Whole-genome analysis of vancomycin-resistant Enterococcus faecium causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavaria, Germany.

OBJECTIVES: Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) represent a major public health concern due to limited treatment options. Among invasive isolates of VREfm, ST117, ST80 and ST78 represent the most frequently detected STs by MLST in Germany. In this study, we investigated the genetic diversity of isolates of VREfm recovered from different nosocomial outbreaks in Bavaria, Germany, by WGS. METHODS: Between January 2018 and April 2019, 99 non-replicate isolates of VREfm originating from nosocomial outbreaks at eight different hospitals in Bavaria were investigated for genetic diversity by WGS. In detail, complex types (CTs) were identified by core-genome MLST. Furthermore, an SNP analysis was performed for all VREfm strains. RESULTS: Most of the isolates of this study (76%) belonged to three major clonal groups, which occurred in at least three hospitals: ST80/CT1065 vanB (n = 45; six hospitals), ST117/CT71 vanB (n = 11; four hospitals) and ST78/CT894like vanA (n = 19; three hospitals). Moreover, isolates of the predominant lineage ST80/CT1065 vanB showed a maximum difference of 36 SNPs as revealed by SNP analysis. CONCLUSIONS: Whole-genome analysis of VREfm causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavarian hospital settings, namely ST80/CT1065 vanB, ST117/CT71 vanB and ST78/CT894like vanA. Further studies are needed for a better understanding of the factors affecting the successful spread of the above-mentioned lineages.

First report of the new emerging global clone ST1193 among clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli from Germany.

OBJECTIVES: Sequence type 1193 (ST1193) is a new emerging global clone of Escherichia coli. The main goal of this study was to determine the prevalence and molecular characteristics of ST1193 among clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing E. coli from University Hospital of Erlangen, Germany. METHODS: Between November 2015 and February 2016, all consecutive non-duplicate clinical E. coli isolates showing resistance to cefotaxime or ceftazidime were further analysed for ESBL production by the combined disk method. ESBL genes were identified by PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, MLST and whole-genome sequencing. RESULTS: ESBL production was confirmed in 51 isolates. The globally dominant ST131 occurred at a frequency of 37.3% (n=19). Major non-ST131 sequence types were ST38 (n=4; 7.8%), ST10 (n=3; 5.9%) and ST1193 (n=3; 5.9%). Among the ESBL-producing E. coli ST1193, two expressed CTX-M-14 and one expressed CTX-M-15 ESBL type. All three ST1193 isolates belonged to serogroup O75:H5, phylogroup B2, and harboured IncFIA and IncFIB plasmids and the virulence factors genes iha, sat, gad, vat and senB. Moreover, they showed ciprofloxacin resistance and exhibited a set of four conserved mutations defining quinolone resistance (gyrA S83L, gyrA D87N, parC S80I and parC L416F). CONCLUSIONS: This study revealed for the first time in Germany the occurrence of ST1193 among clinical isolates of ESBL-producing E. coli. Further national or regional multicentre studies are needed to assess the effective relevance of ESBL-producing E. coli ST1193 as a nosocomial pathogen in Germany.

Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Milk Samples of Dairy Cows with Mastitis in Bavaria, Germany.

The aim of this study was to determine the rate of extended-spectrum ß-lactamase (ESBL)-producing microorganisms among Escherichia coli isolates causing bovine mastitis, including molecular characterization of these isolates. Therefore, a total of 490 bovine E. coli isolates from milk samples of dairy cows with mastitis were investigated for ESBL production by antimicrobial susceptibility testing, PCR-based detection, and sequencing of ESBL encoding genes, which were identified in 22 isolates (4.5%). Moreover, resistance to the fluoroquinolones enrofloxacin and marbofloxacin occurred in 15 of 22 ESBL-producing isolates (68.2%). All ESBL-producing isolates carried a blaCTX-M-like gene, with blaCTX-M-14 (n = 10) as the most prevalent type. Seven isolates producing CTX-M-14 and belonging to phylogenetic group A were further investigated for genetic relatedness by multilocus sequence typing. Five of them could be assigned to four different sequence types (STs): ST10 (n = 2), ST167 (n = 1), ST410 (n = 1), and ST744 (n = 1), whereas the remaining two isolates could not be assigned. To conclude, the rate of ESBL-producing E. coli associated with cattle mastitis was 4.5%. Furthermore, a high proportion of fluoroquinolone coresistance could be detected. Therefore, careful and continuous surveillance of ESBL-producing E. coli in cattle and consequent implementation of prevention measures are needed to avoid a further spread of these multidrug-resistant bacteria.

Screening of ESBL-producing Enterobacteriacae concomitant with low degree of transmission in intensive care and bone marrow transplant units.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) are spreading worldwide in both hospital and community settings. In this study, the molecular epidemiology and the transmission modalities of ESBL-E in intensive care- and bone marrow transplant were investigated. METHODS: All patients included in this study were screened for presence of ESBL-E on admission and weekly. Relevant ß-lactamase genes were identified by PCR and sequencing. RESULTS: A total of 669 patients were included in this study. On admission, ESBL-producing Escherichia coli were detected in 49 (7.3%) patients and ESBL-producing Klebsiella pneumoniae in one patient. The most common ESBL types among E. coli isolates were CTX-M-15 (38.8%) and CTX-M-1 (38.8%). Furthermore, 12 of 49 (24.5%) ESBL-producing E. coli could be assigned to the epidemic clone ST131. A single patient acquired ESBL-producing E. coli during the hospital stay but cross-transmission could not be demonstrated. Among 1095 environmental samples none revealed ESBL. CONCLUSIONS: Our results suggest that early detection of ESBL-producing Enterobacteriaceae and consequent implementation of basic hygiene measures and contact isolation may reduce the transmission rate during the hospital stay.

Prevalence and genetic diversity of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in nursing homes in Bavaria, Germany.

Main goal of this study was to determine the prevalence and molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae among 156 nursing home residents in Bavaria and to compare the results with healthy individuals from the Bavarian community. Intestinal colonisation by ESBL-producing Escherichia coli was detected in 23 nursing home residents (14.7%) using MacConkey agar supplemented with cefotaxime (1mg/L) for screening and the combined disc method for ESBL confirmation. Antimicrobial susceptibility testing revealed co-resistance to ciprofloxacin in 86.9% of the ESBL-producers. All isolates harboured CTX-M-ESBL with CTX-M-15 (65.2%) and CTX-M-27 (21.7%) as the most common types. Moreover, 16 isolates (69.6%) could be assigned by PCR-typing to the epidemic clonal lineage E. coli O25b-ST131. Further typing by rep-PCR and XbaI-macrorestriction with subsequent pulsed-field gel electrophoresis, respectively, revealed that two or more residents shared the same ESBL-producing E. coli clone in four nursing homes. In conclusion, we could show a high prevalence of ESBL-producing E. coli in Bavarian nursing homes (14.7%) compared to the healthy population (6.3%). Although the prevalence of ESBL-type CTX-M-15 in E. coli was similar in nursing home residents (65.2%) and healthy individuals (46%) the presence of E. coli O25b-ST131 clones differed substantially (69.6% and 14.2%, respectively). Furthermore, this study demonstrates that a person-to-person transmission or a common source of infection for ESBL-producing microorganisms may occur in these facilities. Therefore, basic hygiene measures should be assiduously implemented to prevent the further spread of these multidrug-resistant bacteria.

Prevalence, antimicrobial susceptibility, and genetic diversity of Pseudomonas aeruginosa as intestinal colonizer in the community.

In this study we determined the prevalence of intestinal carriage, the antimicrobial susceptibility rates, and the genetic diversity of Pseudomonas aeruginosa in the community. From July 2010 to December 2011, a total of 2110 nonreplicate fecal samples from individuals living in Bavaria were collected. Samples were screened for P. aeruginosa by a selective medium and antimicrobial susceptibility was determined by disc diffusion technique. Genetic diversity was assessed by multilocus sequence typing (MLST). Intestinal colonization was detected in 31 of 2110 (1.47%) individuals. None of the isolates showed resistance to aztreonam, imipenem, meropenem, ciprofloxacin, amikacin or colistin. Twenty-five isolates could be assigned to 20 different sequence types (STs), whereas the remaining 6 could not be assigned. Interestingly, four isolates belonged to ST253. These data show that intestinal colonization by P. aeruginosa occurs in the community with high genetic diversity and low rates of antimicrobial resistance.

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.

Extended-spectrum-ß-lactamase-producing Escherichia coli as intestinal colonizers in the German community.

We determined the presence of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli among 3,344 study participants from the German community. Intestinal colonization was detected in 211 persons (6.3%), without significant differences among the different age groups. The majority (95.2%) of isolates harbored CTX-M-type ESBL, with CTX-M-15 (46%) and CTX-M-1 (24.2%) as the most common types. The finding of ESBL producers and one isolate additionally producing carbapenemase OXA-244 indicates a risk of dissemination of resistant bacteria outside the hospitals.