Antibiotic-resistant bacteria and antimicrobial residues in wastewater and process water from German pig slaughterhouses and their receiving municipal wastewater treatment plants

To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

Antimicrobials are valuable therapeutics whose efficacy is seriously compromised by the emergence and spread of antimicrobial resistance. The provision of antibiotics to food animals encompasses a wide variety of nontherapeutic purposes that include growth promotion. The concern over resistance emergence and spread to people by nontherapeutic use of antimicrobials has led to conflicted practices and opinions. Considerable evidence supported the removal of nontherapeutic antimicrobials (NTAs) in Europe, based on the "precautionary principle." Still, concrete scientific evidence of the favorable versus unfavorable consequences of NTAs is not clear to all stakeholders. Substantial data show elevated antibiotic resistance in bacteria associated with animals fed NTAs and their food products. This resistance spreads to other animals and humans-directly by contact and indirectly via the food chain, water, air, and manured and sludge-fertilized soils. Modern genetic techniques are making advances in deciphering the ecological impact of NTAs, but modeling efforts are thwarted by deficits in key knowledge of microbial and antibiotic loads at each stage of the transmission chain. Still, the substantial and expanding volume of evidence reporting animal-to-human spread of resistant bacteria, including that arising from use of NTAs, supports eliminating NTA use in order to reduce the growing environmental load of resistance genes.

There is extensive genetic substructure within the species Escherichia coli. In 2000 a simple triplex PCR method was described by Clermont and colleagues that enables an E. coli isolate to be assigned to one of the phylo-groups A, B1, B2 or D. The growing body of multi-locus sequence data and genome data for E. coli has refined our understanding of E. coli's phylo-group structure and eight phylo-groups are now recognized: seven (A, B1, B2, C, D, E, F) belong to E. coli sensu stricto, whereas the eighth is the Escherichia cryptic clade I. Here a new PCR-based method is developed that enables an E. coli isolate to be assigned to one of the eight phylo-groups and which allows isolates that are members of the other cryptic clades (II to V) of Escherichia to be identified. The development of the method is described and the method is validated. Over 95% of E. coli isolates can be correctly assigned to a phylo-group. Two collections of human faecal isolates were screened using the new phylo-group assignment method demonstrating that about 13% of E. coli isolates belong to the newly described phylo-groups C, E, F and clade I. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.