Prevalence and genomic characterization of Multi-Drug Resistant Nontyphoidal Salmonella enterica and its control through probiotics

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 37oC 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 blaTEM-1 (59.3%), blaOxA-1 (18%), blaPSE-1 (9.5%), blaCMY-2 (43%), and ampC (8.3%). The second objective was genomic characterization of abundant poultry-associated NTS strains by whole-genome sequencing isolated from chickens and associated food products (meat and eggs) in Pakistan. The genomic DNAs of Non typhoidal Salmonella enterica (n=78) were sequenced using an Illumina MiSeq platform. The generated reads were trimmed, and de novo assembled using CLC Genomics Workbench v.7. The draft genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline and were characterized by in silico Multilocus Sequence Typing (MLST). The antimicrobial-resistance genes (acquired and chromosomal mutations), extrachromosomal plasmids, and Salmonella pathogenicity islands were predicted using ResFinder and CARD, Plasmid Finder, and SPI Finder, respectively. The genome size of NTS ranges from 4.9 to 5.1 Mb with 52.1% GC contents. Eight different serovars Salmonella Agona, S. Typhimurium, S. Enteritidis, S. Infantis, S. Reading, S. Rissen, S. kentucky and S. Cero were identified from 78 genomes through Insilico serotyping. The strains harbor several antibiotic-resistance genes including aac (6')-Iaa, aadA1, aadA2, bla OXA-10, qnrS1, cmlA, floR, tet(A), dfrA12) and point mutation in gyrB, gyrA, ParC conferring antibiotic resistance to fluoroquinolones. The second part of the thesis was to use indigenous Probiotic strains as an alternative therapeutic strategy against MDR NTS. Therefore, first we isolated potential probiotic lactic acid bacteria (LAB) strains from the poultry gut to inhibit fluoroquinolone- and cephalosporin-resistant MDR Salmonella Typhimurium and S. Enteritidis. The safety profile of LAB isolates was evaluated for hemolytic activity, DNase activity, and antibiotic resistance. Based on safety results, three possible probiotic LAB candidates for in vitro Salmonella control were chosen. These LAB isolates were identified by 16S rDNA sequencing as Lactobacillus reuteri PFS4, Enterococcus faecium PFS13, and Enterococcus faecium PFS14. These strains demonstrated good tolerance to gastrointestinal-related stresses, including gastric acid, bile, lysozyme, and phenol. In addition, isolates were able to auto aggregate as well had the ability to co-aggregate with MDR S. Typhimurium and S. Enteritidis. Furthermore, LAB strains competitively reduced the adhesion of pathogens to porcine mucin Type III in co-culture studies. The probiotic combination of the selected LAB isolates inhibited biofilm formation of S. Typhimurium FML15 and S. Enteritidis FML18 by 90% and 92%, respectively. In addition, the cell-free supernatant (CFS) of LAB culture significantly reduced the growth of Salmonella in vitro. Thus, L. reuteri PFS4, E. faecium PFS13, and E. faecium PFS 14 are regarded as potential probiotics that could be used to control MDR S. Typhimurium and S. Enteritidis in poultry. We designed an in vivo trial in poultry model to check the efficacy of potential indigenous probiotic strains against MDR NTS serovars. Effects of indigenous probiotics mix containing Lactobacillus reuteri and Enterococcus faecium and a commercial probiotic mix ProtexinR on the growth performance, histomorphology, serum immunoglobulins and intestinal microflora of broiler chickens challenged with MDR fluroquinolones and extended spectrum cephalosporin resistant Salmonella serovars, Typhimurium and Enteritidis were evaluated in a poultry model. A total of 240 birds were fed with a basal diet supplemented with probiotics (108 CFU/mL) in drinking water and orally challenged with (106 CFU/mL of Salmonella serovars Typhimurium and Enteritidis. Probiotics supplementation significantly (P < 0.05) improved growth performance and feed efficiency as well as reducing (P < 0.05) the population of intestinal and liver Salmonella population, and mortality. The results showed that the chicks infected with S. Typhimurium and Enteritidis were depressed, and their BW reduced. Cecal Lactobacillus and Enterococcus counts was highest (P < 0.05) in the indigenous probiotic group (IPRO-). Salmonella challenge (PC+) significantly effect on the relative weight of liver, intestine, and spleen (P < 0.05). Salmonella challenge decreased the villus height (P < 0.05), the ratio of villus height to crypt depth (P < 0.05). We found that probiotic supplementation in the chick model, improves the gut histological structure and enhance the humoral response serum IgA and IgG to the antigen and protect against Salmonella infection. In summary, the indigenous mixed probiotics L. reuteri and E. faecium effectively reduced the mortality of salmonellosis in chicks, promoted the growth performance, regulated the balance of the intestinal flora, improved the immune function, resisted fluroquinolones, and extended spectrum cephalosporin resistant Salmonella serovars, Typhimurium and Enteritidis, and reduced economy loss in the poultry industry.