Genetic Comparison of ESBL-Producing Escherichia coli from Workers and Pigs at Vietnamese Pig Farms.

We analyzed and compared genomes of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli from pigs and pig farm workers at 116 farms in Vietnam. Analyses revealed the presence of blaCTX-M-55, blaCTX-M-27, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-65, blaCTX-M-24, blaDHA-1, and blaCMY2 in both hosts. Most strains from pigs contained quinolones (qnr) and colistin resistance genes (mcr-1 and mcr-3). Isolates predominantly harbored more than one plasmid replicon and some harbored plasmid replicons on the same contigs as the ESBL genes. Five strains from farm workers of ST38 (2), ST69 (1), and ST1722 (2) were classified as either uropathogenic E. coli (UPECHM)/extraintestinal pathogenic E. coli (ExPECJJ) or UPECHM, and the remaining were genetically distinct commensals. A high heterogeneity was found among the ESBL-producing E. coli from pigs and workers, with most isolates belonging to unrelated phylogroups, serogroups, and sequence types with >4046 Single-Nucleotide Polymorphisms-(SNPs). In comparing the genomes of pig isolates to those from humans, it appeared that ESBL-producing E. coli in workers did not predominantly originate from pigs but were rather host-specific. Nevertheless, the occurrence of ESBL-producing E. coli carrying plasmid-mediated colistin and quinolone resistance genes in pigs could represent a potential source for horizontal transmission to humans through food rather than direct contact.

ESBL and AmpC ß-Lactamase Encoding Genes in E. coli From Pig and Pig Farm Workers in Vietnam and Their Association With Mobile Genetic Elements.

Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing E. coli isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included bla CTX-M-55, bla CTX-M-27, bla CTX-M-65, bla CTX-M-15, bla CTX-M-14, bla CTX-M-3, bla CTX-M-24, and bla CARB-2, and AmpC ß-lactamases included bla CMY-2, bla DHA-1, and bla CMY-42. The most frequent ESBL gene, bla CTX-M-55, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the bla CTX-M-55 gene were different in plasmids carried by different strains. The second most detected gene, bla CTX-M-27, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel bla CTX-M-27 harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL Salmonella Typhimurium plasmid pB71 that might have experienced an insertion of bla CTX-M-27. Our data also point to an emergence of plasmids co-carrying ESBL genes, mcr genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the bla CTX-M-55 encoding plasmids varied considerably, while those encoding bla CTX-M-27 were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC ß-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing E. coli from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.

Research note: Occurrence of mcr-encoded colistin resistance in Escherichia coli from pigs and pig farm workers in Vietnam.

WHO considers colistin as a highest priority critically important drug for human health, and occurrence of colistin-resistant bacteria in livestock is of health concern. The current study determined occurrence of colistin-resistant Escherichia coli in pigs and workers at pig farms in Vietnam, and investigated the genetic background for resistance. Colistin-resistant E. coli were detected from pigs in 53/116 (45.7%) farms, and from workers taking care of the pigs in 21/94 (22.3%) farms. Colistin-resistant isolates showed MIC to colistin between 4-16 mg/L, they were multidrug resistant (99%) and resistance was caused by the presence of mcr-1 genes in 97/102 (95.1%) E. coli from pigs and in 31/34 (91.1%) isolates from humans. mcr-1 is considered a plasmid-encoded gene, but this was not confirmed in the current investigation. In total, one pig isolate carried both mcr-1 and mcr-3 genes, whereas mcr-2, mcr-4 and mcr-5 genes were not detected. Shared resistance profiles between pig and human isolates on the same farm was only observed in four farms. The study showed that commensal E. coli from pigs in Vietnam constitute a reservoir for colistin-resitant E. coli, however, further studies are needed to confirm that mcr genes are associated with plasmids and their importance for human health.

Escherichia coli contamination of fish raised in integrated pig-fish aquaculture systems in Vietnam.

Integrated livestock-fish aquaculture utilizes animal excreta and urine as pond fertilizers to enhance growth of plankton and other microorganisms eaten by the fish. In Vietnam, pigs are commonly integrated with fish and horticulture in household-based VAC systems (vuon = garden; ao = pond; chuong = pigsty), but the level of fecal contamination in the fish produced is unknown. This study was carried out to assess the level Escherichia coli contamination of fish meat and gut contents of grass carp (Ctenopharyngodon idellus), silver carp (Hypophthalmichthys molitrix), and rohu (Labeo rohita) cultured in randomly selected five VAC ponds (with pig manure) and five non-VAC ponds (without pig manure) at sites in periurban Hanoi, Vietnam. Fish muscle tissue samples contained E. coli at <10 or 320 or 820 CFU/g, regardless of the culture system from which they originated. In contrast, the intestinal contents of fish raised in manure-fed ponds contained E. coli at 4.75, 5.25, and 5.07 log CFU/g for silver carp, grass carp, and rohu, respectively, about 100 times higher than the contamination of fish from the control ponds. The results indicate that muscle tissue of fish raised in VAC systems has a low level of fecal contamination despite high levels of E. coli in their gut. Thus, a critical point to control food safety of such fish is the prevention of fecal cross-contamination during degutting and cleaning of the fish at markets and in the home.

Survival of faecal indicator bacteria in treated pig manure stored in clay-covered heaps in Vietnam.

Small-scale pig farmers in Vietnam typically store manure in heaps covered by a layer of clay. However, the hygienic quality of manure applied to agricultural soil is uncertain as storage time is determined by the need for soil fertilizer. This study aimed to assess how the storage of pig manure added straw, lime stone or urea in heaps covered by clay affected the survival of faecal bacteria. E. coli was reduced from 4 log(10)CFU/g to below the detection limit within 2 weeks with 2% (w/w) urea (CO(NH(2))(2)) whereas other amendments were associated with limited E. coli reductions. Enterococcus spp. were not reduced in any heaps indicating possible re-growth and that enterococci should be used as hygiene indicators with caution. Follow-up research should document the practical conditions under which the farmer practice of storing manure with a clay cover can be utilized to obtain a bactericidal effect of ammonia and a safe product with high fertilizer value.

Impact of medicated feed on the development of antimicrobial resistance in bacteria at integrated pig-fish farms in Vietnam.

Integrated livestock-fish aquaculture utilizes animal excreta, urine, and feed leftovers as pond fertilizers to enhance the growth of plankton and other microorganisms eaten by the fish. However, antimicrobial-resistant bacteria may be transferred and develop in the pond due to selective pressure from antimicrobials present in animal feed, urine, and feces. In an experimental pig-fish farm located in periurban Hanoi, Vietnam, nine piglets were provided feed containing 5 µg of tetracycline (TET)/kg pig weight/day and 0.45 µg of enrofloxacin (ENR)/kg pig weight/day during the second and fourth (last) months of the experiment. The aim of this study was to determine the association between the provision of pig feed with antimicrobials and the development of antimicrobial resistance, as measured in a total of 520 Escherichia coli and 634 Enterococcus strains isolated from pig manure and water-sediment pond samples. MIC values for nalidixic acid (NAL) and ENR showed that E. coli and Enterococcus spp. overall exhibited significant higher frequencies of resistance toward NAL and ENR during the 2 months when pigs were administered feed with antimicrobials, with frequencies reaching 60 to 80% in both water-sediment and manure samples. TET resistance for both indicators was high (>80%) throughout the study period, which indicates that TET-resistant E. coli and Enterococcus spp. were present in the piglets before the initiation of the experiment. PCR-based identification showed similar relative occurrences of Enterococcus faecium, Enterococcus faecalis, and other Enterococcus spp. in the water-sediment and manure samples, suggesting that Enterococcus spp. isolated in the ponds originated mainly from the pig manure. The development of antimicrobial resistance in integrated animal husbandry-fish farms and possible transfers and the impact of such resistance on food safety and human health should be further assessed.