Antimicrobial resistance and virulence profiling of Staphylococcus pseudintermedius isolated from cats, Bangladesh.

Staphylococcus pseudintermedius is a significant bacterial pathogen that frequently colonizes different body sites and mucous membranes of pets. The objectives of the cross-sectional study were to estimate the prevalence, antimicrobial resistance pattern, and detection of diverse resistance as well as virulence genes of S. pseudintermedius in cats. A standard bacteriological method, species-specific gene and different antimicrobial resistance as well as virulence genes were confirmed by PCR assay. A total of 233 swab samples were collected from different body sites of 102 cats, among them 146 swabs from 73 healthy cats, and 87 from 29 diseased cats. Overall, prevalence of S. pseudintermedius in cats was 12.01%, while dermatitis and otitis affected cats were 26.08% and 33.33%, respectively. The highest antimicrobial resistance was observed against penicillin (96.42%) followed by streptomycin (85.71%) and erythromycin (78.57%). Moreover, 89.28% of S. pseudintermedius isolates exhibit multi-drug resistance (MDR) (≥ 3 classes' antimicrobial resistant). In addition, 17.86% isolates harbored the mecA gene; thus, were classified as methicillin-resistant S. pseudintermedius (MRSP). Furthermore, the erythromycin resistance genes ermA and ermB were harbored by 25% and 10.71% of isolates, while 42.86% and 17.86% of isolates carried tetK and tetL (tetracycline resistance) genes, respectively. In virulence profiling, 32.14% (sea) and 10.71% (seb) of isolates were found positive for enterotoxin genes, whereas, the toxic shock syndrome toxin-1 (tst-1) gene and the Panton-Valentine leukocidin gene (pvl) were detected in 25% and 14.29% of isolates, respectively. To our knowledge, this is the first report of cats in Bangladesh for MDR S. pseudintermedius, MRSP, and their virulence profiling.

Molecular identification, antimicrobial resistance and virulence gene profiling of Staphylococcus spp. associated with bovine sub-clinical mastitis in Bangladesh.

This study was conducted to investigate the diversity and antimicrobial resistance profiling of Staphylococcus species causing sub-clinical mastitis (SCM) in dairy herds in Bangladesh as well as putative risk factors associated with the infections. Individual quarter milk samples were collected from a total of 284 lactating cows from 30 dairy farms were screened by means of California mastitis test; 178 (62.7%) of them had at least of quarter affected by SCM. After conventional microbiological isolation procedures, PCR tests were used for Staphylococcus species identification and detection of antimicrobial resistance and virulence genes. S. chromogenes (65.7%) was the most predominant species followed by, S. epidermidis (20.2%), S. haemolyticus (19.1%), S. aureus (15.7%), and S. sciuri (5.6%). High levels of antimicrobial resistance to ampicillin and amoxicillin/clavulanic acid were observed in S. aureus (82.1% and 75%) and S. sciuri (80% and 70%), while resistance to cefepime was markedly higher in S. chromogenes (95.7%), S. haemolyticus (94.1%), and S. epidermidis (97.2%). Multidrug resistance isolates were identified in all five species. The mecA gene was detected in S. aureus (32.1%) and S. chromogenes (5.98%). In addition, 20% S. sciuri and 17.7% S. haemolyticus carried the cytotoxin (pvl) gene, while 14.3% S. aureus harbored the toxic shock syndrome toxin (tst) gene. Multivariable logistic regression analysis identified "Old aged" (OR [CI]: 3.5 [1-12.4]); "Early stage of lactation" (OR [CI]: 3.4 [1.2-9.7]) and, "Firm udder condition" (OR [CI]: 4.2 [1.2-14.6]) as risk factors associated with SCM caused by S. aureus, S. chromogenes, and S. haemolyticus, respectively. Moreover, "Use of antimicrobials" (OR [CI]: 10.4 [3.4-32.1] and "History of previous clinical mastitis" (OR [CI]: 4.9 [1.2-19.7] for the carriage of methicillin-resistant Staphylococcus spp.

Phenotypical Identification and Toxinotyping of Clostridium perfringens Isolates from Healthy and Enteric Disease-Affected Chickens.

Clostridium perfringens is a ubiquitous spore-forming anaerobic pathogen that is frequently associated with enteric disease in chickens. Moreover, enterotoxin-producing C. perfringens has high zoonotic potential as well as serious public health concerns due to the emanation of food-borne intoxication. The present study was designed to isolate, identify, and toxinotype C. perfringens from both healthy and cases of necrotic or ulcerative enteritis chickens. A total of 110 samples were collected from July 2019 to February 2021. Among the samples, 38 (34.5%, 95% CI: 26.39-43.83) were positive for C. perfringens and were obtained from broiler 21 (33.3%, 95% CI: 22.91-45.67), Sonali 9 (34.6%, 95% CI: 19.31-53.88), and layer 8 (38%, 95% CI: 20.68-59.20). C. perfringens was highly prevalent (35.7%, 95% CI: 25.48-47.44) in enteritis chickens compared with healthy ones. In multiplex PCR toxinotyping, 34 (89.4%) isolates were identified as C. perfringens type A by the presence of the alpha toxin gene (cpa). Moreover, in addition to the cpa gene, 3 (14.3%, 95% CI: 4.14-35.48) broiler and 1 (11.1%, 95% CI: 0.01-45.67) Sonali isolates harbored the enterotoxin gene (cpe) and were classified as type F. However, none of the isolates carried genes encoding beta (cpb), epsilon (etx), iota (iap), or beta-2 (cpb2) toxins. Multivariable logistic regression analysis identified the following variables such as; "previously used litter materials" (OR 21.77, 95% CI 2.22-212.66, p ≤ 0.008); intestinal lesions, "presence of ulceration" (OR 30.01, 95% CI 3.02-297.91, p ≤ 0.004); "ballooned with gas" (OR 24.74, 95% CI 4.34-140.86, p ≤ 0.001) and "use of probiotics" (OR 5.24, 95% CI 0.74-36.75, p ≤ 0.095) act as risk factors for C. perfringens colonization in chicken gut. This is the first study of molecular toxinotyping of C. perfringens from healthy and enteric-diseased chickens in Bangladesh, which might have a potential food-borne zoonotic impact on human health.

Frequently used therapeutic antimicrobials and their resistance patterns on Staphylococcus aureus and Escherichia coli in mastitis affected lactating cows.

Mastitis is one of the most frequent and costly production diseases of dairy cattle. It is frequently treated with broad-spectrum antimicrobials. The objectives of this work were to investigate the prevalence of Staphylococcus aureus and Escherichia coli, find out the antimicrobials used in mastitis treatment, and explore the antimicrobial resistance profile including detection of resistance genes. Bacterial species and antimicrobial resistance genes were confirmed by the polymerase-chain reaction. A total of 450 cows were screened, where 23 (5.11%) and 173 (38.44%) were affected with clinical and sub-clinical mastitis, respectively. The prevalence of S. aureus was 39.13% (n = 9) and 47.97%(n = 83) while, E. coli was 30.43% (n = 7) and 15.60% (n = 27) in clinical and sub-clinical mastitis affected cows, respectively. The highest antimicrobials used for mastitis treatment were ciprofloxacin (83.34%), amoxycillin (80%) and ceftriaxone (76.67%). More than, 70% of S. aureus showed resistance against ampicillin, oxacillin, and tetracycline and more than 60% of E. coli exhibited resistance against oxacillin and sulfamethoxazole-trimethoprim. Selected antimicrobial resistance genes (mecA, tetK, tetL, tetM, tetA, tetB, tetC, sul1, sul2 and sul3) were identified from S. aureus and E. coli. Surprisingly, 7 (7.61%) S. aureus carried the mecA gene and were confirmed as methicillin-resistant S. aureus (MRSA). The most prevalent resistance genes were tetK 18 (19.57%) and tetL 13 (14.13%) for S. aureus, whereas sul1 16 (47.06%), tetA 12 (35.29%), sul2 11 (32.35%) and tetB 7 (20.59%) were the most common resistance genes in E. coli. Indiscriminate use of antimicrobials and the presence of multidrug-resistant bacteria suggest a potential threat to public health.

Prevalence of coagulase-positive methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in dogs in Bangladesh.

BACKGROUND: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have a significant health impact on people with direct or supportive occupations in veterinary medicine including veterinarians, animal handlers, laboratory personnel and pet owners. OBJECTIVES: This cross-sectional survey was conducted to determine the prevalence of and risk factors for S. aureus, S. pseudintermedius, MRSA and MRSP in dogs in Bangladesh. METHODS: A total of 358 swab samples were collected from different body sites of 150 dogs attending a university teaching veterinary hospital between January and June 2018. Standard bacteriological procedures were followed to isolate Staphylococcus, and identification was confirmed to the species level by PCR to detect the nuc gene. MRSA and MRSP were confirmed by the presence of the mecA gene. RESULTS: The prevalence of coagulase-positive S. aureus and S. pseudintermedius in dogs were 16% and 45.3%, respectively. S. aureus and S. pseudintermedius isolates displayed the highest resistance against nalidixic acid (95.2% and 91%, respectively) and erythromycin (89.3% and 84.7%, respectively). Notably, all the staphylococcal isolates showed resistance to ≥3 antimicrobial classes. The prevalence of MRSA and MRSP in dogs was 8.7% and 6%, respectively. Multivariable logistic regression analysis identified the following variables as risk factors for MRSA colonisation in dogs: dogs with dermatitis (odds ratio [OR], 12.24, 95% CI: 3.12-57.33; p < 0.001) and history of antibiotic use (OR 8.73, 95% CI: 2.23-43.10; p < 0.001). Presence of otitis (OR 14.22; 95% CI: 1.64-103.58; p = 0.008) and oral lesions (OR 9.48, 95% CI: 1.14-64.82; p = 0.002) were identified as the significant risk factors for the carriage of MRSP. CONCLUSIONS: The circulation of multidrug-resistant S. aureus and S. pseudintermedius is a serious concern to dogs and humans. To our knowledge, this is the first report of S. pseudintermedius and MRSP affecting dogs in Bangladesh.

Antimicrobial resistance profiling and burden of resistance genes in zoonotic Salmonella isolated from broiler chicken.

BACKGROUND: Salmonella is frequently found in poultry of which only motile serovars have zoonotic significance due to their potential to induce human gastrointestinal infections. Antimicrobial resistance, being a public health concern, the emergence of multidrug-resistant (MDR) Salmonella serotypes affecting food chain has greater impact worldwide. AIM: Information on circulation of zoonotic Salmonella strains in commercial poultry farm level is limited in many parts of the world. This cross-sectional study was aimed to investigate the zoonotic Salmonella strains circulating in the broiler farm environment with their detailed antimicrobial resistance profiling. METHODS: Pooled faecal samples were collected randomly from commercial broiler farms of Chattogram district, Bangladesh. Standard bacteriological procedure was followed to isolate Salmonella, and identification was confirmed by genus specific polymerase chain reaction (PCR). After phenotypic characterisation of resistance profile against eight antimicrobials by disc diffusion technique, all strains were screened by PCR for some selected resistance genes. RESULTS: Out of the 350 samples, Salmonella was isolated and identified from 86 samples. In antimicrobial sensitivity testing, more than 98.8% isolates showed resistance to ampicillin and 94.2% to tetracycline followed by enrofloxacin (56%) and ciprofloxacin (50%). Notably, 94% isolates were found to be MDR. The results of PCR assays revealed that 81.4% of the isolates were carrying the tetA gene, 19.8% the tetB and 10.47% the tetC gene. The prevalence of the isolates bearing the blaTEM , blaCTX-M and Sul-I gene were 95.4%, 7.0 % and 37.2 %, respectively. CONCLUSION: There is a great risk to secure healthy poultry products due to the circulation of these MDR zoonotic Salmonella.

Lumpy skin disease outbreak in cattle population of Chattogram, Bangladesh.

BACKGROUND: Lumpy skin disease (LSD) is an important viral disease causing significant economic losses in commercial livestock production. In mid-2019, an outbreak of LSD has been reported in cattle population from different parts of Bangladesh including Chattogram division. A cross-sectional surveillance study was undertaken from August 2019 to December 2019 to investigate the prevalence and associated risk factors of LSD in cattle in Chattogram district. METHODS: A total of 3,327 cattle from 19 commercial farms were examined for the LSD specific skin lesions and associated risk factors. A total of 120 skin biopsies were collected from the suspected animal for the confirmation of the disease using molecular detection and histopathological examination. Partial genome sequencing and phylogenetic analyses were performed on selected viral isolates. RESULTS: The overall clinical prevalence of LSD in the study population was 10% (95% confidence interval [CI]: 9.4%-11%) where the highest farm level outbreak frequency was 63.33% (95% CI: 45.51%-78.13%) and the lowest 4.22% (95% CI: 3.39%-5.25%). Crossbred and female cattle showed a significantly higher prevalence of the disease compared to their counterparts. Introduction of new animals in farms was found to be one of the most significant risk factors in the transmission of the disease. All suspected skin biopsies were positive for LSD virus (LSDV) infection with granulomatous and pyogranulomatous dermatitis was revealed on histopathology. Phylogenetic analysis based on the inverted terminal repeat region of the LSDV gene suggested that the locally circulating strain was closely related to the strains isolated from the Middle East and North African countries. CONCLUSIONS: The data generated in this study would be beneficial to the field veterinarians and animal health decision makers in the country as well as it will aid in taking appropriate measures to prevent further relapse or outbreak of this disease in future.

Estimation of the prevalence and determination of risk factors associated with demodicosis in dogs.

OBJECTIVES: Demodicosis is a vital skin problem in dogs. The present study has determined the prevalence and associated risk factors of demodicosis in dogs and the response to treatment. MATERIALS AND METHODS: A total of 100 skin scrapings were collected from dogs having dermatological lesions brought to the Teaching and Training Pet Hospital and Research Center of Chattogram Veterinary and Animal Sciences University for treatment purpose. The collected scrapings were dissolved in 10% potassium hydroxide to detect mites through microscopic examination. Various risk factors like breed, age, sex, hair type, health status, and management system (indoor and outdoor) were analyzed using the logistic regression model. Positive cases were treated with oral ivermectin (Scabo®; at 0.6 mg/kg/day) along with amitraz 12.5% (Ridd®) diluted to 0.05% for rubbing on the body after bathing with chlorhexidine shampoo (PetHex®). Clavulanate amoxicillin (Moxaclav®) and omega-3 fatty acids (OMG-3®) were also suggested to prevent secondary bacterial infection and to maintain skin and hair coat integrity. The recovery rate was observed every 2-4 weeks of treatment upto 11-13 weeks. RESULTS: The overall prevalence of demodicosis was recorded as 27%. Hair type, health status, and management system were significantly (p < 0.05) associated with the occurrence of demodicosis in dogs. Following treatment, the first negative skin scraping for mite was noticed at 8-10 weeks of treatment, and in all cases, clinical signs completely disappeared at 80-90 days of treatment. CONCLUSION: A good percentage of the dogs having dermatological lesions was determined as demodicosis. Long-term oral ivermectin and topical amitraz, an oral antibiotic, and nutritional therapy are effective against canine demodicosis. Keeping long-haired dogs with good body condition and indoor management is highly suggestive of preventing and controlling the disease.

Abundance of Mobilized Colistin Resistance Gene (mcr-1) in Commensal Escherichia coli from Diverse Sources.

Aims: Antimicrobial resistance (AMR) spreads not only by pathogenic but also by commensal bacteria, and the latter can become a reservoir for resistance genes. This study was aimed to investigate the AMR patterns along with the presence of mobilized colistin resistance (mcr) genes in commensal Escherichia coli circulating in chickens, farm environments, street foods, and human patients. Materials and Methods: By a cross-sectional survey, isolates obtained from 530 samples were tested for their AMR profiles against 9 antimicrobials. Minimum inhibitory concentration (MIC) of the phenotypically colistin-resistant isolates was determined and screened for a set of mcr genes followed by sequencing of mcr-1 gene in the multidrug-resistant (MDR) isolates. Results: A total of 313 E. coli strains were isolated and confirmed by polymerase chain reaction. Antimicrobial susceptibility testing revealed that about 98% (confidence interval [95% CI] 95-99) of the isolates were MDR, and 58% (95% CI 52-63) isolates exhibited resistance to colistin. MIC values of colistin against the isolates ranged from 4 to 64 mg/L. Except for human patients, 20.4% colistin-resistant isolates from other sources of isolation had mcr-1 gene. Conclusions: There is abundance of commensal MDR E. coli strains with the acquisition of mcr-1 gene circulating in chickens and farm environments in Bangladesh.

Coagulase-positive methicillin-resistant Staphylococcus aureus circulating in clinical mastitic goats in Bangladesh.

BACKGROUND AND AIM: Staphylococcus aureus is argued as one of the principal organisms responsible for mammary gland infection in lactating goats, causing both clinical and subclinical mastitis. Being highly zoonotic potential, pathogen emergence of methicillin-resistant S. aureus (MRSA) has a significant clinical impact on treatment and management of clinical mastitis. We conducted a cross-sectional study to investigate the prevalence of coagulase-positive S. aureus (CoPS), antimicrobial resistance profile of Staphylococcus spp., prevalence of MRSA, and association between different clinical parameters with CoPS. MATERIALS AND METHODS: A total of 67 clinical mastitic goats were sampled based on clinical examination and California mastitis test. Standard bacteriological methods were performed to isolate and identify Staphylococcus spp. CoPS were confirmed by nuc gene using polymerase chain reaction (PCR). All staphylococcal isolates were further examined for antimicrobial susceptibility testing by disk diffusion method. MRSA was confirmed based on mecA gene-based PCR. RESULTS: Here, 49 (73.13%; 95% confidence interval [CI], 61.41-82.35) samples were positive for Staphylococcus spp., of which 17 (34.69%; 95% CI, 22.88-48.73) isolates were CoPS and rest of the isolates (32; 65.30%; 95% CI, 51.27-77.12) were identified as coagulase-negative Staphylococcus spp. (coagulase-negative staphylococci [CNS]). Both, CoPS and CNS isolates displayed the highest resistance against tetracycline (76.47% and 75%, respectively) and oxacillin (70.58% and 68.75%, respectively). Notably, all staphylococcal isolates were multidrug-resistant (showed resistance to ≥3 classes of antimicrobials). mecA gene was found in 6 (8.96%; 95% CI, 3.84-18.52) CoPS isolates indicating MRSA strains. Among different clinical parameters, presence of high body temperature (p<0.05), firm udder consistency (p<0.01), bloodstained milk (p<0.00), and pus in milk (p<0.00) were significantly associated with the presence of CoPS in mastitic caprine milk. CONCLUSION: To the best of our knowledge, this is the first report of MRSA isolated from clinical caprine mastitis cases in Bangladesh. The findings of this study would help in cautious selection as well as administration of antimicrobials for therapeutic management of mastitic goats.

Acquisition of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli of Livestock Origin in Bangladesh.

Aims: To investigate plasmid-borne colistin resistance mechanism (plasmid-mediated colistin resistance [mcr-1]) in Escherichia coli of human, veterinary, and environmental origin in Bangladesh. Materials and methods: A total of 810 samples were collected from different sources. Isolation and identification of E. coli was performed using classical bacteriology and then tested for antimicrobial susceptibility. Colistin-resistant isolates were further analyzed for mcr-1 gene using PCR. Minimum inhibitory concentration (MIC) was determined using microbroth dilution technique. After sequencing of mcr-1 gene, phylogenetics was conducted to see the relationship with other mcr-1 gene sequences. Results: A total of 358 E. coli were isolated from 810 samples of humans, animals, environment, and food in Bangladesh. Of them 49 (15.9%) isolates were phenotypically resistant to colistin and 254 (70.9%) were resistant to multiple antimicrobials. mcr-1 gene was detected in three E. coli isolates of poultry source. For the three mcr-1 positive isolates the MIC of colistin sulfate was 4, 8, and 128 µg/mL. Gene sequencing of two of the three mcr-1 positive isolates and phylogenetic analysis showed close similarities of one isolate to other mcr-1 sequences available at GenBank while the other appeared to have evolved locally. Conclusion: First-ever report on circulation of mcr-1 E. coli of livestock origin in Bangladesh.

Impact of rice gruel on rumen metabolites and growth performance of sheep.

OBJECTIVES: We investigated the impact of rice gruel as an alternative energy source of molasses as well as measured the effectiveness of rice gruel on the physiology of the rumen environment and the growth performance of growing lamb. MATERIALS AND METHODS: A number of 18 sheep with an average age of 7 months and mean body weight of 5.9 kg were selected and divided into three groups for 60 days long feeding trial with urea molasses straw (UMS), urea rice gruel straw (URS), and concentrate feed. Every fortnight interval, live weight was recorded and rumen liquor from every group was collected four times before and after feeding at 4-h interval to examine the rumen environment. RESULTS: Color, odor, consistency, and protozoal motility remain unchanged in all three groups. The pH of the rumen liquor was highest at 8 h of post feeding among three groups. The bacterial count (6.1 × 1010) was higher in the group that consuming UMS than URS and concentrate feed. The rumen protozoa also showed a similar growth pattern in proportion to a number of rumen bacteria. At the end of the 60 days feeding trial, no significant differences (p ≤ 0.05) were found among the three groups in contrast to body weight gain. CONCLUSION: In the current feeding trial, the close similar effectiveness of rice gruel and molasses was found as a fermentable energy source. However, we suggest that rice gruel can be supplemented as a substitute for molasses under the situation when molasses are not available or expensive in field condition.