Occurrence and multidrug resistance of Campylobacter spp. at duck farms and associated environmental and anthropogenic risk factors in Bangladesh.

BACKGROUND: The alarming rise in multi-drug resistant (MDR) zoonotic pathogens, including Campylobacter spp., has been threatening the health sector globally. In Bangladesh, despite rapid growth in poultry sector little is known about the potential risks of zoonotic pathogens in homestead duck flocks. The aim of this study was to understand the occurrence, species diversity, and multi-drug resistance in Campylobacter spp., and identify the associated risk factors in duck farms in Bangladesh. METHODS: The study involved 20 duck farms at 6 sub-districts of Mymensingh, Bangladesh. Monthly occurrence of Campylobacter spp. in potential sources at the farms during February-September, 2018, was detected by culture and PCR-based methods. Campylobacter isolates were examined for resistance to different antimicrobials. Risk factors, concerning climatic and environmental disposition, farm management, and anthropogenic practices, of Campylobacter infection were estimated by participatory epidemiological tools. RESULTS: Occurrence of Campylobacter spp. was detected in overall 36.90% (155/420) samples, more frequently in drinking water (60%, 30/50), followed by cloacal swab (37.50%, 75/200), egg surface swab (35%, 35/100) and soil of the duck resting places (30%, 15/50) but was not detected in feed samples (n = 20). PCR assays distinguished the majority (61.30%, 95/155) of the isolates as C. coli, while the rest (38.70%, 60/155) were C. jejuni. Notably, 41.7% (25/60) and 31.6% (30/95) strains of C. jejuni and C. coli, respectively, were observed to be MDR. The dynamics of Campylobacter spp., distinctly showing higher abundance during summer and late-monsoon, correlated significantly with temperature, humidity, and rainfall, while sunshine hours had a negative influence. Anthropogenic management-related factors, including, inadequate hygiene practices, use of untreated river water, wet duck shed, flock age (1-6 months), and unscrupulous use of antimicrobials were identified to enhance the risk of MDR Campylobacter infection. CONCLUSION: The present study clearly demonstrates that duck farms contribute to the enhanced occurrence and spread of potentially pathogenic and MDR C. coli and C. jejuni strains and the bacterial dynamics are governed by a combined interaction of environmental and anthropogenic factors. A long-term holistic research at the environment-animal-human interface would be integral to divulge health risk reduction approaches tackling the spread of Campylobacter spp. from duck farms.

Isolation and characterization of strictly anaerobic cellulolytic rumen bacterial species from Sahiwal cattle.

Objective: To isolate and characterize cellulolytic rumen bacteria from the rumen of Sahiwal cattle using rumen bacterial inoculum to increase the nutritional value of rice bran used as broiler feed. Materials and Methods: The ruminal liquid was kept at an optimal pH of 6.9 and a redox potential of less than -300 mV while being incubated anaerobically at 39°C in a medium containing rumen fluid glucose cellobiose agar. By using the Hungate technique, the organisms were detected based on their morphological, physiological, biochemical, and molecular testing. Results: The findings revealed that the isolated Ruminococcus albus, and Ruminococcus flavifaciens were obligate anaerobic, generally Gram-positive, nonmotile cocci or rod, single or pair, occasionally short chain, producing yellow pigment when grown on cellulose, and having a clear zone around the colonies. Both isolate fermented sugars such as cellobiose, glucose, and lactose, as well as decomposed xylan. The results also showed that the isolates recognized as Ruminococcus spp., a cellulolytic rumen bacterium, were catalase-negative, indole-negative, and gelatin liquefaction-positive. Conclusion: Isolation and characterization of Ruminococcus spp. may be helpful for Bangladesh in reducing the cost of producing poultry feed and circumventing restrictions on rice bran use. We can also develop more efficient and long-lasting plans to enhance poultry performance and feed efficiency, as well as increase the nutritional value of rice bran used as broiler feed, by understanding how various Ruminococcus spp. function in this process.

Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh.

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach.

Draft genome sequence of multidrug-resistant Citrobacter portucalensis BAU_133-2 strain isolated from a domestic duck (Anas platyrhynchos domesticus) in Bangladesh.

We announce the genome sequence of the Citrobacter portucalensis BAU_133-2 strain isolated from a domestic duck. Our assembled genome contained a length of 4.8 Mb, 110.0× genome coverage, 51.91% of an average GC content, 1 plasmid, 1 CRISPR array, 8 prophages, 27 antibiotic resistance genes, and 75 virulence factor genes.

Diversity of Streptococcus spp. and genomic characteristics of Streptococcus uberis isolated from clinical mastitis of cattle in Bangladesh.

Introduction: Streptococci are the major etiology in mastitis in dairy cattle, a cause of huge economic losses in the dairy industries. This study was aimed to determine the diversity of Streptococcus spp. isolated from clinical mastitis of cattle reared in Bangladesh. Methods: A total of 843 lactating cattle reared in four prominent dairy farms and one dairy community were purposively included in this study where 80 cattle were positive to clinical mastitis (CM) based on gross changes in the udder (redness, swelling, and sensitive udder) and/or milk (flakes and/or clots). Milk samples were collected from all the eighty cattle with clinical mastitis (CCM) and twenty five apparently healthy cattle (AHC). Samples were enriched in Luria Bertani broth (LB) and one hundred microliter of the enrichment culture was spread onto selective media for the isolation of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli and Corynebacterium spp., the major pathogen associated with mastitis. Isolates recovered from culture were further confirmed by species specific PCR. Results and Discussion: Out of 105 samples examined 56.2% (59/105), 17.14% (18/105), 9.52% (10/105) and 22.9% (24/105) samples were positive for Staphylococcus, Streptococcus, Enterococcus faecalis and E. coli, respectively. This study was then directed to the determination of diversity of Streptococcus spp. through the sequencing of 16S rRNA. A total of eighteen of the samples from CCM (22.5%) but none from the AHC were positive for Streptococcus spp. by cultural and molecular examination. Sequencing and phylogenetic analysis of 16S rRNA identified 55.6, 33.3, 5.6 and 5.6% of the Streptococcus isolates as Streptococcus uberis, Streptococcus agalactiae, Streptococcus hyovaginalis and Streptococcus urinalis, respectively. Considering the high prevalence and worldwide increasing trend of S. uberis in mastitis, in-depth molecular characterization of S. uberis was performed through whole genome sequencing. Five of the S. uberis strain isolated in this study were subjected to WGS and on analysis two novel ST types of S. uberis were identified, indicating the presence of at least two different genotypes of S. uberis in the study areas. On virulence profiling, all the isolates harbored at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all the S. uberis isolated in this study are potential mastitis pathogen. Overall findings suggest that Streptococcus encountered in bovine mastitis is diverse and S. uberis might be predominantly associated with CM in the study areas. The S. uberis genome carries an array of putative virulence factors that need to be investigated genotypically and phenotypically to identify a specific trait governing the virulence and fitness of this bacterium. Moreover, the genomic information could be used for the development of new genomic tools for virulence gene profiling of S. uberis.

Antimicrobial Resistance, Virulence Profiles, and Public Health Significance of Enterococcus faecalis Isolated from Clinical Mastitis of Cattle in Bangladesh.

This study was designed to identify Enterococcus faecalis from clinical mastitis of cattle and determine their antimicrobial resistance and virulence determinants to evaluate their potential public health significance. A total of 105 composite milk samples (80 from cattle with clinical mastitis and 25 from apparently healthy cattle) were analyzed. E. faecalis were isolated by culturing on enterococcal selective media and identified by PCR and sequencing. Antimicrobial resistance phenotype was elucidated by the disc diffusion method, and MIC was determined by broth microdilution method according to CLSI guidelines. Detection of antimicrobial resistance and virulence genes was done by PCR. E. faecalis were isolated from 11.25% (9/80) of the clinical mastitis and 4% (1/25) of the apparently healthy cattle milk samples. The disc diffusion test revealed 40% isolates as resistant to tetracycline and azithromycin, respectively. Among them, 20% (2/10) of isolates showed resistance to both tetracycline and azithromycin. Tetracycline-resistant isolates showed MIC ranging from ≥64 to >128 µg/ml and carried tetracycline-resistant genes tetK, tetL, and tetM in 25%, 25%, and 50% of the resistant isolates, respectively. On the other hand, all the isolates were sensitive to amoxicillin, ampicillin, bacitracin, chloramphenicol, gentamicin, penicillin, and vancomycin. In addition, the isolates carried at least one of the nine virulence genes screened with pil having the highest frequency, followed by fsrB, fsrC, ace, sprE, gelE, and agg genes. Positive correlations were evident between ace, fsrC, gelE, and sprE genes that are associated with the attachment and biofilm formation in E. faecalis. E. faecalis isolated in this study carried antibiotic resistance and virulence determinants which explain their competence to be potential human pathogens.

Molecular detection and antibiotyping of multi-drug resistant Enterococcus faecium from healthy broiler chickens in Bangladesh.

BACKGROUND: Enterococcus faecium is a ubiquitously distributed member of the intestinal microbiota of both humans and animals. Antibiotic resistant E. faecium are a major public health concern. OBJECTIVES: This study aimed to detect multi-drug resistant (MDR) E. faecium and their antibiotic resistance genes from broiler chickens in Bangladesh. METHODS: A total of 100 faecal samples of healthy broilers were screened by conventional methods and polymerase chain reaction (PCR) to detect E. faecium and their resistance genes. Disk diffusion test was employed to determine antibiotic profiles. RESULTS: By PCR, among 100 samples, 45% [95% confidence interval (CI): 35.62%-54.76%] were positive for E. faecium. Based on antibiogram, all the E. faecium isolates were found resistant to ampicillin, and frequently (93.33%-55.56%) resistant to ceftriaxone, cefotaxime, streptomycin, erythromycin, and imipenem; moderate to lower (26.67%-4.44%) resistance to tetracycline, ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and vancomycin. Interestingly, 80% (95% CI: 66.18%-89.10%) E. faecium isolates were MDR in nature. In addition, the indices of multiple antibiotic resistance (MAR) ranged from 0.08 to 0.83. By bivariate analysis, high positive significant correlations were observed between resistance profiles of erythromycin and imipenem, ciprofloxacin and norfloxacin, erythromycin and streptomycin, ceftriaxone and cefotaxime, tetracycline and chloramphenicol, and streptomycin and imipenem. Furthermore, the prevalence of resistance genes of E. faecium was 58.33% (tetA), 33.33% (tetB), 35.56% (blaTEM ), 60% (CITM), 13.33% (aadA1), and 12% (SHV). CONCLUSIONS: To the best of our knowledge, this is the first study in Bangladesh to detect MDR and MAR E. faecium and their associated resistance genes. The detection of MDR and MAR E. faecium and their corresponding resistance genes from healthy broilers is of public health concern because of their potential to enter into the food chain.

Virulence determinants and antimicrobial resistance of E. coli isolated from bovine clinical mastitis in some selected dairy farms of Bangladesh.

E. coli is one of the major significant pathogens causing mastitis, the most complex and costly diseases in the dairy industry worldwide. Present study was undertaken to isolate, detect the virulence factors, phylogroup, antimicrobial susceptibility and antimicrobial resistance genes in E. coli from cows with clinical mastitis. A total of 68 milk samples comprising 53 from clinical mastitis and 15 from apparently healthy cattle were collected from four different established dairy farms in Bangladesh. E. coli was isolated from the milk samples and identified by PCR targeting malB gene and sequencing of 16S rRNA gene. E. coli isolates were screened by PCR for the detection of major virulence genes (stx, eae and cdt) of diarrheagenic E. coli followed by phylogenetic grouping. Antimicrobial susceptibility of the E. coli isolates was determined by disk diffusion test and E. coli showing resistance was further screened for the presence of antimicrobial resistance genes. E. coli was isolated from 35.8% of the mastitis milk samples but none from the apparently healthy cattle milk. All the E. coli isolates were negative for stx, eae and cdt genes and belonged to the phylogenetic groups A and B1 which comprising of commensal E. coli. Antibiotic sensitivity testing revealed 84.2% (16/19) of the isolates as multidrug resistant. Highest resistance was observed against amoxicillin (94.5%) followed by ampicillin (89.5%) and tetracycline (89.5%). E. coli were found resistant against all the classes of antimicrobials used at the farm level. Tetracycline resistance gene (tetA) was detected in 100% of the tetracycline resistant E. coli and blaTEM-1 was present in 38.9% of the E. coli isolates. Findings of this study indicate a potential threat of developing antimicrobial resistance in commensal E. coli and their association with clinical mastitis. Occurrence of multidrug resistant E. coli might be responsible for the failure of antibiotic therapies in clinical mastitis as well as pose potential threat of transmitting and development of antibiotic resistance in human.

Virulence and Antimicrobial Resistance Profiles of Salmonella enterica Serovars Isolated from Chicken at Wet Markets in Dhaka, Bangladesh.

Virulent and multi drug resistant (MDR) Salmonellaenterica is a foremost cause of foodborne diseases and had serious public health concern globally. The present study was undertaken to identify the pathogenicity and antimicrobial resistance (AMR) profiles of Salmonellaenterica serovars recovered from chicken at wet markets in Dhaka, Bangladesh. A total of 870 cecal contents of broiler, sonali, and native chickens were collected from 29 wet markets. The overall prevalence of S. Typhimurium, S. Enteritidis, and untyped Salmonella spp., were found to be 3.67%, 0.57%, and 1.95% respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. S. Enteritidis isolates carried all virulence genes whilst S. Typhimurium isolates carried six virulence genes except sefA and spvC. A diverse phenotypic and genotypic AMR pattern was found. Harmonic descending trends of resistance patterns were observed among the broiler, sonali, and native chickens. Interestingly, virulent and MDR Salmonella enterica serovars were found in native chicken, although antimicrobials were not used in their production cycle. The research findings anticipate that virulent and MDR Salmonella enterica are roaming in the wet markets which can easily anchor to the vendor, consumers, and in the food chain.

Isolation and molecular-based identification of bacteria from unhatched leftover eggs of ducks in selected mini-hatcheries of Kishoreganj, Bangladesh.

OBJECTIVES: The study was designed for isolation and identification of the bacteria present in unhatched leftover eggs of duck in selected mini-hatcheries of Kishoreganj, Bangladesh. MATERIALS AND METHODS: A total of 54 unhatched discarded eggs were collected as samples from different mini-hatcheries of Tarail and Itna Upazilas of Kishoreganj and aseptically carried to the laboratory in the icebox. Surface washings (n = 54) and inner contents (n = 54) were collected and enriched in Luria-Bertani broth followed by the isolation of pure colonies of different bacteria onto eosin methylene blue agar, mannitol salt agar, Salmonella-Shigella agar, and blood agar plates. Identification of the bacterial isolates was done by cultural properties, staining, and biochemical tests followed by molecular detection by Polymerase chain reaction. RESULTS: Of 108 samples, 62 were found positive for Salmonella spp. (76%), 59 for E. coli (54%), 52 for Staphylococcus spp. (48%), and 5 for Clostridium spp. (9%). From the egg surface samples, Staphylococcus spp. were recovered in the highest (67%) followed by Salmonella spp. (59%), E. coli (56%), and Clostridium spp. (9%). From the inner contents of eggs, Salmonella spp. were recovered in the highest (56%), followed by E. coli (53%) and Staphylococcus spp. (30%). CONCLUSION: The isolated bacteria might be associated with the decreased hatchability and embryo mortality in the mini-hatcheries of duck.

Identification and characterization of Salmonella spp. from samples of broiler farms in selected districts of Bangladesh.

BACKGROUND AND AIM: Salmonella spp. are an important group of pathogens responsible for human and animal diseases. This study aimed to estimate the prevalence and identify and characterize of Salmonella spp. isolated from broiler farms of Gazipur, Tangail, and Dhaka districts of Bangladesh. This study also evaluated the difference of Salmonella positivity status between two groups of farms, good practices adapted in broiler rearing at the project intervened farms, and non-project intervened traditional farms. MATERIALS AND METHODS: A total of 352 samples including 128 cloacal swabs, 32 whole carcasses, 64 feed, 64 water, and 64 attendants' hand rinses were collected through convenient sampling technique from 16 poultry food safety project of Food and Agricultural Organization of United Nations Bangladesh intervened farms and other 16 non-project intervened farms in the same location. Various cultural based techniques and biochemical methods were employed for the estimation of prevalence, isolation, and identification of Salmonella spp. which was further evaluated by polymerase chain reaction. Antimicrobial susceptibility test using disk diffusion methods and serogrouping by slide agglutination test was accomplished for additional characterization. RESULTS: Among the samples, an overall prevalence of Salmonella spp. was 31.25% (110/352) (95% confidence interval [CI]=26.44-36.38%). However, the prevalence of Salmonella spp. was 24.43% (43/176) (95% CI=18.28-31.47) in project intervened farms and 38.07% (67/176) (95% CI=30.87-45.68%) in non-intervened farms. Among the 110 isolates, 31.82% (35/110) were fitted under serogroup B, and the rest of the isolates 75 (68.18%) under serogroup D. Of 110 isolates, 82.72%, 77.27%, 81.82%, and 79.09% were susceptible to ciprofloxacin, gentamycin, norfloxacin, and streptomycin, respectively. In addition, 81.82% and 80% isolates were resistant to erythromycin and tetracycline, respectively. Isolated Salmonella spp. presented moderate resistance to both amoxicillin and azithromycin. Alarmingly, 80.91% (89/110) isolates were shown to be multidrug-resistant Salmonella spp. CONCLUSION: The study has presented a significant variation of the prevalence of Salmonella spp. between project intervened and non-project intervened farms, and this indicates project intervened farms are comparatively safer than the non-intervened farms considering public health and food safety grounds. This research outcome also has highlighted a substantial proportion of poultry origin multidrug resistance Salmonella spp. is a potential source of public health hazards. In this regard, proper awareness creation and motivational activities on good agriculture practices in poultry rearing and maintaining good personal hygiene at the farmers' level are warranted through participatory training.

Isolation and molecular detection of Avipoxvirus from field outbreaks in Mymensingh, Bangladesh.

OBJECTIVE: The present study was performed for isolation, identification, and molecular detection of Avipoxvirus [Turkeypox virus (TPV), Fowlpox virus (FPV), and Pigeonpox virus (PPV)] from field outbreaks in some selected areas of Mymensingh division, Bangladesh. MATERIALS AND METHODS: A total of 60 suspected cutaneous nodular samples (10 TPV, 20 PPV, and 30 FPV) were collected. The samples were then subjected to isolation and identification by chicken embryo propagation followed by confirmation using polymerase chain reaction (PCR). RESULTS: The TPV, FPV, and PPV were successfully isolated and identified from the nodular samples using embryo propagation and PCR technique targeting pox virus p4b gene. Out of 10 Turkeypox suspected field samples, five (50%) were positive for TPV. Similarly, among 30 Fowl pox suspected field samples, 12 (40%), and out of 20 Pigeonpox suspected field samples, eight (40%) were found to be positive for FPV and PPV, respectively. The overall prevalence of avipox (TPV, FPV, and PPV) virus infections in Mymensingh division was 41.67% (n = 25/60). CONCLUSION: This study has shown that TPV, FPV, and PPV are circulating in Mymensingh division. The isolated TPV, FPV, and PPV field isolates can be used as vaccine candidates to develop an effective vaccine for effective controlling of the avipox in Mymensingh division and surrounding areas.

Characterization of Staphylococcus aureus isolated from chicken and quail eggshell.

OBJECTIVES: This study was conducted to assess the prevalence and characterization of Staphylococcus aureus from chicken and quail eggshells and to study the antibiogram of the isolates. MATERIALS AND METHODS: A total of 300 eggs (220 chicken eggs and 80 quail eggs) were collected from different retail shops and farms in Mymensingh district. Swabs taken from the egg surfaces were cultured on Mannitol Salt Agar for the isolation of S. aureus. Polymerase chain reaction was conducted for confirmatory identification of the bacterial species targeting nuc gene, followed by confirmation of methicillin-resistant S. aureus by targeting the mecA gene. Antibiotic sensitivity test of the isolated bacteria was done against commonly used antibiotics by the disk diffusion method. RESULTS: The prevalence of Staphylococcus spp. and S. aureus in the chicken eggshell surface was 20.45% and 10.45%, respectively. Similarly, the prevalence of Staphylococcus spp. and S. aureus in quail eggshell surface was 16.25% and 5%, respectively. Overall, 27 isolates were identified as S. aureus, of which 23 were from the chicken eggshell surface and four from quail eggshell surface. Among the seven isolates tested, overall four (57.14%) were positive for the nuc gene. On the other hand, the mecA gene could be detected in three (50%) S. aureus out of six oxacillin resistant isolates. The antibiogram study indicated that most of the isolates were resistant to the antibiotics under ß-lactam group. CONCLUSION: The present study concludes that chicken and quail egg surface harbor multidrug-resistant bacteria which may cause public health hazards, if these antibiotic-resistant bacteria are transferred to a human.