Avian pathogenic Escherichia coli (APEC) are causative agent of extraintestinal infections, collectively known as colibacillosis, which results significant losses in poultry industries. The extraintestinal survival of E. coli is facilitated by numerous virulence factors which are coded by virulence genes. This study was conducted to find out the pattern of antibiotic resistance and virulence genes content in the APEC strains isolated from broiler chickens at National Avian Disease Investigation Laboratory and Veterinary Teaching Hospital, Rampur, Chitwan, Nepal. A total of 50 E. coli strains were isolated from 50 colibacillosis suspected broiler chickens. Out of 50 isolates of E. coli, 47 (94%) showed resistant to three or more antimicrobials. The highest levels (22%) of multidrug-resistant E. coli were observed for five different types of antimicrobials. Antibiogram profiles of 50 E. coli strains showed the maximum resistance to ampicillin (98%), followed by co-trimoxazole (90%), and doxycycline (62%). The highest intermediate resistance was shown by colistin (50%) and the highest sensitivity was against amikacin (84%), followed by nitrofurantoin (55%). Based on the genetic criteria, 45 (90%) E. coli isolates were considered as pathogenic (APEC) which contained more than five virulence genes. Out of total APEC genes detected, we found the combination of iss, iucD, hlyF, ompT, iroN, and iutA genes were mostly associated with the APEC and additionally, to some lesser extent irp2, papC, Cva/cvi, and tsh genes showed the critical role for virulent traits of APEC strains. In this study, high prevalent of antimicrobial resistant pattern was found with avian pathogenic E. coli strains isolated from broiler chickens. To our knowledge, this is the first molecular analysis which confirmed the prevalence of APEC strains in poultry sector in Nepal. These finding suggest the need of surveillance and intervention system to control misuse of antibiotics and APEC outbreak in the poultry farm.

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Antibiotic resistance pattern and virulence genes content in avian pathogenic Escherichia coli (APEC) from broiler chickens in Chitwan, Nepal.

Antimicrobial resistance is a major health problem, particularly in developing countries like Bangladesh, where there is a paucity of information on resistance patterns and prevalence of antimicrobial determinants. Therefore, the aims of this study were to investigate the prevalence of resistance, including multi-drug resistance (MDR), and the associated genetic determinants in Escherichia coli isolates from cloacal swabs of live broiler chickens in Bangladesh. Altogether, 400 cloacal swabs (200 from Rajshahi and 200 from Dhaka divisions) were randomly collected from individual chickens in 50 broiler farms. E. coli was isolated and identified using conventional bacteriological culture and biochemical methods. The isolates were further confirmed using genus-specific 16S rRNA-targeted polymerase chain reaction (PCR) primers. Antimicrobial susceptibilities and MDR of the isolates against nine different antimicrobial agents (ampicillin, erythromycin, tetracycline, gentamicin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, colistin sulphate, and streptomycin) were determined using the Kirby-Bauer disc diffusion method. Resistance determinants of E. coli to ampicillin (blaTEM), streptomycin (aadA1), erythromycin [ere(A)], trimethoprim (dfrA1), and tetracycline [tet(A), tet(B)] were screened using PCR. Our results showed that all swab samples were positive for E. coli. The isolates were uniformly resistant to ampicillin, tetracycline, streptomycin, ciprofloxacin, erythromycin, and trimethoprim-sulphamethoxazole. The isolates exhibited highest susceptibility to colistin sulphate (73.5%), followed by gentamicin (49%), and levofloxacin (17%). All isolates were resistant to three classes of antibiotics, 204 isolates (51%) were resistant to four classes, and 56 isolates (14%) were resistant to five. The highest prevalence of antimicrobial resistance gene was recorded for tetracycline (tet(A):95.25%; tet(B):95.25%) followed by ampicillin (blaTEM:91.25%), streptomycin (aadA1:88.25%), erythromycin (ere(A):84.75%), and trimethoprim (dfrA1:65.5%). In conclusion, surveillance for MDR bacteria in poultry is a critical piece of knowledge, which would be useful for optimizing empiric antimicrobial treatments and exploring alternative antimicrobial agents.

https://www.mdpi.com/2076-0817/8/3/118/pdf

Susceptibility and Multidrug Resistance Patterns of Escherichia coli Isolated from Cloacal Swabs of Live Broiler Chickens in Bangladesh.

Pathogenic Escherichia coli found in humans and poultry carcasses harbor similar virulence and resistance genes. The present study aimed to analyze the distribution of extraintestinal pathogenic E. coli (ExPEC) virulence factors (VF), bla CTX-M groups, fosA3, and mcr-1 genes in E. coli isolated from commercialized chicken carcasses in southern Brazil and to evaluate their pathogenic risk. A total of 409 E. coli strains were isolated and characterized for genes encoding virulence factors described in ExPEC. Results of antimicrobial susceptibility testing confirmed that the strains were resistant to β-lactams, fosfomycin, colistin, and others resistance groups. The highest prevalence of VFs was observed in isolates belonging to the CTX-M groups, especially the CTX-M-2 group, when compared to those in other susceptible strains or strains with different mechanisms of resistance. Furthermore, ESBL strains were found to be 1.40 times more likely to contain three to five ExPEC virulence genes than non-ESBL strains. Our findings revealed the successful conjugation between ESBL-producing E. coli isolated from chicken carcass and the E. coli recipient strain J53, which suggested that genetic determinants encoding CTX-M enzymes may have originated from animals and could be transmitted to humans via food chain. In summary, chicken meat is a potential reservoir of MDR E. coli strains harboring resistance and virulence genes that could pose serious risks to human public health.

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Distribution of ExPEC Virulence Factors, blaCTX-M, fosA3, and mcr-1 in Escherichia coli Isolated From Commercialized Chicken Carcasses

Antimicrobial resistance is a major global issue for human and animals. Increased use of antimicrobials in livestock and poultry has become one of the causes of antimicrobial resistance development in microorganisms. The aim of the study was to characterize antimicrobial resistant bacteria from raw buffalo and chicken meat in standard in vitro condition. A total of 140 raw meat samples were collected from different retail shops of Bhaktapur Metropolitan City, Nepal. Among them, 70 were raw buffalo meat and 70 were raw chicken meat samples. Bacterial growth, identification, and antimicrobial susceptibility test were performed according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Out of 140 samples, bacterial growth was seen in 67 raw buffalo meat and 59 raw chicken meat samples, i.e., bacterial growth was observed in 90.0% of the samples. A total of 161 bacterial isolates were detected. Escherichia coli (35.4%) and Klebsiella spp. (30.4%) were found to be the most prevalent bacteria followed by Citrobacter spp. (11.8%), Staphylococcus aureus (9.3%), Salmonella spp. (7.4%), and Proteus spp. (5.5%). Chicken meat isolates showed higher antimicrobial resistance rates in comparison to buffalo meat isolates, particularly against antimicrobials like Amoxicillin, Tetracycline, Cotrimoxazole and Nalidixic acid, p value<0.05 when compared between buffalo and chicken meat. Overall, 32.7% Multidrug-Resistant (MDR) isolates were found, in which 50.0% MDR isolates were found from chicken raw meat and 21.9% were found from buffalo raw meat. MDR isolates of Escherichia coli, Proteus spp. and Staphylococcus aureus constituted 52.5%, 77.7% and 40.0%, respectively, of both buffalo and chicken raw meat. This study indicates antimicrobials resistant bacteria existing at an alarming rate, higher in chicken meat than in buffalo meat.

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Multidrug-Resistant Bacteria from Raw Meat of Buffalo and Chicken, Nepal.

Antibiotic-resistant Escherichia coli (E. coli) are common in retail poultry products. In this study, we aimed to isolate and characterize multidrug resistant (MDR) E. coli in raw chicken meat samples collected from poultry shops in Sylhet division, Bangladesh, as well as to determine correlation between resistance phenotype and genotype. A total of 600 chicken meat swabs (divided equally between broiler and layer farms, n = 300 each) were collected and the isolates identified as E. coli (n = 381) were selected. Disc diffusion antimicrobial susceptibility assay showed resistance of these isolates to ampicillin, erythromycin, tetracycline, streptomycin, trimethoprim-sulfamethoxazole, chloramphenicol, and gentamicin. Polymerase chain reaction (PCR) identified several antibiotic resistance genes (ARGs) in our isolates. Among these ARGs, the prevalence of tetA (for tetracycline) was the highest (72.58%) in broiler chicken isolates, followed by sul1 (for sulfonamide; 44.16%), aadA1 (for streptomycin; 33.50%), ereA (for erythromycin; 27.41%), aac-3-IV (for gentamicin; 25.38%), and the two genes cmlA (24.87%) and catA1 (8.63%) for chloramphenicol. On the other hand, the respective prevalence in layer chicken isolates were 82.06%, 47.83%, 35.87%, 35.33%, 23.91%, 19.02%, and 5.43%. Furthermore, 49.23% of the isolates from broiler chicken were MDR, with the presence of multiple antibiotic resistance genes, including 3 (40.11%) and 4 (9.13%) genes. On the other hand, 51.09% of layer chicken E. coli isolates were MDR, with 3, 4 or 5 ARGs detected in 36.41%, 14.13%, and 0.54% of the isolates, respectively. We also found that 12.8% of broiler chicken E. coli isolates and 7.61% of layer chicken isolates carried genes coding for extended-spectrum SHV beta-lactamases. Lastly, we report the presence of the AmpC beta-lactamase producing gene (CITM) in 4.56% and 3.26% of broiler and layer chicken E. coli isolates, respectively. We found significant correlations between most of the antimicrobial resistant phenotypes and genotypes observed among the investigated E. coli isolates. Our findings highlight the need for the prudent use of antimicrobials in chickens to minimize the development of antibiotic-resistant bacterial strains.

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Isolation and molecular characterization of multidrug-resistant Escherichia coli from chicken meat

Multidrug resistance (MDR) and extended spectrum beta lactamase (ESBL) producer Gram negative bacteria are considered as a major health problem, globally. ESBL enzyme hydrolyses the beta lactam ring of third generation cephalosporins, which alters the structure of the antibiotic. Due to the modification in structure of the antibiotic, bacteria show resistance to these antibiotics. Resistant bacterial strains are transmitted to humans from animals through consumption of uncooked meat, through contact with uncooked meat and meat surfaces. This study aims to assess bacteriological profile and analyze the situation of antibiotic resistance, multidrug resistance, and ESBL producing Gram negative bacteria in chicken meat. A total of 38 chicken meat samples were studied in which 103 Gram negative bacteria were isolated. Species of Gram negative bacteria were identified as Citrobacter spp. (44.7%), Salmonella spp. (26.2%), Proteus spp. (18.4%), Escherichia coli (4.8%), Shigella spp. (3.9%), Pseudomonas spp. (1.9%), and Klebsiella spp. (1.0%). The prevalence of MDR isolates was found to be 79.6%. Total ESBL producer was 36.9% and ESBL producer among MDR was 34.9%. This concludes wide range of antibiotic resistance bacteria is prevalent in raw chicken meat.

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Raju Shah Turha

Papers

Multi-drug resistance and extended spectrum beta lactamase producing Gram negative bacteria from chicken meat in Bharatpur Metropolitan, Nepal.