Antimicrobial Susceptibility and Frequency of bla and qnr Genes in Salmonella enterica Isolated from Slaughtered Pigs

Abstract

Salmonella enterica is known as one of the most common foodborne pathogens worldwide. While salmonellosis is usually self-limiting, severe infections may require antimicrobial therapy. However, increasing resistance of Salmonella to antimicrobials, particularly fluoroquinolones and cephalosporins, is of utmost concern. The present study aimed to investigate the antimicrobial susceptibility of S. enterica isolated from pork, the major product in Philippine livestock production. Our results show that both the qnrS and the blaTEM antimicrobial resistance genes were present in 61.2% of the isolates. While qnrA (12.9%) and qnrB (39.3%) were found less frequently, co-carriage of blaTEM and one to three qnr subtypes was observed in 45.5% of the isolates. Co-carriage of blaTEM and blaCTX-M was also observed in 3.9% of the isolates. Antimicrobial susceptibility testing revealed that the majority of isolates were non-susceptible to ampicillin and trimethoprim/sulfamethoxazole, and 13.5% of the isolates were multidrug-resistant (MDR). MDR isolates belonged to either O:3,10, O:4, or an unidentified serogroup. High numbers of S. enterica carrying antimicrobial resistance genes (ARG), specifically the presence of isolates co-carrying resistance to both β-lactams and fluoroquinolones, raise a concern on antimicrobial use in the Philippine hog industry and on possible transmission of ARG to other bacteria.

Virulence and Antimicrobial Resistance Gene Profiling of Salmonella Isolated from Swine Meat Samples in Abattoirs and Wet Markets of Metro Manila, Philippines

“…Following standardized protocols [19], each meat sample bag was aseptically opened, and 25 g of meat sample was sliced with sterilized scissors and forceps and weighed in sterilized aluminum foil. Samples were then transferred in 225 ml of buffered peptone water (BPW) (BD Diagnostics System, USA) contained in Whirl-Pak ® bags (Nasco, USA), followed by agitation for 2 min and incubation at 37℃ for 18−24 h. A 100 μl of resulting BPW cultures was then transferred to 10 ml Rappaport Vassiliadis (RV) broth (BD Diagnostics System) and incubated at 42℃ for 18−24 h. Subsequently, 10 μl of resulting RV cultures was streaked into xylose lysine deoxycholate (XLD) agar (BD Diagnostics System) and incubated at 37℃ for 18−24 h. Colonies that grew with typical phenotypic characteristics (black colonies on red media) on XLD were then subcultured to nutrient agar (NA) (BD Diagnostics System) and incubated at 37℃ for 18−24 h before DNA extraction and other downstream processes.…”

“…DNA extraction was performed through a boil-lysis method as previously described [18,19]. Then, 2−3 colonies of presumptive Salmonella in NA were suspended in 50 μl 1× Tris-EDTA buffer and subjected to boiling temperature at 100℃ for 10 min.…”

“…Each multiplex PCR reaction mixture was 12.5 μl in volume and contained 6.25 μl 5× MyTaq HS Red Mix (Bioline), 2.75 μl of nuclease-free water, 0.25 μl of forward and reverse primers wherein bla TEM primers at 10 μM, bla CTX-M and bla SHV at 30 μM, and 2 μl of DNA template. Following previous studies [19,36], multiplex PCR protocol for bla genes involved 95℃ for 3 min (initial denaturation), then 30 cycles of three steps, namely, 95℃ for 30 s (denaturation), 60℃ for 30 s (annealing), and 72℃ for 1 min (extension), and finally, 72℃ for 10 min (final extension). For positive controls, Klebsiella pneumoniae (ATCC 700603) was used for bla SHV , whereas in in-house laboratory controls from Calayag et al [19], positive samples were used for bla TEM and bla CTX-M .…”

“…Following previous studies [19,36], multiplex PCR protocol for bla genes involved 95℃ for 3 min (initial denaturation), then 30 cycles of three steps, namely, 95℃ for 30 s (denaturation), 60℃ for 30 s (annealing), and 72℃ for 1 min (extension), and finally, 72℃ for 10 min (final extension). For positive controls, Klebsiella pneumoniae (ATCC 700603) was used for bla SHV , whereas in in-house laboratory controls from Calayag et al [19], positive samples were used for bla TEM and bla CTX-M .…”

“…High phenotypic resistances including multidrug resistance (MDR) or non-susceptibility to at least one drug from three or more classes of antibiotics, were observed in Salmonella isolated from slaughtered swine in abattoirs [17−19] and raw chicken from wet markets [20] within Metro Manila, Philippines. In genotypic studies, Calayag et al [19] also reported high prevalence of some β-lactamase (bla) and quinolone resistance (qnr) genes in Salmonella from slaughtered swine. Since the discovery of penicillin, β-lactam antibiotics continue to be developed and used in different settings [13].…”