In-silico insights of ESBL variants and tracking the probable sources of ESBL-producing Escherichia coli in a small-scale poultry farm.

Commercial broiler farms face challenges of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli transmitted from both vertical and horizontal routes. Understanding the dynamics of ESBL-E. coli transmission in compromised biosecurity settings of small-scale rural poultry farms is essential. This study aimed to elucidate the probable transmission pathways of ESBL-E. coli in such settings, employing phylogenetic analysis and molecular docking simulations to explore the catalytic properties of ß-lactamase variants. Sampling was conducted on a small-scale poultry farm in West Bengal, India, collecting 120 samples at three intervals during the broiler production cycle. E. coli isolates underwent resistance testing against eight antimicrobials, with confirmation of ESBL production. Genotypic analysis of ESBL genes and sequencing were performed, alongside molecular docking analyses and phylogenetic comparisons with publicly available sequences. Among 173 E. coli isolates, varying resistance profiles were observed, with complete resistance to cefixime and high resistance to amoxicillin and tetracycline. The incidence of ESBL-E. coli fluctuated over the production cycle, with dynamic changes in the prevalence of blaCTX-M-type and blaSHV-type genes. Phylogenetic analysis indicated partial clonal relationships with human clinical strains and poultry strains from the Indian subcontinent. Molecular docking confirmed the catalytic efficiencies of these ESBL variants. The study highlights probable vertical transmission of ESBL-E. coli and emphasizes drinking water as a potential source of horizontal transmission in small-scale poultry farms. Strict biosecurity measures could prevent the spread of antimicrobial-resistant bacteria in birds and their products in a small scale poultry farm.

Isolation of Reticuloendotheliosis Virus from Chorio-allantoic Membrane of SPF Chicken Eggs inoculated with Fowl Pox Virus.

Fowl Pox Viruses (FPV) infect chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth etc. The birds, affected with FPV, also show anemia and ruffled appearance which are clinical symptoms of Reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of Reticuloendotheliosis Virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the Chorio-allantoic Membrane (CAM) of 10 days old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. But the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV virus, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Docking analysis of circulating CTX-M variants in multi-drug resistant, beta-lactamase and biofilm-producing E. coli isolated from pet animals and backyard livestock.

The generation of antimicrobial-resistant bacteria largely depends on the use of antimicrobials not only in humans but also in pet animals and livestock. The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing- E.coli in healthy pet and backyard livestock. The study also intended on molecular docking experiments to confirm the nature of the catalytic mechanism in ß-lactamase enzymes, encoded by the various blaCTX-M genotypes and phylogenetic analysis to reveal clonal relationship of the animal origin E. coli isolates with human clinical strains. The rectal swabs were collected from healthy dogs (n = 254), cats (n = 108), sheep (n = 119) and goats (n = 143) in India. In total 247 (76.47%) E. coli strains were identified as ESBL producers. The possession of ESBL-producers was significantly more (p < 0.05) in pets than in the backyard livestock. Most of the strains possessed blaCTX-M-15 like clones. E. coli strains possessing blaCTX-M-15.2, blaCTX-M-157, blaCTX-M-181 and blaCTX-M-218 like clones, isolated from pets were not reported earlier. The study detected 56.65% of E. coli strains as moderate or strong biofilm producers possessing biofilm-associated genes (csgA, rcsA, rpoS, sdiA). ESBL-producing E. coli showed phenotypical resistance to tetracycline (93.1%), azithromycin (89.8%), ampicillin (84.2%), cefotaxime (80.9%), doxycycline (82.5%), co-trimoxazole (80.9%), ampicillin/cloxacillin (76.9%). The CTX-M variants obtained in this study were modelled by the SWISS-MODEL and verified. Ligand having minimum binding energy, show the highest affinity of ß-lactamases for cefotaxime and cefpodoxime. The Gibbs free energy release for all 14 different complex ranges between -6.9 (CTX-M-15.2+cefpodoxime) to -5.3 (CTX-M-218+cefpodoxime) Kcal/mol. Phylogenetic analysis of the animal origin ESBL-E. coli strains revealed a partial clonal relationship with the clinical isolates of local human patients. The present study described the significant presence of biofilm and ß-lactamase producing, multi-drug resistant E. coli in pet animals having public health importance.

Exploring broilers and native fowls of Andaman and Nicobar Islands as a source of ß-lactamase-producing Enterobacteriaceae even with limited anthropogenic activities and docking-based identification of catalytic domains in novel ß-lactamase variants.

Objectives: The present study was conducted to detect the occurrence of ß-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the ß-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database. Materials and methods: A total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical ß-lactamase producers were identified by a double-disc test. The genes (bla CTX, bla SHV, bla TEM , and bla AmpC) were screened, and selected sequences of ß-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different ß-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the ß-lactamase variants. The selected ß-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank. Results: In total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61-76%) and the lowest against gentamicin (15-20%). Phenotypical production of ß-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of ß-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the ß-lactamase-producing isolates revealed the maximum possession of bla TEM, followed by bla SHV and bla CTX - M. The nucleotide sequences were found to be similar with bla CTX - M-15, bla SHV - 11, bla SHV - 27, bla SHV - 228, bla TEM - 1, and bla AmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from -8.1 (SHV-27 + cefotaxime) to -7 (TEM-1 + cefotaxime) kcal/mol. Conclusion and relevance: The study revealed ß-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed bla TEM - 1, followed by bla CTX - M-15. Possession of bla SHV - 11, bla SHV - 27, and bla SHV - 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of ß-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Detection, characterization, and antibiogram of extended-spectrum beta-lactamase Escherichia coli isolated from bovine milk samples in West Bengal, India.

BACKGROUND: Milk is considered as complete food and an important part of human diet throughout the world including India. Bacterial contamination of milk such as Escherichia coli due to unhygienic condition and poor udder health can cause infections, especially in infants and elders or in immunocompromised persons. Possession of antimicrobial resistance genes by commensal bacteria present in milk makes the issue more serious. AIM: The study was aimed to isolate and characterize extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk samples collected from different parts of West Bengal, India, to assess the potential risk associated with the food. MATERIALS AND METHODS: Around 182 milk samples were collected from apparently healthy cows reared by organized dairy farms in West Bengal. E. coli was isolated from collected samples as per standard methods followed by serotyping. The detection of ESBL-producing E. coli was done both phenotypically and genotypically by detecting the presence of bla CTX-M gene. Antibiogram of the ESBL-positive isolates was done using common 12 antibiotics by disc diffusion method. RESULTS: A total of 22 (12.1%) samples were found to be positive for E. coli in this study. Different serotypes such as O11, O20, O22, O34, O35, O128, O149, and UT were isolated from the collected samples. 12 (54.5%) E. coli strains showed the capability of producing ESBL, both phenotypically and genotypically with the presence of bla CTX-M gene. Antibiogram of these ESBL-positive isolates revealed the drugs such as colistin (100%), levofloxacin (83.33%), and imipenem (66.67%) to be highly sensitive against this pathogen but drugs such as cefotaxime (100%), ceftazidime (91.67%), amoxicillin/clavulanic acid (83.33%), tetracycline (75.00%), and gentamicin (58.33%) to be very much resistant. CONCLUSION: More than 50% of the E. coli strains prevalent in the bovine milk samples were positive for ESBL production and are resistant to most of the common antimicrobials which may be alarming for human health.

Characterization of Salmonella Gallinarum isolates from backyard poultry by polymerase chain reaction detection of invasion (invA) and Salmonella plasmid virulence (spvC) genes.

AIM: The aim was to characterize Salmonella enterica serovar Gallinarum isolated from backyard poultry by polymerase chain reaction (PCR) detection of virulence genes invasion (invA) and Salmonella plasmid virulence C (spvC). MATERIALS AND METHODS: Two strains of Salmonella serovar Gallinarum isolates used in this study were obtained from an outbreak of fowl typhoid in backyard Vanaraja fowl. PCR technique was used for detection of invA and spvC genes using standard methodology. The invA PCR product from one representative isolate was sequenced and compared with other related Salmonella serovars in GenBank data. RESULTS: Salmonella Gallinarum produced expected amplicons of invA and spvC gene products. Nucleotide sequence of 285 bp invA gene was deposited in GenBank with accession no. KX788214. Sequence analysis of invA gene was found conserved in Salmonella serovars and demonstrated 100% homology with closely related serovars of Salmonella. CONCLUSION: Invasion gene (invA) was found to be highly conserved in Salmonella Gallinarum and highly similar with closely related serovars. The isolates also contained plasmid-mediated spvC gene indicating possession of virulence plasmid.

Seroprevalence of bluetongue in ruminants of Jharkhand.

AIM: This study was carried out to assess the presence of anti-bluetongue (BT) antibodies in sheep, goat and cattle of different agro-climatic zones of Jharkhand. MATERIALS AND METHODS: Serum samples were collected from apparently healthy as well as suspected sheep, goat and cattle from different districts of Jharkhand covering different agro-climatic zones. Serum samples were screened by indirect enzyme linked immunosorbent assay (iELISA) for detecting anti-BT antibodies. RESULTS: Out of a total of 480 animal serum samples (sheep-190, goats-210 and cattle-80) screened, 83 (43.68%) of sheep, 91 (43.33%) of goat and 46 (57.50%) of cattle sera were found positive. The % positivity ranged between 41% and 51% in different agro-climatic zones. The results showed slight higher seroprevalence, although not significantly, in cattle than sheep and goats in different agro-climatic zones of Jharkhand. CONCLUSIONS: The above data indicate widespread prevalence of BT virus antibodies in studied areas. The incidence of BT is not detected officially, so far. The present seroprevalence status of BT in Jharkhand indicates presence of BT infection in the state for the first time.