Antimicrobial Resistance Profiling of Biofilm Forming Non Typhoidal Salmonella enterica Isolates from Poultry and Its Associated Food Products from Pakistan

Abstract

Salmonellosis caused by non-typhoidal Salmonella enterica from poultry products is a major public health concern worldwide. This study aimed at estimating the pathogenicity and antimicrobial resistance in S. enterica isolates obtained from poultry birds and their food products from different areas of Pakistan. In total, 95/370 (25.67%) samples from poultry droppings, organs, eggs, and meat were positive for Salmonella. The isolates were further identified through multiplex PCR (mPCR) as Salmonella Typhimurium 14 (14.7%), Salmonella Enteritidis 12 (12.6%), and other Salmonella spp. 69 (72.6%). The phenotypic virulence properties of 95 Salmonella isolates exhibited swimming and/or swarming motility 95 (100%), DNA degrading activity 93 (97.8%), hemolytic activity 92 (96.8%), lipase activity 87 (91.6%), and protease activity 86 (90.5%). The sopE virulence gene known for conferring zoonotic potential was detected in S. Typhimurium (92.8%), S. Enteritidis (100%), and other Salmonella spp. (69.5%). The isolates were further tested against 23 antibiotics (from 10 different antimicrobial groups) and were found resistant against fifteen to twenty-one antibiotics. All isolates showed multiple drug resistance and were found to exhibit a high multiple antibiotic-resistant (MAR) index of 0.62 to 0.91. The strong biofilm formation at 37 °C reflected their potential adherence to intestinal surfaces. There was a significant correlation between antimicrobial resistance and the biofilm formation potential of isolates. The resistance determinant genes found among the isolated strains were bla_TEM-1 (59.3%), bla_OxA-1 (18%), bla_PSE-1 (9.5%), bla_CMY-2 (43%), and ampC (8.3%). The detection of zoonotic potential MDR Salmonella in poultry and its associated food products carrying cephalosporin and quinolone resistance genes presents a major threat to the poultry industry and public health.

Keywords: MAR index; NTS; Pakistan; Salmonella enterica; antibiotic resistance; eggs; poultry.

Figure 1

Salmonella strains (95) showing phenotypic antibiotic resistance profiles of 12 antibiotics, their source, origin, serotypes, and MAR index. Black squares indicate resistance; white squares indicate susceptibility; red squares indicate the presence of AMR genes; blue color presents isolates from poultry droppings, green; poultry food products (meat and eggs), yellow; poultry organs. Abbreviations: C: chloramphenicol (30 μg); AMC: amoxicillin-clavulanic acid (10 μg): CIP; ciprofloxacin (10 μg); CN: gentamicin (10 μg); SXT: sulfamethoxazole/trimethoprim (25 μg); K: kanamycin(30 μg): AMP: ampicillin (30 μg); MEM: meropenem (10 μg); IPM: imipenem (10 μg); CEF: cefepime (30 μg); CFM: cefixime (5 μg); ENR: enrofloxacin (10 ug); MAR: multiple antibiotic resistance; a*: No. of antibiotics resistant; b*: No. of antibiotics tested.

Figure 2

Results of biofilm formation assay. X-axis: Salmonella isolates from poultry droppings; poultry food products; poultry organs at two temperatures, (A): 37 °C; (B): 30 °C evaluated by crystal violet assay. Biofilm formation was assessed by staining the attached bacteria with 0.2% CV and measuring the OD values at 620 nm after 48 h growth. Error bars represent standard deviations between three replicates.