Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of blaCMY-, blaCTX-M-, and blaOXA-48/VIM-1-Containing Plasmids in Escherichia coli

Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from −4.5% to 7.2%. Transfer of a single blaCMY-16-carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants’ genomes after 500 generations give interesting insights into aspects of plasmid–host adaptationsPeer Reviewe

Genome-Based Analysis of Klebsiella spp. Isolates from Animals and Food Products in Germany, 2013–2017

The increase in infections with multidrug-resistant and virulent Klebsiella pneumoniae (K. pneumoniae) strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for Klebsiella spp. that cause infections in humans and in livestock animals are not well understood. In this study, we aimed to analyze the distribution of antibiotic resistance genes and important virulence determinants (ybt, clb, iro, iuc, rmpA/A2) among 94 Klebsiella spp. isolates from different animal and food sources isolated between 2013 and 2017 in Germany. Antibiotic susceptibility testing was performed, and the genomes were sequenced by Illumina and Nanopore technology. Genetic relationships were assessed by conducting core genome multilocus sequence typing (cgMLST). Kleborate was used to predict resistance and virulence genes; Kaptive was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the K. pneumoniae sensu lato complex. Within this complex, 44 known sequence types (STs), 18 new STs, and 38 capsule types were identified. Extended-spectrum beta-lactamase (ESBL) genes were detected in 16 isolates (17.0%) and colistin resistance in one (1.1%) K. pneumoniae isolate. Virulence genes were found in 22 K. pneumoniae isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes ybt and iuc, respectively. Notably, aerobactin (iuc lineage 3) was only detected in K. pneumoniae isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of Klebsiella spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in K. pneumoniae strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for iuc lineage 3.Peer Reviewe

Complete Genome Sequencing of Acinetobacter sp. Strain LoGeW2-3, Isolated from the Pellet of a White Stork, Reveals a Novel Class D Beta-Lactamase Gene

Whole-genome sequencing of Acinetobacter sp. strain LoGeW2-3, isolated from the pellet of a white stork (Ciconia ciconia), reveals the presence of a plasmid of 179,399 bp encoding a CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated genes) system of the I-F type, and the chromosomally encoded novel class D beta-lactamase OXA-568

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

Klebsiella pneumoniae carbapenemases (KPC) are considered a serious threat to antibiotic therapy as they confer resistance to carbapenems, which are used to treat Extended-spectrum beta-lactamase (ESBL) producing bacteria. Here, we describe the development and evaluation of a DNA microarray for detection and genotyping of KPC genes (blaKPC) within 5 hours. To test the whole assay procedure (DNA extraction + DNA microarray assay) directly from clinical specimen, we compared two commercial DNA extraction kits (QIAprep Spin Miniprep Kit (Qiagen), Urine Bacterial DNA Isolation Kit (Norgen)) for the direct DNA extraction from urine samples (dilution series spiked in human urine). A reliable SNP typing from 1×105 CFU/mL urine was demonstrated for Escherichia coli (Qiagen and Norgen) and 80 CFU/mL urine on average for K. pneumoniae (Norgen). The study presents for the first time the combination of a new KPC-microarray with commercial sample preparation for the detection and genotyping of microbial pathogens directly from clinical specimen which paves the way towards tests providing epidemiological and diagnostic data enabling better antimicrobial stewardship

Sepsis Caused by Extended-Spectrum Beta-Lactamase (ESBL)-Positive K. pneumoniae and E. coli: Comparison of Severity of Sepsis, Delay of Anti- Infective Therapy and ESBL Genotype

Infections with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) are associated with increased mortality. Outcome differences due to various species of ESBL-E or ESBL genotypes are not well investigated. We conducted a cohort study to assess risk factors for mortality in cases of ESBL-E bacteremia (K. pneumoniae or E. coli) and the risk factors for sepsis with organ failure. All consecutive patients of our institution from 2008 to 2011 with bacteremia due to ESBL-E were included. Basic epidemiological data, underlying comorbidities, origin of bacteremia, severity of sepsis and delay of appropriate anti-infective treatment were collected. Isolates were PCR- screened for the presence of ESBL genes and plasmid-mediated AmpC β-lactamases. Cox proportional hazard regression on mortality and multivariable logistic regression on risk factors for sepsis with organ failure was conducted. 219 cases were included in the analysis: 73.1% due to E. coli, 26.9% due to K. pneumoniae. There was no significant difference in hospital mortality (ESBL-E. coli, 23.8% vs. ESBL-K. pneumoniae 27.1%, p = 0.724). However, the risk of sepsis with organ failure was associated in cases of K. pneumoniae bacteremia (OR 4.5, p<0.001) and patients with liver disease (OR 3.4, p = 0.004) or renal disease (OR 6.8, p<0.001). We found significant differences in clinical presentation of ESBL-E bacteremia due to K. pneumoniae compared to E. coli. As K. pneumoniae cases showed a more serious clinical presentation as E. coli cases and were associated with different risk factors, treatment and prevention strategies should be adjusted accordingly

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in Enterobacteriaceae in Critically III Patients

Carbapenem-resistant Enterobacteriaceae (CRE) cause health care-associated infections worldwide, and they are of severe concern due to limited treatment options. We report an outbreak of KPC-2-producing CRE that was caused by horizontal transmission of a promiscuous plasmid across different genera of bacteria and hospitals in Germany. Eleven isolates (8 Citrobacter freundii, 2 Klebsiella oxytoca, and 1 Escherichia coli) were obtained from seven critically ill patients during the six months of the outbreak in 2016. One patient developed a CRE infection while the other six patients were CRE-colonized. Three patients died in the course of the hospital stay. Six of the seven patients carried the same C. freundii clone; one K. oxytoca clone was found in two patients, and one patient carried E. coli and C. freundii. Molecular analysis confirmed the presence of a conjugative, blaKPC-2-carrying 70 kb-IncN plasmid in C. freundii and E. coli and an 80 kb-IncN plasmid in K. oxytoca. All transconjugants harbored either the 70 or 80 kb plasmid with blaKPC-2, embedded within transposon variant Tn4401g. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a blaKPC-2-carrying plasmid of a large outbreak in another German hospital two years earlier. Differences in plasmid sizes and open reading frames point to the presence of inserted mobile genetic elements. There are few outbreak reports worldwide on the transmission of blaKPC-2-carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of blaKPC-2-carrying IncN-plasmids harboring the Tn4401g isoform in Germany.</p

Mortality and molecular epidemiology associated with extended-spectrum β-lactamase production in Escherichia coli from bloodstream infection

Background: The rate of infections due to extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is growing worldwide. These infections are suspected to be related to increased mortality. We aimed to estimate the difference in mortality due to bloodstream infections (BSIs) with ESBL-positive and ESBL-negative E. coli isolates and to determine the molecular epidemiology of our ESBL-positive isolates. Materials and methods: We performed a cohort study on consecutive patients with E. coli BSI between 2008 and 2010 at the Charité University Hospital. Collected data were ESBL production, basic demographic parameters, and underlying diseases by the Charlson comorbidity index (CCI). The presence of ESBL genes was analyzed by polymerase chain reaction (PCR) and sequencing. Phylogenetic groups of ESBL-positive E. coli were determined by PCR. Risk factors for mortality were analyzed by multivariable regression analysis. Results: We identified 115 patients with BSI due to E. coli with ESBL phenotype and 983 due to ESBL-negative E. coli. Fifty-eight percent (n=67) of the ESBL-positive BSIs were hospital-acquired. Among the 99 isolates that were available for PCR screening and sequencing, we found mainly 87 CTX-M producers, with CTX-M-15 (n=55) and CTX-M-1 (n=21) as the most common types. Parameters significantly associated with mortality were age, CCI, and length of stay before and after onset of BSI. Conclusion: The most common ESBL genotypes in clinical isolates from E. coli BSIs were CTX-M-15 (58%) and CTX-M-1 (22%). ESBL production in clinical E. coli BSI isolates was not related to increased mortality. However, the common occurrence of hospital-acquired BSI due to ESBL-positive E. coli indicates future challenges for hospitals

Retrospective Analysis of Bacterial Cultures Sampled in German Chicken- Fattening Farms During the Years 2011–2012 Revealed Additional VIM-1 Carbapenemase-Producing Escherichia coli and a Serologically Rough Salmonella enterica Serovar Infantis

Carbapenems are last-resort antibiotics used in human medicine. The increased detection of carbapenem-resistant Enterobacteriaceae (CRE) is therefore worrying. In 2011 we reported the first livestock-associated VIM-1-producing Salmonella (S.) enterica serovar Infantis (R3) isolate from dust, sampled in a German chicken fattening farm. Due to this observation we retrospectively investigated more than 536 stored bacterial cultures, isolated from 45 chicken fattening farms during the years 2011 and 2012. After a non-selective overnight incubation, the bacteria were transferred to selective media. Escherichia (E.) coli and Salmonella growing on these media were further investigated, including antibiotic susceptibility testing, carbapenemase gene screening and whole genome sequencing (WGS). In total, four CRE were found in three out of 45 investigated farms: Besides R3, one additional Salmonella (G-336-1a) as well as two E. coli isolates (G-336-2, G-268-2). All but G-268-2 harbored the blaVIM-1 gene. Salmonella isolates R3 and G-336-1 were closely related although derived from two different farms. All three blaVIM-1-encoding isolates possessed identical plasmids and the blaVIM-1- containing transposon showed mobility at least in vitro. In isolate G-268-2, the AmpC beta-lactamase gene blaCMY-2 but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the mcr-1 gene, combining phenotypical carbapenem- as well as colistin resistance in one isolate. Carbapenem-resistant Enterobacteriaceae have been found in three out of 45 investigated chicken flocks. This finding is alarming and emphasizes the importance of intervention strategies to contain the environmental spread of resistant bacteria in animals and humans