Antimicrobial resistance of microorganisms isolated from the swine chain

Abstract

Antimicrobial resistant bacteria represent a global health issue due to their dissemination through different stages of livestock production and acquisition of resistance mechanisms towards critical antimicrobials for human and veterinary medicine. Italy is one of the largest producers of livestock, meat, and meat-based products in the European Union. However, antibiotic resistant bacteria are repeatedly detected in pig farms and slaughterhouse environment. Some bacterial populations disseminated among livestock displaying a multi-drug resistant phenotype represent a cause of concern due to their transmission to humans. Therefore, this work was aimed to improve the knowledge of antimicrobial resistance properties of bacterial isolates from different stages of livestock farming, as well as their distribution among pigs. The first objective was thus to evaluate the molecular characteristics of Methicillin resistant Staphylococcus aureus (MRSA) isolated from 50 finishing units located in Lombardy, a region of northern Italy. A total of 400 samples originating from cutaneous samples at slaughter and environmental samples at farm was collected. Isolates were identified as MRSA by phenotypical (enrichment, growth on selective media, and Gram staining) and biochemical (miniaturized system Api Staph, coagulase, and urease fermentation) tests, and confirmed as such by multiplex-PCR with three set of primers (nuc, mecA, and mecC). In addition, molecular characterization was carried out by means of MLST (targeting seven housekeeping genes) and sub-typing of the staphylococcal protein A. Finally, phylogenetic and neighbour-joining tree analyses were performed on confirmed MRSA isolates. MRSA was detected in 25 out of 400 samples analysed (6.25 %), whereas 17 finishing units were positive for MRSA (34 %). In particular, MRSA was detected in 12 samples from farms and 13 samples at slaughter. MLST analysis assigned most of the MRSA isolates to ST398, followed on less extent by ST97 and ST30. In addition, a new ST, namely ST5422, was detected in one isolate from slaughter. A higher diversity was recorded among spa- types, with t899, t011, t18494, t1200 and t1939 being the most frequent among MRSA isolates; of note, all were related to ST 398. Less frequent spa-types were represented by t4795 and t1730 (ST97), t1730 (ST5422), t318 (ST30), t18494 (ST4894), and at last t034 (ST398). Different spa-types were detected within the same farm in 6 out of 21 farms surveyed, whereas unrelated spa-types clustered by BURP analysis were detected in five farms. The second objective was to provide a more in-depth insight into the antimicrobial resistance properties both at phenotypic and genotypic level, population structure, enterotoxigenic potential and biofilm formation of MRSA isolates obtained from heavy swine farms, located in Lombardy (northern Italy). To this purpose, a total of 440 samples from animals at slaughter and 150 environmental samples originating from 138 farms were analysed. Phenotypic analyses were conducted following the ISO 6888-2 norm, and further subjected to Gram staining, coagulase, and urease tests; finally, molecular confirmation was performed by duplex PCR with two set of primers (nuc and mecA). Confirmed MRSA were tested for susceptibility to a panel of 14 antimicrobials, and the MIC towards CIAs was determined for the resulting multi-drug resistant isolates. Subsequently, five in-house customised PCR protocols were used to determine the presence of genes conferring resistance to nine classes of antimicrobials in MRSA isolates. Further, all MRSA isolates were subjected to a multiplex PCR protocol to detect the presence of genes related with staphylococcal enterotoxins by using five set of primers (sea, seb, sec, sed and see). In addition, molecular characterization was carried out by means of MLST and sub-typing of the staphylococcal protein A, followed by phylogenetic and neighbour-joining tree analyses. In order to have a better picture regarding the phenotypical properties of the MRSA isolates, the biofilm producing ability was determined on polystyrene plates at 37°C for 24h through crystal violet staining. A two-sided chi-square (X2) test was performed to determine the correlation between biofilm forming capacity and the antimicrobial multi-resistance phenotype of MRSA isolates. A total of 87 MRSA isolates were obtained, with an overall incidence of 14.75 % among samples. The majority of MSRA isolates belonged to ST398/t899 and ST398/t011 lineages, while other relevant sequences such as ST1 or ST97 were also identified. Resistance to penicillins, tetracycline and ceftiofur was very common among MRSA isolates, while lower rates of resistance to doxycycline (32.18 %), enrofloxacin (27. 59 %), and gentamicin (25.29 %) were observed. A noteworthy level of antibiotic multi-resistance (77.01 %) was noticed among MSRA isolates. In addition, multi-drug resistant isolates displayed high values of MIC towards tetracycline (MICTET ≥ 6 mg/L), doxycycline (MICDXT ≥ 8 mg/L), trimethoprim/sulfamethoxazole (MICSXT ≥ 4 mg/L), gentamicin (MICCN ≥ 1.5 mg/L), which were all above the available resistance breakpoint values. Outcomes from genetic assays revealed that 97. 70% of the isolates harboured at least one antibiotic resistant gene and 77.01 % of the obtained isolates carried at least one enterotoxin gene, highlighting a high isolate virulence potential. Additionally, 65.52 % of MRSA isolates proved to produce quantifiable amounts of biofilm at 24h. Correlation was not found between multi-drug resistant isolates and biofilm forming capacity of MRSA isolates. In spite of the current programmes for antibiotic reduction in intensively farming, a still on-going high level of AMR and virulence potential in MRSA was demonstrated, making this pathogen a serious risk in swine production chain, highlighting once more the need to develop efficient, pathogen-specific control strategies. The microbiota present in slaughter premises is a major risk for carcass contamination and direct (from food to non-food surfaces) as well as indirect (formation of bioaerosol) transmission of resistant bacteria can occur throughout the slaughter line. Therefore, the last study aimed to assess the level of microbial contamination and resistance of bacterial isolates from a high-throughput heavy pig slaughterhouse towards antimicrobials deemed as critically important for human, veterinary or both chemotherapies. To this purpose, TVC and Enterobacteriaceae counts were performed on samples from carcasses, surfaces, and air, collected from two defined zones: a “wet” room (slaughter) and a “clean” (dressing) room, before and after the initiation of the slaughterhouse operations. Following bacterial enumeration from PCA and VRBGA plates, 60 Gram-negative bacterial isolates were recovered from VRBGA plates and confirmed via Gram staining, oxidase, and biochemical tests. Gram-negative bacteria (GNB) were tested for susceptibility by determining the MIC to a panel of 15 antimicrobials, followed by biofilm production determination on polystyrene plates at 24h through crystal violet staining assay. Statistical tests were performed to compare TVC and GNB values between locations, significance between pre-operative and operative samples; finally, the correlation between the biofilm formation and antimicrobial multi-resistance of GNB was carried out. Overall, the initiation of the slaughter operations increased TVC and GNB mean values in samples from air and surfaces, while no variations in TVC and neither in GNB were observed for samples collected from carcasses. GNB identified in this study belonged to 20 species, 15 genera and 10 families, of which Enterobacteriaceae, Moraxellaceae, and Pseudomonadaceae were the most represented ones. Among GNB, 37 isolates presented resistance to at least one antimicrobial and a multi-drug resistant phenotype was common for 17 isolates. High MIC values towards trimethoprim, ampicillin, tetracycline, and sulfamethoxazole were observed in Escherichia coli isolates, recording MICMAX of 16, 32, 32, and 512 mg/L, respectively. Additionally, Pseudomonas spp. isolates displayed MICMAX values of 32 mg/L to ampicillin and 64 mg/L towards azithromycin. Furthermore, fifteen Gram-negative isolates were classified as strong biofilm producers, yet the ability to produce quantifiable biomass levels was a common feature for n= 55 isolates. Although some isolates were both multi-drug resistant and biofilm producers, no statistical correlation was found between the two phenotypical properties. Results demonstrated that a high diversity of bacteria containing antibiotic resistant and multi-resistant species is present in the sampled abattoir. Considering these findings, it could be hypothesised that the processing environment could be a potential diffusion determinant of antibiotic resistant bacteria through the food chain and operators.