Dulí, and, having descended and reascended again we obtained a most beautiful view near the village Gúroré, which lies on rising ground, surrounded by a good many large monkey-bread-trees, or bodóje (sing. bokki). For from this elevated spot we enjoyed a prospect over the beautiful meadow-lands sloping gently down towards the river, which from this spot is not much more than five miles distant, taking its course between Mounts Bengo and Bágelé, and washing the foot of the latter, but not visible to us. The country continued beautiful and pleasant, and was here enlivened by numerous herds of cattle, while in the villages which we had passed I had seen none, as the Fúlbe drive their cattle frequently to very distant grazing grounds.
While marching along at a good pace, Mohámmedu walked up to me, and with a certain feeling of pride showed me his fields, “gashí gonakína.” Though a poor man, he was master of three slaves, a very small fortune in a conquered and newly colonized country, like Ádamáwa, based entirely upon slavery, where many individuals have each more than a thousand slaves. I was greatly surprised to see here a remarkable specimen of a bokki or monkey-bread-tree, branching off from the ground into three separate trunks; at least, I never remember to have seen anything like it, although the tree is the most common representative of the vegetable kingdom through
the whole breadth of Central Africa. All the ground to the right of the path is inundated during the height of the flood.
We had now closely approached the Bágelé, the summit of which, though not very high, is generally enveloped in clouds, a fact which, when conveyed to me in the obscure language of the natives, had led me to the misconception, while writing in Kúkawa my report of the provisional information I had obtained of the country whither I was about to proceed, that this mountain was of volcanic character. It seems to consist chiefly of granite, and has a very rugged surface, strewn with great irregular blocks, from between which trees shoot up. Nevertheless, stretching out to a length of several miles from south-south-east to north-north-west, it contains a good many spots of arable land, which support eighteen little hamlets of independent pagans. These, protected by the inaccessible character of their strongholds, and their formidable double spears, have not only been able hitherto to repulse all the attacks which the proud Mohammedans, the centre of whose government is only a few miles distant, have made against them, but, descending from their haunts, commit almost daily depredations upon the cattle of their enemies. [65] One of their little hamlets, perched on the top of steep cliffs, we could plainly distinguish by the recently thatched roofs of the huts, the snow-white colour of which very conspicuously shone forth from the dark masses of the rock. The country was always gaining in interest as we advanced, the meadow-lands being covered with living creatures of every description, such as cattle, horses, asses, goats, and sheep, and we reached the easternmost cluster of huts of the large straggling village or district of Ribáwo or Ribágo,[66] stretching out on our left on a little rising ground. The district is not only rich in corn and pasturage, but also in fish, which are most plentiful in a large inlet or backwater, “illágul,” as it is called by the Fúlbe, branching off from the river along the north-east foot of the Bágelé, and closely approaching the village. In this shallow water the fish are easily caught.
Numbers of inquisitive people of every age and sex gathered round us from the neighbouring hamlets; but while hovering round me and the camels with great delight, they behaved very decently
and quietly They followed us till we took up our quarters a little before ten o’clock, with a friend of Bíllama’s, in a large group of huts lying close to the path, and shaded by most luxuriant trees. Although there were several clean huts, I preferred the cool and ventilated entrance hall of the same description as I have mentioned above, and remained here even during the night, although a most terrible storm, which broke out at six o’clock in the evening, and lasted full four hours, flooded the whole ground, and rendered my resting-place rather too cool. I would advise other travellers not to follow my example during the rainy season, but rather to make themselves comfortable in the warm interior of a well-protected hut.
In our last march through these rich low grounds, which are every year flooded by the river, I had not observed the least traces of the cultivation of rice, for which they seem to be so marvellously adapted, the cultivation round Ribágo being almost exclusively limited to maiwa or maiwári, a peculiar species of sorghum called “matëa” in Kanúri. On inquiring why these people did not grow rice, I learnt that the Fúlbe hereabouts had all migrated from Bórnu after the downfall of their jemmára and dominion in that country, when not only were the new political intruders repulsed, but even the old settlers, who had been established in that country from very ancient times, were obliged to emigrate. In Bórnu, however, as I have had occasion to mention before, no rice is cultivated, so that these people, although at present established in regions where rice would probably succeed much better than millet and Indian corn, abstain entirely from its cultivation. On the other hand, in the western parts of Adamáwa and in Hamárruwa, whither the Fúlbe had migrated from Háusa, rice is cultivated to a considerable extent. On a former occasion I have already touched on the question, whether rice be indigenous in Negroland or not. It has evidently been cultivated from time immemorial in the countries along the middle course of the Ísa, or Kwára, from Kébbi up to Gágho, or Gógó; but this might seem to be in consequence of a very ancient intercourse between those regions and Egypt, which I hope to be able to establish in the course of my narrative. It grows, however, wild in many parts, from the southern provinces of Bórnu, Bagírmi, and Wadáy, as far north as el Haúdh and Bághena, on the border of the western desert.
Another important point of which I here became aware was, that the Bátta language, which, among the numerous languages of Ádamáwa, or rather Fúmbiná, is the most extensively spoken, has two very different dialects; for, being anxious to finish my small vocabulary of this language, which I had commenced in Kúkawa with the assistance of Mohámmedu, I soon found that the dialect spoken here differed considerably from that of which I had previously written specimens. The Bátta language, as I have stated above, is intimately related to the Marghí and Záni idiom, and bears several points of resemblance to the Músgu language, which is itself related to the various dialects of Kótoko. All these languages have some general points of affinity to the South African languages.
At present, however, the indigenous population is almost totally extinct in this district, which is exclusively inhabited by the conquerors, who have here found an abode remarkably suited to their mode of living. The whole place has not less than six thousand inhabitants.
Friday, June 20.—We started early in order to reach the capital, if possible, before noon, and passed through several hamlets, all belonging to the extensive village or district of Ribágo, and interrupted here and there by projecting masses of schistose rock, while the concavity between this rising ground and Mount Bágelé was fast filling with the flood from the river, and presented already a considerable sheet of water. The country, after we had passed this populous district, became thickly wooded, which I had not expected to find so near the capital; and, on account of some ravines which intersect it, and of the neighbourhood of the inlet of the river, it certainly cannot afford a very easy passage towards the end of the rainy season. Here also the rock projects above the plain in many places.
About eight o’clock, when we had travelled round the southwestern foot of Mount Bágelé, we passed through a number of small hamlets, which however did not exhibit any traces of cultivation, and then again entered upon a wild tract, while we obtained a glance at a picturesquely seated place before us, which I unhesitatingly took for Yóla, but which proved to be a small village situated at a
considerable distance from the capital. Before we reached it, we had to cross a sheet of water nearly five feet deep, and called by my companions “Máyo Bínti,” which caused us a great deal of trouble and delay, and wetted almost all my luggage. The water, which at present had no current, skirts the foot of the rocky slope on which the village is situated, the name of which is Yebbórewó. Here our camels created an extraordinary interest, and a great many women, although we did not attend to their wish to stop, managed to pass under the bellies of these tall creatures, in the hope of obtaining their blessing, as they thought them sacred animals.
Having kept along the rising ground, and passed several little hamlets adorned with monkey-bread-trees, we had to cross very difficult swampy ground, which, a little later in the season, must be avoided by a long circuit. Two months later Mount Bágelé must look almost like an island, so surrounded is it on all sides by deep inlets and swamps. The detached cone of Mount Takabéllo, rising to a height of about a thousand feet above the plain, for some time formed a conspicuous object in front of us on our winding path, till at length, a little before noon, we reached the outskirts of the capital in a state of mind not exempt from anxious feeling.
