Linda Falgenhauer

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Multiresistant extended-spectrum β-lactamase- producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BackgroundMultiresistant Gram-negative bacteria producing extended-spectrum β-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of β-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses.ResultsIn this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(‘6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates.ConclusionsIsolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

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.

Circulation of clonal populations of fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany

Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum β-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.

Molecular characterisation of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from hospital and ambulatory patients in Germany

The increase of Escherichia coli producing extended-spectrum β-lactamases (ESBL) in hospitals and their emergence as intestinal colonisers of healthy humans is of concern. Transmission ways and the extent of spread of distinct E. coli clones or ESBL genes among humans and animals via the food chain or the environment is a matter of debate. In this study we determined ESBL genotypes in E. coli isolates (n=233) resistant to 3rd generation cephalosporins from hospitals and medical practices using PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, multilocus sequence typing (MLST) and a ST131-specific PCR. Results showed that CTX-M-15 (50.4%), CTX-M-1 (28.4%) and CTX-M-14 (5.6%) were the most common ESBL types. Especially, CTX-M-15 was associated with E. coli ST131 of phylogenetic group B2, which was the dominant sequence type among our isolates (35.8%). MLST typing revealed 40 different sequence types (STs), with ST131, ST410, ST10 and ST38 as the most prevalent ones. Our findings give an overview of the current distribution of ESBL-producing E. coli isolates from humans in Germany. E. coli O25b:H4-ST131 was confirmed to be the most common clone, which is known for its successful dissemination worldwide. Although heterogeneity among the isolates was found, several successful clones previously described in animals (ST410, ST10) also occurred in our isolate collection. Further detailed investigations of ESBL-producing isolates from different habitats are needed to evaluate possible transfer ways.

Multiple ESBL-Producing Escherichia coli Sequence Types Carrying Quinolone and Aminoglycoside Resistance Genes Circulating in Companion and Domestic Farm Animals in Mwanza, Tanzania, Harbor Commonly Occurring Plasmids

The increased presence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in humans, animals, and their surrounding environments is of global concern. Currently there is limited information on ESBL presence in rural farming communities worldwide. We performed a cross-sectional study in Mwanza, Tanzania, involving 600 companion and domestic farm animals between August/September 2014. Rectal swab/cloaca specimens were processed to identify ESBL-producing Enterobacteriaceae. We detected 130 (21.7%) animals carrying ESBL-producing bacteria, the highest carriage being among dogs and pigs [39.2% (51/130) and 33.1% (43/130), respectively]. The majority of isolates were Escherichia coli [93.3% (125/134)] and exotic breed type [OR (95%CI) = 2.372 (1.460–3.854), p-value < 0.001] was found to be a predictor of ESBL carriage among animals. Whole-genome sequences of 25 ESBL-producing E. coli were analyzed for phylogenetic relationships using multi-locus sequence typing (MLST) and core genome comparisons. Fourteen different sequence types were detected of which ST617 (7/25), ST2852 (3/25), ST1303 (3/25) were the most abundant. All isolates harbored the blaCTX-M-15 allele, 22/25 carried strA and strB, 12/25 aac(6′)-lb-cr, and 11/25 qnrS1. Antibiotic resistance was associated with IncF, IncY, as well as non-typable plasmids. Eleven isolates carried pPGRT46-related plasmids, previously reported from isolates in Nigeria. Five isolates had plasmids exhibiting 85–99% homology to pCA28, previously detected in isolates from the US. Our findings indicate a pan-species distribution of ESBL-producing E. coli clonal groups in farming communities and provide evidence for plasmids harboring antibiotic resistances of regional and international impact.

Comparative genome analysis of IncHI2 VIM-1 carbapenemase-encoding plasmids of Escherichia coli and Salmonella enterica isolated from a livestock farm in Germany

Carbapenem-resistant Enterobacteriaceae are not any more isolated only from human settings, but also from livestock. We reported for the first time the presence of VIM-1 carbapenemases in a livestock farm in Germany. The VIM-1 resistance gene found in these farms was located on IncHI2 plasmids. In order to be able to analyse these plasmids in more detail, two different plasmids from a single farm (pRH-R27 from Salmonella enterica and pRH-R178 from Escherichia coli) were completely sequenced and analysed for the presence of antibiotic and heavy metal resistances. The plasmids showed to harbour blaVIM-1, aacA4, aadA1, sul1, qacEΔ (encoded in an In110 class 1 integron), as well as blaACC-1, strA/strB, and catA1 genes together with resistance to heavy metals (ter-, mer-, sil-, ars-, rcn-, and pco). Comparison with other IncHI2 plasmid revealed that while pRH-R27 is a mosaic IncHI2 plasmid with both high homology to the plasmid pSTm-A54650 and R478, both isolated from humans, pRH-R178 is a deletion derivative of pRH-R27, presumably caused by several IS-mediated deletions indicating genetic evolution of plasmids in this environment.

Complete Genome Sequence of Phage-Like Plasmid pECOH89, Encoding CTX-M-15

ABSTRACT A nonconjugative and nontypable plasmid of a clinical Escherichia coli isolate expressing resistance to extended-spectrum cephalosporins (ESCs) was isolated and sequenced. The plasmid pECOH89 contains a CTX-M-15 resistance cassette and comprises 111,741 bp, with strong homology to bacteriophage-like plasmids and to the Salmonella-specific bacteriophage SSU5.

Zum Vorkommen von Extended-Spektrum- und AmpC-Beta-Laktamase-produzierenden Escherichia coli in Nutztierbeständen: Ergebnisse ausgewählter europäischer Studien

Extended-Spektrum-Beta-Laktamase (ESBL) und plasmidkodierte Cephamycinase (pAmpCs) produzierende Escherichia ( E.) coli in Nutztierbestanden sind in jungster Zeit Gegenstand einer erhohten wissenschaftlichen und gesellschaftlichen Aufmerksamkeit. In diesem Artikel werden ausgewahlte europaische Studien zum Vorkommen und den Risikofaktoren fur das Auftreten dieser Resistenzen zusammengefasst. Diese Studien sind durch ihre unterschiedliche Methodik nicht unmittelbar vergleichbar, dennoch kann insgesamt eine sehr hohe Pravalenz feststellt werden. Fur Broiler liegt die Betriebspravalenz bei uber 40 % und die Einzeltierpravalenz bei ca. 30 %. Fur schweinehaltende Betriebe schwanken die Ergebnisse zur Pravalenz sehr stark. So wurden Betriebspravalenzen von 1 bis 80 % und Einzeltierpravalenzen von 15 bis 100 % berichtet. Bei der Untersuchung rinderhaltender Betriebe spielen die Art des Betriebes sowie die jeweilige Lebensphase der Tiere eine wesentliche Rolle. So wurden die hochsten Pravalenzen bei Kalbern beobachtet, wahrend diese bei alteren Mastrindern deutlich geringer waren. Bei Milchkuhen wurden nach der Kalbung mehr positive Proben gefunden als davor. Die ermittelten Risikofaktoren fur das Auftreten ESBL/pAmpC-produzierender E. coli unterscheiden sich je nach Tierart. In verschiedenen Studien konnte ein Zusammenhang zwischen dem Auftreten ESBL-produzierender E. coli und Faktoren wie dem Einsatz von Antibiotika sowie Managementfaktoren, z. B. Mastdauer und Zukauf von Tieren unterschiedlicher Herkunfte, festgestellt werden. Zum jetzigen Zeitpunkt fehlen landerubergreifende systematische und standardisierte epidemiologische Untersuchungen zum Vorkommen von ESBL/ pAmpC-produzierenden E. coli in Nutztierbestanden. Um die weitere Verbreitung und die Effektivitat von Praventionsmasnahmen kontrollieren zu konnen, sind flachendeckende, speziesubergreifende Monitoring- und Surveillance-Systeme mit harmonisierter Methodik essenziell. Moderne, insbesondere sequenzbasierte Typisierungsverfahren konnen dabei weitere Informationen zur Aufklarung von Ubertragungswegen liefern.

Environmental emission of multiresistant Escherichia coli carrying the colistin resistance gene mcr-1 from German swine farms.

Objectives Pigs have been the focus of the worldwide spread of colistin resistance. However, there is little information on the transmission of mcr-1 -containing bacteria into the environment of pig farms. We therefore rescreened environmental Escherichia coli isolates from the surrounding farm areas of three previously mcr-1 -positive swine herds in Germany. Methods Thirty-five mixed bacterial cultures obtained from boot swabs, flies, dog faeces and manure from three pig farms in Germany in 2011-12 were non-selectively recultivated and the presence of the mcr-1 gene was checked by real-time PCR. After separation, single E. coli colonies were subsequently isolated and the presence of mcr-1 was confirmed by PCR and sequencing. In addition, phenotypic antimicrobial resistance screening and WGS followed by phylogenetic analysis and resistance genotyping as well as plasmid typing were performed. Results Seven mcr-1 -positive E. coli strains originating from environmental boot swabs, dog faeces, stable flies and manure were found. The isolates belonged to five different STs (ST10, ST1011, ST1140, ST5281 and ST342) and harboured extensive additional resistance genes. Comparative plasmid analysis predominantly located mcr-1 on IncX4 plasmids, which are strongly related to a recently described plasmid of human clinical origin (pICBEC72Hmcr). Conclusions WGS-based analysis of the environmental E. coli isolates of farm surroundings showed clear links to mcr-1 -harbouring E. coli recovered from pig production in Europe as well as from human clinical isolates worldwide, presenting another piece of the puzzle, which further complicates the rapidly evolving epidemiology of plasmid-mediated colistin-resistant E. coli strains.