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

Background By using whole genome sequence data we aimed at describing a population snapshot of carbapenemase-producing K. pneumoniae isolated from hospitalized patients in Germany between 2008 and 2014. Methods We selected a representative subset of 107 carbapenemase-producing K. pneumoniae 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, wzi gene/cps type, virulence genes). NGS data from the present study were also compared with 238 genome data from two previous international studies on K. pneumoniae. 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 K. pneumoniae strain types; most of them possessing IncL/M plasmid replicons suggesting a preferred dissemination of blaOXA-48 via this well-known plasmid type. Clusters ST15, ST147, ST258, and ST512 demonstrated an intermingled subset structure consisting of German and other European K. pneumoniae 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” K. pneumonaie 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 K. pneumoniae isolates outside Asia is highly worrisome and requires intense molecular surveillance.Peer Reviewe

Dissemination of carbapenem-resistant Pseudomonas aeruginosa isolates and their susceptibilities to ceftolozane-tazobactam in Germany

Pseudomonas aeruginosa (PA) is a major cause of healthcare-associated infections. Antipseudomonal carbapenems are among the antimicrobial agents used to treat PA infections, but several mechanisms of resistance, including the production of a carbapenemase (CP), may compromise their clinical efficacy. The objectives of this study were to determine: (i) the dissemination of carbapenem-resistant CP-negative and CP-positive PA isolates; and (ii) the in-vitro activity of ceftolozane-tazobactam (CTT) against carbapenemsusceptible and carbapenem-resistant isolates. Isolates were collected prospectively from January 2016 to April 2017 at 20 German medical laboratories. Each centre was asked to provide 50 consecutive isolates from hospitalized patients. Overall, 985 isolates were collected, of which 34% were obtained from intensive care patients. Seven hundred and thirty-eight (74.9%) isolates were susceptible to both imipenem and meropenem (Subgroup I), and 247 (25.1%) isolates were resistant to carbapenems (Subgroup II): 125 (12.7%) were imipenem-resistant but meropenem-susceptible, 12 (1.2%) were meropenem-resistant but imipenem-susceptible, and 110 (11.2%) were resistant to both carbapenems (Subgroup III). A CP was detected in 28 (2.8%) isolates (predominantly VIM-2). Nine hundred and fifty (96.4%) isolates were CTTsusceptible. Susceptibility to CTT was seen in 99.6% of Subgroup I isolates, 87% of Subgroup II isolates and 74.5% of Subgroup III isolates. Overall, 2.8% of PA produced a CP, while 22.2% were carbapenemresistant, CP-non-producing isolates. Based on these findings, CTT may be considered for treatment of PA infections, particularly those caused by multi-drug-resistant CP-non-producing isolates. (C) 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved

In vitro activity of cefiderocol against aerobic Gram-negative bacterial pathogens from 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% (MIC50/90) 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. MIC50/90 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. MIC50/90 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. (C) 2020 The Authors. Published by Elsevier Ltd

Significance of the d-Serine-deaminase and d-Serine metabolism of staphylococcus saprophyticus for virulence

Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.Published versio

Evaluation of six commercial products for colistin susceptibility testing in Enterobacterales

Objectives: The recommended technique for colistin susceptibility testing by both EUCAST and CLSI is broth microdilution (BMD). However, many routine laboratories still use other methods such as gradient strips or semi-automated systems. The objective of this study was to compare six of the most widespread commercial products for colistin susceptibility testing in Europe with in-house prepared BMD. Methods: A collection of 325 carbapenemase-producing Enterobacterales was tested for colistin susceptibility with three semi-automated systems (Vitek 2, BD Phoenix, MicroScan WalkAway), one gradient-strip test (Etest (R)) and two commercial BMD products (MICRONAUT-S, TREK Sensititre). BMD, in-house prepared according to ISO standard 20776-1, served as reference. Results: The MICRONAUT-S BMD performed best with only one false-resistant (major error, ME) and four false-susceptible (very major error, VME) results while the TREK BMD performed poorer with 16 ME and seven VME. The semi-automated systems Vitek 2 and Phoenix performed poorly with 31 and 26 VME, respectively. The WalkAway semi-automated system showed 16 and 13 false results, depending on the inoculation method. The Etest (R) showed six ME and 10 VME. Conclusions: This study shows that colistin susceptibility testing remains a challenging task for laboratories. It emphasizes the need for selecting the most reliable test method to advocate proper treatment and shows that critical evaluation and precautious usage of colistin susceptibility testing results is constantly required. (C) 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved

Additional file 2: of Genome-based analysis of Carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008-2014

Table S1. Metadata of the 107 German carbapenemase-producing, clinical Klebsiella isolates, 2008-2014. Isolates were ordered by carbapenemase type. For more information see main text document. (XLSX 54 kb

Additional file 3: of Genome-based analysis of Carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008-2014

Table S2. Names and sequences of used primers for MLST and PCR amplification of virulence genes and β-lactamase genes. Table S3. Identified virulence genes in K. pneumoniae isolate no. 316/15 (ST23, OXA-48). Table S4. Identified wzi alleles from NGS data of 107 carbapenemase-producing K. pneumoniae from Germany. (DOCX 35 kb

Additional file 1: of Genome-based analysis of Carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008-2014

Figure S1. Origin of the 107 German carbapenemase-producing K. pneumoniae isolates. Regions are shown, where isolates originated from. Isolates from Saxony could not be elucidated further due to the lack of additional geographic information. Number of isolates is given by the size of the circle (see legend). Image is from: © Bundesamt für Kartographie und Geodäsie, Frankfurt am Main, Germany. Figure S2. Virulence gene content in 107 carbapenemase-producing K. pneumonaie isolates from Germany. Data are given in % of isolates showing possession of the corresponding gene cluster. The graph shows four most frequent virulence genes identified in more than one single isolate. Figure S3. Detailed view of the ML tree concerning ST258/ST512 – carbapenemase-producing K. pneumoniae isolates from Germany, 2008-2014. The image shows a subtree of Fig. 3 containing 52 isolates of ST258 (light violett) and ST512 (grey). Colour codes of the inner ring designate the corresponding carbapenemase type, the outer designates the wzi allele (see legend). Figure S4. ML tree of NGS-based analysis of German K. pneumoniae isolates and isolates from an international collection - detailed view of the cluster ST258/ST512 isolates. The image shows a subtree of Fig. 4 containing 66 isolates of ST258 and ST512. Colour codes of the inner ring correspond to the origin of strains, the middle ring to the carbapenemase KPC-2 or KPC-3, and the outer ring demonstrates the wzi allele type. (PPTX 1367 kb