The increasing incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia (E.) coli in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic E. coli (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL E. coli were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as E. coli, while 74 (45.1%) were characterized as ESBL E. coli. The frequency of isolation of ESBL E. coli was highest in Aseel chickens (35.1%). Of the 164 confirmed E. coli, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were bla_CTX-M (54.1 %, 40/74), bla_TEM, (12.2%, 9/74) and co-existence (bla_CTX-M and bla_TEM) were shown in 33.8% (25/74). The bla_CTX-M gene sequence showed homology to bla_CTX-M-15 from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL E. coli (0.25) when compared to non-ESBL E. coli (0.17). Both free-range husbandry management system (p = 0.02, OR: 30.00, 95% CI = 1.47–611.79) and high antimicrobial usage in the last 6 months (p = 0.01, OR: 25.17, 95% CI = 1.81–348.71) were found significantly associated with isolation of ESBL-producing E. coli in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL E. coli in the Jhang district, Punjab, Pakistan.

Extended-spectrum β-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the “Trimmu Barrage” in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby–Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was bla_CTX-M (51.55%), bla_TEM (20.62%), bla_OXA (6.18%) and bla_SHV (2.06%). In addition, gene combinations bla_CTX-M + bla_TEM, bla_TEM + bla_OXA and bla_CTX-M + bla_SHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

Colibacillosis associated with colistin-resistant avian pathogenic Escherichia coli (E. coli) poses a threat to both food security and public health. The potential horizontal transmission of mobilized colistin-resistant (mcr) genes facilitates the co-emergence of Klebsiella pneumoniae. This study aimed to determine the prevalence, molecular detection, analyze the antibiogram and identify associated risk factors for colistin-resistant E. coli and Klebsiella pneumoniae isolated from broiler chicken in three districts of Punjab province, Pakistan. In total, 230 visceral organ samples were collected from 13 different chicken farms located in Sargodha, Jhang and Toba Tek Singh in Pakistan. Following isolation, the broth microdilution test was used to confirm phenotypic colistin resistance. Polymerase chain reaction was used to detect mcr-1 and mcr-2 genes associated with colistin resistance. Antimicrobial susceptibility test against 11 antibiotics was performed using the Kirby-Bauer disk diffusion method. Risk factors associated with colistin-resistant bacteria, including host attributes, farm management practices, environmental and agent characteristics, were analyzed. The prevalence of colistin-resistant E. coli and K. pneumoniae was 24.78% (95% CI, 19.6–30.7%) and 3.04% (95% CI, 1.5–6.1%), respectively. The prevalence of colistin-resistant E. coli varied between cities at 42, 23.61 and 5.55% for Jhang, Sargodha and Toba Tek Singh, respectively. The detection frequency of mcr-1 gene, 42.1% (24/57), was significantly (p < 0.01) higher than that of the mcr-2 gene, 14.03% (8/57). Phylogenetic analysis of lipid A phosphoethanolamine transferase sequences revealed greater similarity with mcr-1.5 variant. Isolates were found resistant to amoxicillin-clavulanic acid (84.21%), cefotaxime (70.17%), and trimethoprim-sulfamethoxazole (73.68%). The multivariate logistic regression predicted preceding viral infection of the respiratory tract as a significant association (OR = 4.808, p < 0.01), whereas daily removal/culling of dead/diseased chicken (OR = 0.308, p = 0.01) was a protective factor against the emergence of colistin-resistant strains. These findings indicate that the emergence of colistin-resistant strains deteriorate colibacillosis control efforts in poultry and serves as a possible reservoir for zoonotic infections.

Members of Enterobacteriaceae are known to produce extended-spectrum beta-lactamases (ESBL) which hydrolyze the beta-lactam group of antibiotics. The existence of ESBL-producing Salmonella enterica (S. enterica) and Escherichia coli (E. coli) harbored by urban avifauna was investigated in this study. Dropping samples (n= 180) were collected from six different bird spe-cies in the district Jhang, Punjab province, Pakistan. Isolation and identification of ESBL isolates were made by using cefotaxime-(4 mg/L) supplemented MacConkey agar and double disc synergy test (DDST). Polymerase chain reaction (PCR) was performed for the detection of four different ESBL genes including bla(CTX-M), bla(TEM), bla(SHV) and bla(OXA). A total of 42.69% isolates were confirmed as ESBL via DDST including 30.64% S. enterica and 49.54% E. coli. The inci-dence of ESBL S. enterica and ESBL E. coli was found highest in egret (Ardea alba) and pigeon (Columba livia) as 64.28% and 78.95%, respectively. The blaCTX-M gene was detected in 57.89% and 64.81% of isolates of S. enterica and E. coli, respectively. Among other genes in S. enterica and E. coli, blaTEM (21.05%, 20.4%); blaSHV (15.78%, 9.26%), and blaOXA (5.26%, 5.56%) were detected, respectively. All of the tested isolates were found resistant to at least one of the thirteen antimicrobial agents except meropenem. To the best of our knowledge, this is the first study reporting the incidence and genetic diversity of ESBL bacteria associated with urban avifauna in Pakistan. The urban avifauna can serve as a potential subject of bio-surveillance to monitor the emergence of antimicrobial-resistant bacteria.

Objective: This study aimed to investigate the prevalence of paratuberculosis in cattle and buffaloes at twelve public dairy farms in Punjab, Pakistan.Methods: A total of 2,181 more than two-year-old animals (1,242 cattle and 939 buffaloes) were tested by avian tuberculin, i.e., killed purified protein derivative of Mycobacterium avium paratuberculosis and indirect enzyme linked immunosorbent assay (ELISA). Blood and fecal samples were collected from tuberculin positive animals. These samples were further processed by indirect ELISA. The data were analyzed using frequency analysis and logistic analysis procedures.Results: The prevalence of paratuberculosis at public dairy farms was 3.8%, as determined by tuberculin+ELISA test. It varied from 0.71% to 13.5% with a 100% herd prevalence. Multivariate logistic regression analysis revealed that species, milk production, total animals, total small ruminants, and total buffaloes were significantly associated with the occurrence of paratuberculosis. Odd ratio analysis revealed that with a one-kilogram increase in body weight, there will be a 0.006% increase in disease occurrence. With the increase in one animal in small ruminants and buffaloes, there will be 0.008% and 0.42% greater chances of developing paratuberculosis, respectively. Bivariate logistic regression analysis of cattle and buffaloes revealed that farm number, age, and total number of cattle were significantly associated with the occurrence of paratuberculosis. A one-month increase in lactation length increases the chance of tuberculosis by 0.004%; similarly, a one-liter increase in milk production increases the chance of disease by 10%. With each additional buffalo in the herd, there will be a 0.007% greater chance for the occurrence of paratuberculosis.Conclusion: This study concluded that tuberculin testing can be used in conjunction with ELISA to screen animals for paratuberculosis in countries with scarce resources, such as Pakistan. The efficacy of disease diagnosis can be improved by combining multiple tests.