Publications by authors named "Yvonne Pfeifer"

95 Publications

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

Pathogens 2021 May 8;10(5). Epub 2021 May 8.

Division Nosocomial Pathogens and Antibiotic Resistances, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, 38855 Wernigerode, Germany.

The increase in infections with multidrug-resistant and virulent () strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for 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 (, , , , ) among 94 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). was used to predict resistance and virulence genes; was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the 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%) isolate. Virulence genes were found in 22 isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes and , respectively. Notably, aerobactin ( lineage 3) was only detected in isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for lineage 3.
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http://dx.doi.org/10.3390/pathogens10050573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170897PMC
May 2021

High Prevalence of Carbapenemase-Producing in Wound Infections, Ghana, 2017/2018.

Microorganisms 2021 Mar 5;9(3). Epub 2021 Mar 5.

Institute for Medical Microbiology and Göttingen International Health Network, University Medical Center Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.

Three years after a prospective study on wound infections in a rural hospital in Ghana revealed no emergence of carbapenem-resistant bacteria we initiated a new study to assess the prevalence of multidrug-resistant pathogens. Three hundred and one samples of patients with wound infections were analysed for the presence of resistant bacteria in the period August 2017 till March 2018. Carbapenem-resistant were further characterized by resistance gene sequencing, PCR-based bacterial strain typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST "Oxford scheme"). was detected in wound infections of 45 patients (15%); 22 isolates were carbapenem-resistant. Carbapenemases NDM-1 and/or OXA-23 were detected in all isolates; two isolates harboured additionally OXA-420. PFGE and MLST analyses confirmed the presence of one strain in 17 patients that was assigned to the worldwide spread sequence type ST231 and carried NDM-1 and OXA-23. Furthermore, two new STs (ST2145 and ST2146) were detected in two and three patients, respectively. Within three years the prevalence of carbapenem-resistant increased dramatically in the hospital. The early detection of multidrug-resistant bacteria and prevention of their further spread are only possible if continuous surveillance and molecular typing will be implemented.
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http://dx.doi.org/10.3390/microorganisms9030537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998214PMC
March 2021

Detection of a New Resistance-Mediating Plasmid Chimera in a -Positive Strain at a German University Hospital.

Microorganisms 2021 Mar 31;9(4). Epub 2021 Mar 31.

Institute for Medical Microbiology, University Medical Center Göttingen, 37075 Göttingen, Germany.

Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven gene-carrying isolates, which were isolated between 2013 and 2014 at the University Medical Center in Göttingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively; and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in in Germany.
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http://dx.doi.org/10.3390/microorganisms9040720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066831PMC
March 2021

Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of -, -, and -Containing Plasmids in .

Antibiotics (Basel) 2021 Jan 19;10(1). Epub 2021 Jan 19.

Robert Koch Institute, Department Infectious Diseases, Division Nosocomial Pathogens and Antimicrobial Resistances, Wernigerode Branch, 38855 Wernigerode, Germany.

(1) 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 . (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to 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 -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 adaptations.
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http://dx.doi.org/10.3390/antibiotics10010090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832316PMC
January 2021

Hypervirulent of Lineage ST66-K2 Caused Tonsillopharyngitis in a German Patient.

Microorganisms 2021 Jan 8;9(1). Epub 2021 Jan 8.

Division Nosocomial Pathogens and Antibiotic Resistance, Department of Infectious Diseases, Robert Koch Institute, Wernigerode Branch, 38855 Wernigerode, Germany.

Hypervirulent (hvKp) is a novel pathotype that has been rarely described in Europe. This study characterizes a hvKp isolate that caused a community-acquired infection. The hypermucoviscous () strain 18-0005 was obtained from a German patient with tonsillopharyngitis in 2017. Antibiotic susceptibility testing was performed and the genome was sequenced by Illumina and Nanopore technology. Whole genome data were analyzed by conducting core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP) analysis. Virulence genes were predicted by applying Kleborate. Phenotypic and whole genome analyses revealed a high similarity of the study isolate 18-0005 to the recently reported antibiotic-susceptible hvKp isolate SB5881 from France and the "ancestral" strain Kp52.145; both were assigned to the ST66-K2 lineage. Comparative genomic analysis of the three plasmids showed that the 18-0005 plasmid II differs from SB5881 plasmid II by an additional 3 kb integrated fragment of plasmid I. Our findings demonstrate the genetic flexibility of hvKp and the occurrence of a strain of the clonal group CG66-K2 in Germany. Hence, it emphasizes the need to improve clinical awareness and infection monitoring of hvKp.
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http://dx.doi.org/10.3390/microorganisms9010133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827599PMC
January 2021

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Populations.

Front Vet Sci 2020 16;7:614513. Epub 2020 Dec 16.

Robert Koch Institute, Berlin, Germany.

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 μg/ml ZnCl We further provided evidence that enhanced zinc tolerance in porcine intestinal populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, < 0.001 and = 0.002, resp.). Strikingly, harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 μg/ml ZnCl MIC, < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased.
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http://dx.doi.org/10.3389/fvets.2020.614513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772137PMC
December 2020

Genome sequences of two clinical isolates harboring the novel colistin-resistance gene variants - and -.

Gut Pathog 2020 3;12:40. Epub 2020 Sep 3.

Division of Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Wernigerode, Germany.

Background: Colistin is still a widely used antibiotic in veterinary medicine although it is a last-line treatment option for hospitalized patients with infections caused by multidrug-resistant Gram-negative bacteria. Colistin resistance has gained additional importance since the recent emergence of mobile colistin resistance () genes. In the scope of a study on colistin resistance in clinical isolates from human patients in Germany we characterized the - gene variants.

Results: Our PCR-based screening for -carrying from German patients revealed the presence of -- genes in 60 isolates. Subsequent whole-genome sequence-based analyses detected one non-synonymous mutation in the - gene for two isolates. The mutations were verified by Sanger sequencing and resulted in amino acid changes Met1Thr (isolate 803-18) and Tyr9Cys (isolate 844-18). Genotyping revealed no relationship between the isolates. The two clinical isolates were assigned to sequence types ST155 (isolate 803-18) and ST69 (isolate 844-18). Both - variants were found to be located on IncX4 plasmids of 33 kb size; these plasmids were successfully conjugated into sodium azide resistant J53 Azi in a broth mating experiment.

Conclusions: Here we present the draft sequences of isolate 803-18 carrying the novel variant - and isolate 844-14 carrying the novel variant -. The results highlight the increasing issue of transferable colistin resistance.
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http://dx.doi.org/10.1186/s13099-020-00375-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472697PMC
September 2020

In vitro activity of cefiderocol against aerobic Gram-negative bacterial pathogens from Germany.

Int J Antimicrob Agents 2020 Oct 3;56(4):106128. Epub 2020 Aug 3.

Robert Koch Institute, Department of Infectious Diseases, FG13 Nosocomial Pathogens and Antibiotic Resistances, Wernigerode, Germany.

Objectives: Cefiderocol (CID), also known as S-649266, a novel siderophore cephalosporin, possesses potent activity against multidrug-resistant aerobic Gram-negative bacteria (GNB). This study aimed to determine the in vitro activity of CID against two different sets of GNB: i) a random sample of 213 clinical isolates, including 17 extended-spectrum beta-lactamase (ESBL) producers, obtained from intensive care unit patients with nosocomial infections collected during a multicentre surveillance study (set I); and ii) a group of 59 challenge GNB producing various types of carbapenemases (CP; set II).

Methods: Minimum inhibitory concentrations (MICs) were determined using the microdilution method according to the standard ISO 20776-1. Iron-depleted medium was used for testing CID.

Results: CID inhibited 97.2% of set I isolates at the EUCAST susceptibility breakpoint of ≤ 2 mg/L. The concentrations of CID inhibiting 50% and 90% (MIC) of the Enterobacterales isolates (n = 146) were 0.12/1.0 mg/L, with ESBL-positive isolates tending to exhibit higher MICs than ESBL-negative isolates to CID. MIC values of CID for isolates of the Acinetobacter baumannii group (n = 13) and Pseudomonas aeruginosa (n = 54) were 0.06/0.12 mg/L and 0.12/0.5 mg/L, respectively. Further, CID inhibited 88.1% of set II CP-producing isolates at ≤ 2 mg/L. All seven class D CP-producing Acinetobacter baumannii were inhibited at ≤ 0.25 mg/L. MIC values for CP-producing Enterobacterales (n = 30) and Pseudomonas aeruginosa (n = 22) were 1/4 mg/L and 0.5/2 mg/L, respectively.

Conclusion: CID showed potent activity against Acinetobacter baumannii, Enterobacterales and Pseudomonas aeruginosa, including CP-producing isolates. Overall, CID inhibited 259 of 272 (95.2%) GNB at ≤ 2 mg/L.
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http://dx.doi.org/10.1016/j.ijantimicag.2020.106128DOI Listing
October 2020

Low Occurrence of in Gulls and Songbirds.

Pol J Microbiol 2020 ;69:1-6

Robert Koch Institute , Wernigerode Branch, Wernigerode , Germany.

is a worldwide occurring nosocomial pathogen, the natural habitats of which remain to be defined. Recently, white stork nestlings have been described as a recurring source of . Here, we challenged the hypothesis of a general preference of for avian hosts. Taking advantage of campaigns to ring free-living birds, we collected cloacal swab samples from 741 black-headed gulls () in Poland, tracheal and cloacal swabs from 285 songbirds in Poland as well as tracheal swabs from 25 songbirds in Slovenia and screened those for the growth of on CHROMagar Acinetobacter. Of the 1,051 samples collected only two yielded isolates. Each carried one variant of the gene, i.e. OXA-71 and OXA-208, which have been described previously in clinical isolates of . In conclusion, our data do not support a general preference of for avian hosts.

is a worldwide occurring nosocomial pathogen, the natural habitats of which remain to be defined. Recently, white stork nestlings have been described as a recurring source of . Here, we challenged the hypothesis of a general preference of for avian hosts. Taking advantage of campaigns to ring free-living birds, we collected cloacal swab samples from 741 black-headed gulls () in Poland, tracheal and cloacal swabs from 285 songbirds in Poland as well as tracheal swabs from 25 songbirds in Slovenia and screened those for the growth of on CHROMagar Acinetobacter. Of the 1,051 samples collected only two yielded isolates. Each carried one variant of the gene, i.e. OXA-71 and OXA-208, which have been described previously in clinical isolates of . In conclusion, our data do not support a general preference of for avian hosts.
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http://dx.doi.org/10.33073/pjm-2020-011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256842PMC
December 2020

IS-Mediated Transfer of as the Main Route of Resistance Transmission During a Polyclonal, Multispecies Outbreak in a German Hospital.

Front Microbiol 2019 17;10:2817. Epub 2019 Dec 17.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

One of the most demanding challenges in infection control is the worldwide dissemination of multidrug-resistant (MDR) bacteria in clinical settings. Especially the increasing prevalence of carbapenemase producing Gram-negative pathogens poses an urgent threat to public health, as these enzymes confer resistance to almost all β-lactam antibiotics including carbapenems. In this study, we report a prolonged nosocomial outbreak of various NDM-1-producing species due to clonal spread and cross-species exchange of plasmids and possibly transposons. Between July 2015 and September 2017, a total of 51 carbapenemase-positive isolates were collected from 38 patients and three environmental sources in a single German hospital. Combining molecular typing methods and whole genome sequencing, the metallo-β-lactamase gene was found to be present in 35 isolates of which seven additionally carried the carbapenemase gene . Core genome MLST (cgMLST) revealed different clusters of closely related isolates of , , , or indicating clonal spread. The detailed reconstruction of the plasmid sequences revealed that in all outbreak-associated isolates was located on similar composite transposons, which were also very similar to Tn previously described for . In contrast to Tn, these structures were flanked by IS elements, which could facilitate horizontal gene transfer. Moreover, the identical plasmid was found to be shared by and isolates. Our results highlight the importance of detailed genome-based analyses for complex nosocomial outbreaks, allowing the identification of causal genetic determinants and providing insights into potential mechanisms involved in the dissemination of antibiotic resistances between different bacterial species.
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http://dx.doi.org/10.3389/fmicb.2019.02817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929489PMC
December 2019

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

Front Microbiol 2019 28;10:2734. Epub 2019 Nov 28.

Robert Koch Institute, Berlin, Germany.

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 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]: = 99) and a corresponding "control group" ([CG]: = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, 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 isolates showed three different ZnCl MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl (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" ZnCl MICs and isolates representing the HZG as well as "lower ZnCl MICs" with isolates of the CG ( = 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 B, A, and . In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant , 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.
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http://dx.doi.org/10.3389/fmicb.2019.02734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892955PMC
November 2019

Extensively drug-resistant ST307 outbreak, north-eastern Germany, June to October 2019.

Euro Surveill 2019 Dec;24(50)

National Reference Centre for multidrug-resistant Gram-negative bacteria, Ruhr University Bochum, Bochum, Germany.

From June to October 2019, 17 patients (six infected, 11 colonised) with an extensively drug-resistant (XDR) strain were notified from four Western Pomerania medical facilities. The XDR produced carbapenemases NDM-1 and OXA-48, and was only susceptible to chloramphenicol, tigecycline and cefiderocol. Synergistic activity was observed for the combination of aztreonam plus ceftazidime-avibactam. Genomic analyses showed all isolates belonged to sequence type 307. Control measures and further investigations are ongoing.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.50.1900734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918589PMC
December 2019

Extended-Spectrum Beta-Lactamase (ESBL)-Producing Isolated from Flies in the Urban Center of Berlin, Germany.

Int J Environ Res Public Health 2019 04 30;16(9). Epub 2019 Apr 30.

Institute of Hygiene and Environmental Medicine, Charité-Universitätsmedizin, 12203 Berlin, Germany.

: The monitoring of antimicrobial resistance (AMR) in microorganisms that circulate in the environment is an important topic of scientific research and contributes to the development of action plans to combat the spread of multidrug-resistant (MDR) bacteria. As a synanthropic vector for multiple pathogens and a reservoir for AMR, flies can be used for surveillance. : We collected 163 flies in the inner city of Berlin and examined them for extended-spectrum β-lactamase (ESBL)-producing genotypically and phenotypically. : The prevalence of ESBL-producing in flies was 12.9%. Almost half (47.6%) of the ESBL-positive samples showed a co-resistance to ciprofloxacin. Resistance to carbapenems or colistin was not detected. The predominant ESBL-type was CTX-M-1, which is associated with wildlife, livestock, and companion animals as a potential major source of transmission of MDR to flies. : This field study confirms the permanent presence of ESBL-producing in an urban fly population. For continuous monitoring of environmental contamination with multidrug-resistant (MDR) bacteria, flies can be used as indicators without much effort.
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http://dx.doi.org/10.3390/ijerph16091530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539871PMC
April 2019

Fluoroquinolone-Resistant , spp., and from Local and Imported Poultry Meat in Kumasi, Ghana.

Foodborne Pathog Dis 2019 05 23;16(5):352-358. Epub 2019 Mar 23.

1 Department Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine (BNITM), Hamburg, Germany.

and are important gastroenteric pathogens. is an emerging enteric pathogen. Data on the frequencies of these poultry-associated pathogens on meat products sold in sub-Saharan Africa are scarce. This study aimed to analyze the frequency of , , and antibiotic resistance and underlying mechanisms of resistance to fluoroquinolones in locally produced and imported poultry sold in urban Ghana. Chicken meat was collected and cultured on standard media. Bacterial strains were identified by biochemical methods and by mass spectrometry. Antibiotic susceptibility was tested by disk diffusion. Ciprofloxacin-resistant strains were assessed for molecular mechanisms of resistance. Among 200 samples, comprising 34% ( = 68) from the Ghanaian poultry industry and 66% ( = 132) from imports, 9% ( = 17) contained , 11% ( = 22) , and 26.5% ( = 53) . Higher overall contamination frequencies were found in local meat. Most common serovars identified were Kentucky (/ = 5/16; 31%) and Poona (/ = 4/16; 25%). were (/ = 10/19; 53%) and (/ = 9/19; 47%). Resistance to fluoroquinolones was high with 63% ( = 10), 75% ( = 15), and 52% ( = 25) in , , and , respectively. A link between Kentucky [sequence type (ST) 198] and a ciprofloxacin minimum inhibitory concentration of 16 μg/mL was found. Poona-ST308 revealed transferable fluoroquinolone resistance genes. Markedly high frequencies of resistant , , and predominant in locally produced meat represent a probable transmission reservoir for human infections. These findings highlight the need for implementation of surveillance systems that focus on food hygiene, use of antibiotics in animal husbandry, and continuous monitoring of the quality of meat products from imports.
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http://dx.doi.org/10.1089/fpd.2018.2562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529854PMC
May 2019

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in in Critically Ill Patients.

Front Microbiol 2019 19;10:276. Epub 2019 Feb 19.

Robert Koch Institute, FG13 Nosocomial Pathogens and Antibiotic Resistances, Wernigerode, Germany.

Carbapenem-resistant (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 , 2 , and 1 ) 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 clone; one clone was found in two patients, and one patient carried and . Molecular analysis confirmed the presence of a conjugative-carrying 70 kb-IncN plasmid in and and an 80 kb-IncN plasmid in All transconjugants harbored either the 70 or 80 kb plasmid with embedded within transposon variant Tn. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a -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 -carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of -carrying IncN-plasmids harboring the Tn isoform in Germany.
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http://dx.doi.org/10.3389/fmicb.2019.00276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390000PMC
February 2019

No evidence of carbapenemase-producing Enterobacteriaceae in stool samples of 1,544 asylum seekers arriving in Rhineland-Palatinate, Germany, April 2016 to March, 2017.

Euro Surveill 2019 Feb;24(8)

These two authors have contributed equally to this manuscript and share last authorship.

Introduction: Since 2015, increased migration from Asia and Africa to Europe has raised public health concerns about potential importation of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE), specifically those producing carbapenemases (C-PE), into European hospitals.

Aims: To inform infection control practices about ESBL-PE prevalence in asylum seekers and to investigate whether C-PE prevalence exceeds that in the German population.

Methods: Cross-sectional study from April 2016-March 2017. Routinely collected stool samples from asylum seekers were tested for antibiotic resistant Enterobacteriaceae. Country/region of origin and demographic characteristics were explored as risk factors for faecal colonisation.

Results: Of 1,544 individuals, 294 tested positive for ESBL-PE colonisation (19.0%; 95% confidence intervals (CI): 17.0-21.0). Asylum seekers originating from Afghanistan/Pakistan/Iran had a prevalence of 29.3% (95% CI: 25.6-33.2), from Syria 20.4% (95% CI: 16.1-25.2) and from Eritrea/Somalia 11.9% (95% CI: 8.7-15.7). CTX-M-15 (79%) and CTX-M-27 (10%) were the most common ESBL determinants. Highest ESBL-PE prevalences were observed in boys under 10 years and women aged 20-39 years (interaction: p = 0.03). No individuals tested positive for C-PE. Faecal C-PE colonisation prevalence in asylum seekers was not statistically significantly different from prevalence reported in German communities.

Conclusion: In absence of other risk factors, being a newly arrived asylum seeker from a region with increased faecal ESBL-PE colonisation prevalence is not an indicator for C-PE colonisation and thus not a reason for pre-emptive screening and isolation upon hospital admission.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.8.1800030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446954PMC
February 2019

Overestimation of an Outbreak of Enterobacter cloacae in a Neonatal Intensive Care Unit in Germany, 2015.

Pediatr Infect Dis J 2019 06;38(6):631-637

Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany.

Background: In August 2015, 17 neonates with Enterobacter cloacae (E. cloacae) colonization were identified in a neonatal intensive care unit (NICU) in Germany. Two developed severe brain abscesses. Despite temporary NICU closure in September, another infant with E. cloacae colonization was detected in October 2015.

Methods: We defined potential cases as inpatients treated in the NICU or any pediatric/maternity ward in 2015 with E. cloacae in any specimen before molecular typing. Cases were at first confirmed by arbitrarily-primed-polymerase-chain-reaction and later by XbaI-macrorestriction/pulsed-field gel electrophoresis and next-generation-sequencing. Enhanced barrier precautions and cohorting were implemented for all potential cases and microbiologic screening was extended from NICU to all pediatric/maternity wards.

Results: Of 41 potential cases (occurring between 08/04/2015 and 15/11/2015 in 4 wards), the isolates of 23 shared identical arbitrarily-primed-polymerase-chain-reaction patterns; 3 without plausible epidemiologic link. Pulsed-field gel electrophoresis analyses verified only 10 cases (all in the NICU); next-generation-sequencing analysis confirmed these results. In addition 6 cases without isolates available for genotyping were closely linked in place and time.

Conclusions: Forty-one suspected patients were cohorted and the NICU was temporarily closed. Further analyses revealed that only 16 cases belonged to the outbreak. Only close interdisciplinary collaboration and highly discriminatory genotyping methods allowed to clearly differentiate between cases and noncases in this E. cloacae outbreak.
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http://dx.doi.org/10.1097/INF.0000000000002264DOI Listing
June 2019

Evaluation of the automated BD Phoenix CPO Detect panel in combination with the β-CARBA assay for detection and classification of carbapenemase-producing Enterobacterales.

J Microbiol Methods 2019 01 3;156:29-33. Epub 2018 Dec 3.

Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany.

Recently, the CPO Detect panel for the detection of carbapenemase-producing, Gram-negative bacteria was introduced for the Phoenix semi-automated antimicrobial susceptibility testing system. The CPO Detect assay aims to detect carbapenemase activity (P/N test) and to type carbapenemase producers according to the Ambler classification (Ambler test). The P/N test-based detection of carbapenemase producers was 100% sensitive and 55.3% specific in the assessment of 57 carbapenemase-producing and 38 non-carbapenemase-producing Enterobacterales. False-positive test isolates in the P/N test arose from carbapenemase-non-producing, but carbapenem-non-susceptible isolates. In contrast, using the Ambler test-based approach for carbapenemase detection resulted in a specificity of 100% and a sensitivity of 79%. In order to improve the overall performance, we established an algorithm that additionally included the colorimetric β-CARBA assay as downstream test for P/N test-positive isolates, which remained un-typed in the Ambler test. This algorithm displayed an overall sensitivity and specificity of 98.3% and 100%, respectively. Our data demonstrate that the combination of the CPO Detect assay with the β-CARBA test allows for rapid detection and classification of carbapenemase-producing Enterobacterales.
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http://dx.doi.org/10.1016/j.mimet.2018.11.024DOI Listing
January 2019

Profiling antimicrobial peptides from the medical maggot Lucilia sericata as potential antibiotics for MDR Gram-negative bacteria.

J Antimicrob Chemother 2019 01;74(1):96-107

Fraunhofer Institute for Molecular Biology and Applied Ecology, Department of Bioresources, Gießen, Germany.

Background: The ability of MDR Gram-negative bacteria to evade even antibiotics of last resort is a severe global challenge. The development pipeline for conventional antibiotics cannot address this issue, but antimicrobial peptides (AMPs) offer an alternative solution.

Objectives: Two insect-derived AMPs (LS-sarcotoxin and LS-stomoxyn) were profiled to assess their suitability for systemic application in humans.

Methods: The peptides were tested against an extended panel of 114 clinical MDR Gram-negative bacterial isolates followed by time-kill analysis, interaction studies and assays to determine the likelihood of emerging resistance. In further in vitro studies we addressed cytotoxicity, cardiotoxicity and off-target interactions. In addition, an in vivo tolerability and pharmacokinetic study in mice was performed.

Results: LS-sarcotoxin and LS-stomoxyn showed potent and selective activity against Gram-negative bacteria and no cross-resistance with carbapenems, fluoroquinolones or aminoglycosides. Peptide concentrations of 4 or 8 mg/L inhibited 90% of the clinical MDR isolates of Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Salmonella enterica isolates tested. The 'all-d' homologues of the peptides displayed markedly reduced activity, indicating a chiral target. Pharmacological profiling revealed a good in vitro therapeutic index, no cytotoxicity or cardiotoxicity, an inconspicuous broad-panel off-target profile, and no acute toxicity in mice at 10 mg/kg. In mouse pharmacokinetic experiments LS-sarcotoxin and LS-stomoxyn plasma levels above the lower limit of quantification (1 and 0.25 mg/mL, respectively) were detected after 5 and 15 min, respectively.

Conclusions: LS-sarcotoxin and LS-stomoxyn are suitable as lead candidates for the development of novel antibiotics; however, their pharmacokinetic properties need to be improved for systemic administration.
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http://dx.doi.org/10.1093/jac/dky386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322280PMC
January 2019

Inactivation of multidrug-resistant pathogens and Yersinia enterocolitica with cold atmospheric-pressure plasma on stainless-steel surfaces.

Int J Antimicrob Agents 2018 Dec 31;52(6):811-818. Epub 2018 Aug 31.

Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation (TiHo), Bischofsholer Damm 15, D-30173 Hannover, Germany. Electronic address:

The objective of this study was to investigate the impact of cold atmospheric-pressure plasma (CAP) produced by a surface micro-discharge plasma source as a new strategy to combat the transmission of five multidrug-resistant (MDR) pathogens and Yersinia enterocolitica on typical hospital- and food-producing surfaces, e.g. stainless-steel. Approximately 10 CFU/cm of vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Y. enterocolitica were inoculated on a 3.14-cm stainless-steel surface. Bovine serum albumin (BSA) (3%) was used as a disruptive factor simulating natural organic material. The inoculated surfaces were subsequently exposed to CAP, generated by a peak-to-peak voltage of 10 kV with sinusoidal waveform and a frequency of 2 kHz, for 5, 10 and 20 min, respectively. Fluorescent staining with propidium iodide and SYTO 9 was used to demonstrate the manner of bacterial cell damage. Significant (P < 0.05) inactivation of 1.68 ± 0.17 up to 2.80 ± 0.17 log steps was achieved after 5 min of CAP treatment. However, bacterial reduction could be increased to 3.35 ± 0.1 up to 5.17 ± 0.67 log steps after 20 min of CAP treatment. Bacterial cells covered with BSA were statistically significantly less inactivated by CAP. Fluorescent staining showed a predominant level of orange-stained, sublethally damaged bacterial cells after 10 min of CAP treatment. In conclusion, CAP has the ability to inactivate MDR bacterial pathogens on stainless-steel surfaces. Further research is required to investigate the clinical features of CAP.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.08.023DOI Listing
December 2018

Whole genome analyses of CMY-2-producing Escherichia coli isolates from humans, animals and food in Germany.

BMC Genomics 2018 Aug 9;19(1):601. Epub 2018 Aug 9.

Robert Koch-Institute, FG 13 Nosocomial Pathogens and Antibiotic Resistance, Burgstr, 37 38855, Wernigerode, Germany.

Background: Resistance to 3rd-generation cephalosporins in Escherichia coli is mostly mediated by extended-spectrum beta-lactamases (ESBLs) or AmpC beta-lactamases. Besides overexpression of the species-specific chromosomal ampC gene, acquisition of plasmid-encoded ampC genes, e.g. bla, has been described worldwide in E. coli from humans and animals. To investigate a possible transmission of bla along the food production chain, we conducted a next-generation sequencing (NGS)-based analysis of 164 CMY-2-producing E. coli isolates from humans, livestock animals and foodstuff from Germany.

Results: The data of the 164 sequenced isolates revealed 59 different sequence types (STs); the most prevalent ones were ST38 (n = 19), ST131 (n = 16) and ST117 (n = 13). Two STs were present in all reservoirs: ST131 (human n = 8; food n = 2; animal n = 6) and ST38 (human n = 3; animal n = 9; food n = 7). All but one CMY-2-producing ST131 isolates belonged to the clade B (fimH22) that differed substantially from the worldwide dominant CTX-M-15-producing clonal lineage ST131-O25b clade C (fimH30). Plasmid replicon types IncI1 (n = 61) and IncK (n = 72) were identified for the majority of bla-carrying plasmids. Plasmid sequence comparisons showed a remarkable sequence identity, especially for IncK plasmids. Associations of replicon types and distinct STs were shown for IncK and ST57, ST429 and ST38 as well as for IncI1 and ST58. Additional β-lactamase genes (bla, bla, bla, bla) were detected in 50% of the isolates, and twelve E. coli from chicken and retail chicken meat carried the colistin resistance gene mcr-1.

Conclusion: We found isolates of distinct E. coli clonal lineages (ST131 and ST38) in all three reservoirs. However, a direct clonal relationship of isolates from food animals and humans was only noticeable for a few cases. The CMY-2-producing E. coli-ST131 represents a clonal lineage different from the CTX-M-15-producing ST131-O25b cluster. Apart from the ST-driven spread, plasmid-mediated spread, especially via IncI1 and IncK plasmids, likely plays an important role for emergence and transmission of bla between animals and humans.
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http://dx.doi.org/10.1186/s12864-018-4976-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085623PMC
August 2018

Genome-based analysis of Carbapenemase-producing isolates from German hospital patients, 2008-2014.

Antimicrob Resist Infect Control 2018 2;7:62. Epub 2018 May 2.

1Robert Koch Institute, Berlin, Germany.

Background: By using whole genome sequence data we aimed at describing a population snapshot of carbapenemase-producing isolated from hospitalized patients in Germany between 2008 and 2014.

Methods: We selected a representative subset of 107 carbapenemase-producing clinical isolates possessing the four most prevalent carbapenemase types in Germany (KPC-2, KPC-3, OXA-48, NDM-1). Isolates were processed via illumina NGS. Data were analysed using different SNP-based mapping and de-novo assembly approaches. Relevant information was extracted from NGS data (antibiotic resistance determinants, gene/ type, virulence genes). NGS data from the present study were also compared with 238 genome data from two previous international studies on

Results: NGS-based analyses revealed a preferred prevalence of KPC-2-producing ST258 and KPC-3-producing ST512 isolates. OXA-48, being the most prevalent carbapenemase type in Germany, was associated with various strain types; most of them possessing IncL/M plasmid replicons suggesting a preferred dissemination of via this well-known plasmid type. Clusters ST15, ST147, ST258, and ST512 demonstrated an intermingled subset structure consisting of German and other European isolates. ST23 being the most frequent MLST type in Asia was found only once in Germany. This latter isolate contained an almost complete set of virulence genes and a K1 capsule suggesting occurrence of a hypervirulent ST23 strain producing OXA-48 in Germany.

Conclusions: Our study results suggest prevalence of "classical" strain types associated with widely distributed carbapenemase genes such as ST258/KPC-2 or ST512/KPC-3 also in Germany. The finding of a supposed hypervirulent and OXA-48-producing ST23 isolates outside Asia is highly worrisome and requires intense molecular surveillance.
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http://dx.doi.org/10.1186/s13756-018-0352-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930415PMC
July 2019

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

Front Microbiol 2018 27;9:538. Epub 2018 Mar 27.

Institute for Animal Hygiene and Environmental Health, Freie Universitaet Berlin, Berlin, Germany.

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 (.) 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. and 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 (G-336-1a) as well as two isolates (G-336-2, G-268-2). All but G-268-2 harbored the gene. isolates R3 and G-336-1 were closely related although derived from two different farms. All three -encoding isolates possessed identical plasmids and the containing transposon showed mobility at least . In isolate G-268-2, the AmpC beta-lactamase gene but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the 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.
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http://dx.doi.org/10.3389/fmicb.2018.00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880886PMC
March 2018

[Effective management of an outbreak with multiresistent Klebsiella pneumoniae in a neurorehabilitation unit].

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2018 May;61(5):543-552

Abteilung für Infektionsepidemiologie, Robert Koch-Institut (RKI), Berlin, Deutschland.

Background: In addition to acute care hospitals, rehabilitation centres are increasingly confronted with multi-resistant pathogens. Long durations of stay and intensive treatments impose special hygienic challenges.

Material And Methods: We investigated an extended spectrum beta-lactamase-Klebsiella pneumoniae (ESBL-K. pneumoniae) outbreak in a neurorehabilitation centre. We defined confirmed cases as patients who stayed in the centre during the outbreak period and from whom ESBL-K. pneumoniae was isolated with the outbreak sequence type. Probable cases had an epidemiological link to at least one confirmed case but no isolate for typing. Next generation sequencing (NGS) was performed on 53 isolates from patients. Environmental sampling was performed. Systematic microbiological screening was implemented and ESBL-K. pneumoniae-positive patients were cohorted in a designated ward.

Results: We identified 30 confirmed and 6 probable cases. NGS revealed three genetic clusters: Cluster 1 - the outbreak cluster - with isolates of 30 cases (sequence type ST15), Cluster 2 with 7 patients (ST405) and Cluster 3 with 8 patients (ST414). In two patients, the outbreak strain developed further antibiotic resistance, one with colistin resistance and the other carbapenem resistance. The outbreak ceased after strict isolation measures.

Discussion: Epidemiology and NGS results paired with the effectiveness of cohorting suggest that transmission occurred mainly from person to person in this outbreak. There was an apparent association of the probability to acquire ESBL-K. pneumoniae and treatment intensity, whereas infection rate was related to morbidity. The identification of the outbreak clone and additional clusters plus the development of additional antibiotic resistance shows the relevance of NGS and highlights the need for timely and efficient outbreak management.
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http://dx.doi.org/10.1007/s00103-018-2728-9DOI Listing
May 2018

Whole Genome Sequence Analysis of CTX-M-15 Producing Isolates Allowed Dissecting a Polyclonal Outbreak Scenario.

Front Microbiol 2018 23;9:322. Epub 2018 Feb 23.

Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany.

Extended-spectrum β-lactamase (ESBL) producing pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant in a German neurorehabilitation center from April 2015 to April 2016 to dissect the benefit of whole genome sequencing (WGS) for outbreak analyses. In total, 53 isolates were obtained from 52 patients and examined using WGS. Two independent analysis strategies (reference-based and -free) revealed the same distinct clusters of two CTX-M-15 producing clones (ST15, = 31; ST405, = 7) and one CTX-M-15 producing strain (ST414, = 8). Additionally, we determined sequence variations associated with antimicrobial resistance phenotypes in single isolates expressing carbapenem and colistin resistance, respectively. For rapid detection of the major outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.
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http://dx.doi.org/10.3389/fmicb.2018.00322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829066PMC
February 2018

Reply to Godbole et al.

Clin Infect Dis 2018 06;66(12):1977-1978

Department of Internal Medicine I, University Hospital of Bonn.

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http://dx.doi.org/10.1093/cid/ciy033DOI Listing
June 2018

Escherichia coli ST1421 harbouring the hybrid extended-spectrum β-lactamase CTX-M-64 from a German patient.

J Glob Antimicrob Resist 2018 03 8;12:167-168. Epub 2018 Feb 8.

Antiinfectives Intelligence GmbH, Rheinbach, Germany; Rheinische Fachhochschule Köln gGmbH, Cologne, Germany.

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http://dx.doi.org/10.1016/j.jgar.2018.01.025DOI Listing
March 2018

Carbapenemase detection using the β-CARBA test: Influence of test conditions on performance and comparison with the RAPIDEC CarbaNP assay.

J Microbiol Methods 2018 04 7;147:17-19. Epub 2018 Feb 7.

Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg, Regensburg, Germany.

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http://dx.doi.org/10.1016/j.mimet.2018.02.005DOI Listing
April 2018

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

Genome Announc 2018 Jan 11;6(2). Epub 2018 Jan 11.

Robert Koch-Institut, Bereich Wernigerode, Wernigerode, Germany

Whole-genome sequencing of sp. strain LoGeW2-3, isolated from the pellet of a white stork (), 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.
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http://dx.doi.org/10.1128/genomeA.01405-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764934PMC
January 2018