Recurrent detection of VIM-1-producing Escherichia coli clone in German pig production

Sir,

Carbapenems are declared as ‘critically important’ antibiotics by the WHO.¹ Recently, carbapenemase-producing Enterobacteriaceae (CPE) arose as a major concern in human medicine, where they are increasingly isolated from patients in hospitals. However, they are only sporadically reported from non-human sources.^2,3 In Europe, carbapenems are not licensed for veterinary use and no maximum residue levels are defined. Therefore, the isolation of VIM-1-producing Escherichia coli and Salmonella spp. in 2011–12 from German swine and poultry farms raised concerns that livestock might emerge as a reservoir for CPE and that pan-resistant isolates could be transmitted from this reservoir to humans.⁴ In fact, spread and persistence of the β-lactamase gene (bla) VIM-1 over a whole fattening period was demonstrated in the VIM-1-positive swine farm. Here we describe the presence of VIM-1-positive E. coli isolates that appear closely related to the isolates from 2011, isolated in December 2015 from a different German swine farm.^4,5

In Germany, monitoring of antimicrobial resistance (AMR) in commensal E. coli is linked to the national monitoring of zoonotic agents.⁶ According to the Commission Implementing Decision 2013/652/EU, susceptibility is determined using the microdilution method following CLSI guidelines (CLSI M07-A9). Isolates with an ESBL/AmpC phenotype or phenotypic resistance to carbapenems are further characterized by PCR and subsequent sequencing of the PCR products.^5,7

So far, no carbapenemase-producing E. coli isolates had been recorded within the scope of the national monitoring programmes. This study describes the first detection of a commensal E. coli isolate showing resistance to meropenem (MIC ≥0.5 mg/L), ertapenem (MIC ≥0.12 mg/L) and imipenem (MIC ≥2 mg/L) (isolate R1176, isolated in December 2015 from the colon content of a slaughter pig) within the monitoring programme. PCR and subsequent sequencing analysis revealed the presence of a bla_VIM-1 gene. XbaI PFGE revealed a highly similar restriction pattern of R1176 to E. coli isolates described by Fischer et al.^4,5 from samples collected in 2011 from a swine farm (Figure 1; R29 and R178). This indicates a clonal relationship of these VIM-1-positive E. coli, although the affected livestock farms are regionally clearly separated. In contrast to E. coli isolates R29 and R178, in R1176 neither the bla_ACC-1 gene nor the typical 220 kb VIM-1 plasmid of the former isolates was detected through PCR and S1 nuclease PFGE (Figure S1, available as Supplementary data at JAC Online). Supported by the failure of bla_VIM-1 hybridization experiments on S1 nuclease PFGE (Figure S2) and transformation experiments, a chromosomal location of the bla_VIM-1 gene in R1176 is assumed. This might be driven by an association of the bla_VIM-1 gene with mobile genetic elements, as described for R178 as well.

Figure 1.

XbaI PFGE analysis. PFGE plugs of R1176 I and II were cast independently to verify reproducibility. R1176 was isolated in December 2015, R1177–R1180 were isolated in March 2016, and R178 and R29 are representatives of E. coli isolated in 2011–12. S. Br., Salmonella Braenderup (H9812) as size marker.

To verify a potential clonal persistence of VIM-1-positive E. coli within the farm that the pig originated from, in March 2016 colon content from five healthy animals in another slaughter batch from this farm was examined. This resulted in the isolation of four additional carbapenem-resistant E. coli (isolates R1177–R1180) from one of the samples. Again, XbaI PFGE patterns of these four isolates were very similar to those mentioned above (Figure 1). This hints at the presence of a specific clone on this farm and a link with isolates obtained from the farm investigated in 2011.⁴ S1 nuclease PFGE with subsequent hybridization experiments, described by Rodriguez et al.⁷ in 2009, revealed bla_VIM-1 localization on 180–200 kb IncHI2 plasmids in all four isolates (Figure S1). And, indeed, the VIM-1-plasmid-harbouring isolates were also positive for the bla_ACC-1 gene, further resistance genes strA and strB and class-I-integron-associated resistance genes aadA1 and aacA4 and sul1, as shown for pRH-178, assuming the presence of a highly similar plasmid in these isolates compared with the ones from 2011.^4,5

All five isolates described in this study belonged to ST88 and phylogenetic group A and harboured the bla_VIM-1 gene on a class 1 integron with gene cassettes that were identical to those described for R29 and R178, independently of its localization on the plasmid or the chromosome.⁵

In the above-mentioned national AMR monitoring programmes no carbapenemase-producing E. coli had been detected until the end of 2015, indicating a very low prevalence of such bacteria in the German livestock population. However, detection of highly related VIM-1-producing E. coli isolates from an additional swine farm in Germany in this study indicates persistence of a VIM-1-producing E. coli clone in the swine population for at least 4 years. Further investigations on the persistence of this clone are currently under way. Detailed genomic analysis will be carried out to reveal a potential reason for stable maintenance of this clone and to uncover potential transmission pathways of these isolates. The understanding of transmission pathways and the persistence of CPEs among different populations to limit the spread of CPEs in livestock is of major relevance for public health. Finally, results of this study underline the importance of the carbapenemase monitoring recommended by the European Food Safety Authority (EFSA) and the European Commission.