Alexandra Irrgang

Prevalence of mcr-1 in E. coli from Livestock and Food in Germany, 2010–2015

Since the first description of a plasmid-mediated colistin resistance gene (mcr-1) in November 2015 multiple reports of mcr-1 positive isolates indicate a worldwide spread of this newly discovered resistance gene in Enterobacteriaceae. Although the occurrence of mcr-1 positive isolates of livestock, food, environment and human origin is well documented only few systematic studies on the prevalence of mcr-1 are available yet. Here, comprehensive data on the prevalence of mcr-1 in German livestock and food isolates are presented. Over 10.600 E. coli isolates from the national monitoring on zoonotic agents from the years 2010–2015 were screened for phenotypic colistin resistance (MIC value >2 mg/l). Of those, 505 resistant isolates were screened with a newly developed TaqMan-based real-time PCR for the presence of the mcr-1 gene. In total 402 isolates (79.8% of colistin resistant isolates) harboured the mcr-1 gene. The prevalence was depending on the food production chain. The highest prevalence was detected in the turkey food chain (10.7%), followed by broilers (5.6%). A low prevalence was determined in pigs, veal calves and laying hens. The mcr-1 was not detected in beef cattle, beef and dairy products in all years investigated. In conclusion, TaqMan based real-time PCR provides a fast and accurate tool for detection of mcr-1 gene. The overall detection rate of 3.8% for mcr-1 among all E. coli isolates tested is due to high prevalence of mcr-1 in poultry production chains. More epidemiological studies of other European countries are urgently needed to assess German prevalence data.

Chromosomal Locations of mcr-1 and blaCTX-M-15 in Fluoroquinolone-Resistant Escherichia coli ST410

To the Editor: Recently, Yi-Yun Liu et al. reported on the discovery of mcr-1, a plasmidborne resistance gene mediating resistance to colistin, in isolates obtained from humans and animals (1). Since the original publication, mcr-1 with or without the insertion element ISApl1 has been detected on plasmids of different incompatibility groups, including IncI2, IncHI2, and IncX4, and in many different countries (1–3). Because colistin is a last-resort parenteral antimicrobial drug, the transfer of mcr-1 by conjugation or through mobilizable plasmids raises concern about the emergence of pan-resistant Enterobacteriaceae.

Spread and persistence of VIM-1 Carbapenemase-producing Enterobacteriaceae in three German swine farms in 2011 and 2012

The occurrence of carbapenemase-producing Enterobacteriaceae in livestock is considered as a threat for public health. In Germany, VIM-1-producing Escherichia (E.) coli sequence type (ST) 88 and Salmonella Infantis isolates harbouring blaVIM-1IncHI2 plasmids have been isolated from swine and poultry farms. A retrospective study was performed to determine if there was a broader distribution of VIM-1-positive isolates in any of the carbapenemase-positive swine farms. Selective incubation (carbapenem-containing broth) of 249 conserved cultures collected in three farms (2011-2012), allowed the detection of 40 blaVIM-1-positive isolates. Apart from the already known non-motile Salmonella Infantis isolate R25 (farm S1) and R27 (S2), a third isolate was recovered from farm S3. For E. coli, additional to isolates R29 and R178 (S2), 35 new isolates were identified in the same farm during all the sampling periods (three dates, 2011) and in samples from different animals, farm environment, manure and flies. The newly identified E. coli and Salmonella isolates showed similar genetic and phenotypic characteristics (XbaI-PFGE profiles, antimicrobial resistance patterns, plasmid content, phylogroups, antigenic formula) to those in the previously described strains, suggesting microevolution within the clonal lines within one fattening period. The study shows that persistence of carbapenemase-producing clonal lines in livestock farms is possible, and underlines the need for harmonised monitoring and surveillance studies to follow up the occurrence of such bacteria in European livestock.

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii.

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

CTX-M-15-Producing E. coli Isolates from Food Products in Germany Are Mainly Associated with an IncF-Type Plasmid and Belong to Two Predominant Clonal E. coli Lineages

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA−1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation.

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

this last case, the description here of blaNDM-1-carrying A. pittii in France is made in a patient with no recent history of travel. The diagnosis was made fortuitously from a rectal swab sample. The context of this diagnosis suggests that the circulation of ACB species carrying blaNDM may be underestimated in France. This case raises questions about the management of patients with carbapenemase-producing A. pittii carriage in hospitals. Here, all the patients hospitalized in the same ward and screened for carbapenemase carriage were negative. Dissemination of NDM1-producing A. pittii has been noted in an ICU. NDM-producing A. pittii had then been isolated in the air within the ICU, being suspected to contribute to the dissemination of the bacterium. Therefore, early detection of carbapenemases in Acinetobacter species seems critical to control the dissemination of carbapenemase-producing isolates. Here, this first French case of NDM-producing A. pittii in a patient with no history of travel enhances the problem of carbapenemaseproducing ACB species and their management in hospitals.

Evaluation of a LAMP-based assay for the rapid detection of plasmid-encoded colistin resistance gene mcr-1 in Enterobacteriaceae isolates

Colistin, which belongs to the class of polypeptide antibiotics known as polymyxins, is a “last-resort” antibiotic often used for the treatment of patients infected with extensively multidrug-resistant (MDR) bacteria such as carbapenem-resistant members of the family Enterobacteriaceae. The

Complete Genome Sequence of a blaCTX-M-1-Harboring Escherichia coli Isolate Recovered from Cattle in Germany

ABSTRACT We describe here the whole-genome sequence and basic characteristics of Escherichia coli isolate 15-AB01393, recovered from German beef within a national monitoring program in 2015. This isolate was identified as an extended-spectrum-β-lactamase-producing E. coli strain of multilocus sequence type (MLST) ST58 harboring the antimicrobial resistance genes blaCTX-M-1, mph(A), sul2, dfrA5, strA, and strB.

Diversity of CTX-M-1-producing E. coli from German food samples and genetic diversity of the blaCTX-M-1 region on IncI1 ST3 plasmids

Antimicrobial resistance to cephalosporins is commonly mediated by extended-spectrum β-lactamases (ESBL) or plasmidic AmpC β-lactamases (pAmpC). In livestock blaCTX-M-1 is the most frequently detected ESBL-encoding gene. As transmission to consumers through contaminated food is often proposed, this study characterized ESBL/pAmpC-producing E. coli collected from food samples. Therefore, samples from food products of animal origin and vegetables were screened for phenotypically resistant E. coli by selective cultivation. The ESBL genotype was confirmed for 404 isolates with the majority of them (n = 212) harboring the blaCTX-M-1 gene. PFGE and MLST analyses as well as plasmid characterization were carried out for 89 isolates, selected under epidemiological aspects. In addition, 44 isolates were investigated by whole genome sequencing and/or sequencing of their plasmids on an Illumina Miseq platform. MLST and PFGE indicated a diverse population of CTX-M-1-producing E. coli in German food samples with no spread of single clonal lineages. The majority of the isolates harbored the blaCTX-M-1 gene on IncI1 plasmids. Frequently, the gene was associated with the ISEcp1 element and located on a ∼100 kb IncI1 plasmid depicting the plasmid multilocus sequence type (ST) 3. The blaCTX-M-1 gene and its flanking sequences were located within the shufflon of the type IV pilus region in diverse orientations. In conclusion, dissemination of the CTX-M-1 β-lactamase within food samples of animal origin is driven by the transmission of a ∼100 kb large IncI1 ST3 plasmid. Apart from conjugal transfer of IncI1 ST3 plasmids the transmission of the blaCTX-M-1 gene might be further promoted through mobilization due to its location within a recombination hot-spot of IncI1 plasmids.