Prolonged carriage of OXA-244-carbapenemase-producing Escherichia coli complicates epidemiological investigations.

The rapid increase of OXA-244-producing Escherichia coli, predominantly driven by genetically clustered isolates of sequence type (ST)38, has been observed in at least nine European countries, including Germany. However, the reasons for the spread of OXA-244-producing E. coli remain unclear. Here, we aim to evaluate the possibility of prolonged carriage. We identified a total of six different patients with repeated detection of OXA-244-producing E. coli isolates, which were subjected to both short and long-read whole-genome sequencing (WGS). Besides allelic differences using core genome multilocus sequence typing (cgMLST) analyses, we obtained numbers of single-nucleotide polymorphisms (SNPs) to calculate individual base-pair substitution (BPS) rates. To assess possible re-exposure and risk factors for prolonged carriage, case interviews were conducted. The time between detections ranged from eleven months to more than three years. Initial isolates originated in three+ out of six cases from clinical samples, whereas remaining samples were from screening, mostly in the inpatient setting. As expected, cgMLST analyses showed low numbers of allelic differences between isolates of each case ranging from 1 to 4, whereas numbers of SNPs were between 2 and 99 (mean = 36), thus clearly highlighting the discrepancy between these different bacterial typing approaches. For five out of six cases, observed BPS rates suggest that patients can be colonized with OXA-244-producing E. coli, including ST38 cluster isolates, for extensively long times. Thus, we may have previously missed the epidemiological link between cases because exposure to OXA-244-producing E. coli could have occurred in a time frame, which has not been evaluated in previous investigations. Our results may help to guide future epidemiological investigations as well as to support the interpretation of genetic diversity of OXA-244-producing E. coli, particularly among ST38 cluster isolates.

Presence of hypervirulence-associated determinants in Klebsiella pneumoniae from hospitalised patients in Germany.

BACKGROUND: Klebsiella (K.) pneumoniae is a ubiquitous Gram-negative bacterium and a common coloniser of animals and humans. Today, K. pneumoniae is one of the most persistent nosocomial pathogens worldwide and poses a severe threat/burden to public health by causing urinary tract infections, pneumonia and bloodstream infections. Infections mainly affect immunocompromised individuals and hospitalised patients. In recent years, a new type of K. pneumoniae has emerged associated with community-acquired infections such as pyogenic liver abscess in otherwise healthy individuals and is therefore termed hypervirulent K. pneumoniae (hvKp). The aim of this study was the characterisation of K. pneumoniae isolates with properties of hypervirulence from Germany. METHODS: A set of 62 potentially hypervirulent K. pneumoniae isolates from human patients was compiled. Inclusion criteria were the presence of at least one determinant that has been previously associated with hypervirulence: (I) clinical manifestation, (II) a positive string test as a marker for hypermucoviscosity, and (III) presence of virulence associated genes rmpA and/or rmpA2 and/or magA. Phenotypic characterisation of the isolates included antimicrobial resistance testing by broth microdilution. Whole genome sequencing (WGS) was performed using Illumina® MiSeq/NextSeq to investigate the genetic repertoire such as multi-locus sequence types (ST), capsule types (K), further virulence associated genes and resistance genes of the collected isolates. For selected isolates long-read sequencing was applied and plasmid sequences with resistance and virulence determinants were compared. RESULTS: WGS analyses confirmed presence of several signature genes for hvKp. Among them, the most prevalent were the siderophore loci iuc and ybt and the capsule regulator genes rmpA and rmpA2. The most dominant ST among the hvKp isolates were ST395 capsule type K2 and ST395 capsule type K5; both have been described previously and were confirmed by our data as multidrug-resistant (MDR) isolates. ST23 capsule type K1 was the second most abundant ST in this study; this ST has been described as commonly associated with hypervirulence. In general, resistance to beta-lactams caused by the production of extended-spectrum beta-lactamases (ESBL) and carbapenemases was observed frequently in our isolates, confirming the threatening rise of MDR-hvKp strains. CONCLUSIONS: Our study results show that K. pneumoniae strains that carry several determinants of hypervirulence are present for many years in Germany. The detection of carbapenemase genes and hypervirulence associated genes on the same plasmid is highly problematic and requires intensified screening and molecular surveillance. However, the non-uniform definition of hvKp complicates their detection. Testing for hypermucoviscosity alone is not specific enough to identify hvKp. Thus, we suggest that the classification of hvKp should be applied to isolates that not only fulfil phenotypical criteria (severe clinical manifestations, hypermucoviscosity) but also (I) the presence of at least two virulence loci e.g. iuc and ybt, and (II) the presence of rmpA and/or rmpA2.

Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023.

BackgroundThe war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients.AimTo investigate the genomic epidemiology of New Delhi metallo-ß-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries.MethodsWhole-genome sequencing of 66 isolates sampled in 2022-2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire.ResultswgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam-avibactam. Patients with data on age (n = 41) were 19-74 years old; of 49 with information on sex, 38 were male.ConclusionXDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

Performance evaluation of the UMIC® Cefiderocol to determine MIC in Gram-negative bacteria.

BACKGROUND: Cefiderocol is a catechol-substituted cephalosporin with potent in vitro activity against carbapenem-resistant (CR) Gram-negative bacteria (GNB). Cefiderocol susceptibility testing is complex because iron concentrations need to be taken into consideration. Here, we assessed the clinical performance of Bruker's UMIC® Cefiderocol and corresponding iron-depleted CAMHB to determine MIC by broth microdilution (BMD) for clinically relevant GNB. METHODS: MICs of cefiderocol for 283 GN clinical isolates were determined by BMD using iron-depleted CAMHB. Frozen panels were used as a reference. The concentration range of cefiderocol was 0.03-32 mg/L. The isolates, with different degrees of susceptibility to cefiderocol, included Enterobacterales (n = 180), Pseudomonas aeruginosa (n = 49), Acinetobacter baumannii (n = 44) and Stenotrophomonas maltophilia (n = 10). RESULTS: The rates of categorical agreement (CA), essential agreement (EA) and bias were calculated to evaluate the performance of the UMIC® Cefiderocol, as compared with the reference method. Overall, the UMIC® Cefiderocol showed 90.8% EA (95% CI: 86.9%-93.7%) with a bias of -14.5% and a CA of 90.1% (95% CI: 86.1%-93.1%). For Enterobacterales, the UMIC® Cefiderocol showed 91.7% EA (95% CI: 86.7%-94.9%) with a bias of -25.0% and a CA of 87.8% (95% CI: 82.2%-91.8%). For non-fermenters, the UMIC® Cefiderocol showed 89.3% EA (95% CI: 81.9%-93.9%) (not significantly different from 90.0%, Student t-test) with a bias of -3.9% and a CA of 94.2% (95% CI: 87.7%-97.3%). CONCLUSIONS: UMIC® Cefiderocol is a valid method for the determination of cefiderocol MICs even if higher than expected discrepancies were observed with NDM-producing Enterobacterales, which presented in most cases MIC values close to the breakpoint.

Molecular surveillance reveals the emergence and dissemination of NDM-5-producing Escherichia coli high-risk clones in Germany, 2013 to 2019.

BackgroundCarbapenemase-producing Enterobacterales (CPE) are rapidly increasing worldwide, also in Europe. Although prevalence of CPE in Germany is comparatively low, the National Reference Centre for Multidrug-resistant Gram-negative Bacteria noted annually increasing numbers of NDM-5-producing Escherichia coli isolates.AimAs part of our ongoing surveillance programme, we characterised NDM-5-producing E. coli isolates received between 2013 and 2019 using whole genome sequencing (WGS).MethodsFrom 329 identified NDM-5-producing E. coli, 224 isolates from known geographical locations were subjected to Illumina WGS. Analyses of 222 sequenced isolates included multilocus sequence typing (MLST), core genome (cg)MLST and single-nucleotide polymorphism (SNP)-based analyses.ResultsResults of cgMLST revealed genetically distinct clusters for many of the 43 detected sequence types (ST), of which ST167, ST410, ST405 and ST361 predominated. The SNP-based phylogenetic analyses combined with geographical information identified sporadic cases of nosocomial transmission on a small spatial scale. However, we identified large clusters corresponding to clonal dissemination of ST167, ST410, ST405 and ST361 strains in consecutive years in different regions in Germany.ConclusionOccurrence of NDM-5-producing E. coli rose in Germany, which was to a large extent due to the increased prevalence of isolates belonging to the international high-risk clones ST167, ST410, ST405 and ST361. Of particular concern is the supra-regional dissemination of these epidemic clones. Available information suggest community spread of NDM-5-producing E. coli in Germany, highlighting the importance of epidemiological investigation and an integrated surveillance system in the One Health framework.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.

OBJECTIVES: Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria. METHODS: Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels. RESULTS: Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE. CONCLUSIONS: Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates.

Characterization of GMB-1, a novel metallo-ß-lactamase (MBL) found in three different Enterobacterales species.

OBJECTIVES: To identify novel carbapenem resistance mechanisms and their potential to spread among clinical isolates. METHODS: Four clinical isolates of Citrobacter freundii, Serratia marcescens and Raoultella planticola (n = 2) from one hospital in Central Germany were sent to the German National Reference Centre for Multidrug-resistant Gram-negative Bacteria for carbapenemase detection. Phenotypic tests indicated the presence of a metallo-ß-lactamase (MBL), but PCR for various MBL genes could not identify any. Using WGS data, a putative bla gene was identified. Its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain, with subsequent MIC determination by broth microdilution, as well as by in vitro hydrolysis assays using purified enzyme. RESULTS: WGS indicated the presence of a putative ß-lactamase with 48% amino acid identity to the subclass B1 MBL SPM-1. MIC studies confirmed that the novel enzyme formed a functional MBL, which was therefore designated as GMB-1 (German MBL). In vitro hydrolysis assays showed a lack of activity not only against aztreonam but also against ertapenem. WGS revealed that in all three species the blaGMB-1 gene was located on the chromosome as part of a genetic island with multiple ISs. CONCLUSIONS: The finding of GMB-1 once again shows that novel carbapenemases continue to emerge and make their way into clinically relevant species. The occurrence of GMB-1 in three different species demonstrates the extraordinary mobility of such genetic islands and their potential to spread carbapenemase genes into diverse genetic environments.

Detection of OXA-181-producing Pseudomonas aeruginosa in Germany.

OBJECTIVES: To report the detection of the class D carbapenemase OXA-181 in an MDR clinical Pseudomonas aeruginosa isolate in Germany. METHODS: Carbapenemase detection was performed by using several phenotypic tests such as the modified Hodge test, a combined disc test with boronic acid, EDTA or cloxacillin, a lysate-based inhibition assays and by PCR for common and rare carbapenemase genes. Antibiotic susceptibilities were determined by broth microdilution. The genetic environment of blaOXA-181 in the clinical P. aeruginosa isolate was characterised by Illumina and MinION sequencing. RESULTS: An multidrug-resistant P. aeruginosa was isolated from a tracheal swab in 2019 and was sent to the German National Reference Centre for multidrug-resistant Gram-negative bacteria for carbapenemase detection. Several phenotypic tests indicated the presence of a carbapenemase which was not inhibited by EDTA nor by boronic acid. PCRs for common and rare carbapenemase genes revealed the presence of a blaOXA-181 gene. WGS data confirmed that the gene was located on the chromosome as part of a Tn2013 transposon. The genetic organisation of blaOXA-181 has already been described in a P. aeruginosa isolate from England, but both isolates differed significantly in their sequence types (ST111/ST235). Analysis of the genetic environment of the blaOXA-181 gene also revealed high homology to a plasmid from a Klebsiella pneumoniae isolate. CONCLUSIONS: To our knowledge, this is the first report of blaOXA-181 in a clinical P. aeruginosa isolate in Germany which emphasises the ongoing spread of yet unusual carbapenemases among different Gram-negative species and therefore complicating their detection in routine laboratories.

Increase in NDM-1 and NDM-1/OXA-48-producing Klebsiella pneumoniae in Germany associated with the war in Ukraine, 2022.

In 2022, German surveillance systems observed rapidly increasing numbers of NDM-1- and NDM-1/OXA-48-producing Klebsiella pneumoniae, which may in part reflect recurring pre-pandemic trends. Among these cases, however, a presence in Ukraine before diagnosis was frequently reported. Whole genome sequencing of 200 isolates showed a high prevalence of sequence types ST147, ST307, ST395 and ST23, including clusters corresponding to clonal dissemination and suggesting onward transmission in Germany. Screening and isolation of patients from Ukraine may help avoid onward transmission.

Phenotypic Detection and Differentiation of Carbapenemase Classes Including OXA-48-Like Enzymes in Enterobacterales and Pseudomonas aeruginosa by a Highly Specialized Micronaut-S Microdilution Assay.

The objective of this study was to evaluate the Micronaut-S carbapenemase detection microtiter plate assay for the detection of carbapenemases and Ambler class determination. The Micronaut-S carbapenemase detection microtiter plate was tested using a challenging collection of 154 carbapenemase-producing and 150 carbapenemase-negative clinical strains of Enterobacterales and Pseudomonas aeruginosa The Micronaut-S carbapenemase detection assay was able to detect 148/154 carbapenemase producers correctly, whereas 5/150 non-carbapenemase-producing isolates tested as false positive. This resulted in an overall sensitivity of 96% and a specificity of 97%. Regarding the detection of the carbapenemase class, the sensitivities and specificities were 93%/100%, 96%/100%, and 97%/99% for class A (n = 27), class B (n = 54), and class D (n = 73) carbapenemases, respectively. The Micronaut-S carbapenemase detection microtiter plate represents an easy-to-use and valuable tool for accurate and reliable detection of carbapenemases. In addition, it provides identification of the class of carbapenemase in most cases which can provide significant therapy guidance.

GPC-1, a novel class A carbapenemase detected in a clinical Pseudomonas aeruginosa isolate.

OBJECTIVES: To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate. METHODS: A carbapenem-resistant P. aeruginosa isolate was recovered from a tracheal swab from a patient of a general ward in central Germany. Various phenotypic tests confirmed production of a carbapenemase that could not be identified further by PCR. A novel bla gene was identified by WGS and its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain. Kinetic parameters of the novel ß-lactamase were determined by spectrophotometric measurements using purified enzyme. RESULTS: WGS confirmed the presence of a novel class A carbapenemase. The novel bla gene was named GPC-1 (GPC standing for German Pseudomonas Carbapenemase) and exhibited 77% amino acid identity to BKC-1. WGS also showed that blaGPC-1 was located on the chromosome surrounded by multiple ISs as part of a 26 kb genetic island. Heterologous expression of GPC-1 in E. coli TOP10 led to increased MICs of penicillins, oxyimino-cephalosporins, aztreonam and imipenem, but not of meropenem or ertapenem. Spectrophotometric measurements supported the MIC studies, but detected a slight hydrolysis of ertapenem and meropenem when using high concentrations of purified enzyme. CONCLUSIONS: The biochemical characterization of GPC-1 emphasizes the ongoing emergence of novel carbapenemases. Strains expressing a weak carbapenemase like GPC-1 might go unrecognized by routine diagnostics due to low MICs for the bacterial strains producing such enzymes.

Rapid spread of OXA-244-producing Escherichia coli ST38 in Germany: insights from an integrated molecular surveillance approach; 2017 to January 2020.

Annually, increasing numbers of OXA-244-producing Escherichia coli in 13 German federal states prompted us to initiate an outbreak investigation. Whole genome sequencing revealed that among 148 isolates analysed, most belonged to sequence type 38 with 62 isolates forming a genetically distinct cluster. Although no epidemiological link could be identified between cases, ongoing investigations suggest non-healthcare associated transmission. A screening-PCR was developed facilitating early detection of ST38 cluster isolates to identify the source and transmission route.

Rapid and Easy Detection of Carbapenemases in Enterobacterales in the Routine Laboratory Using the New GenePOC Carba/Revogene Carba C Assay.

The novel, real-time PCR-based GenePOC Carba assay on the microfluidic revogene platform (GenePOC, Québec, Canada; now Meridian Bioscience, Cincinnati, OH, USA) was recently designed for the detection of blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP The goals of this study were to evaluate the performance of this assay, to assess its suitability for the routine microbiology laboratory, and to compare it to the Xpert Carba-R assay for the detection of carbapenemase-producing Enterobacterales (CPE) strains. The Xpert Carba-R assay (Cepheid) and the GenePOC Carba assay were challenged with a collection of 176 clinical Enterobacterales isolates. The collection included 133 CPE strains producing a total of 139 carbapenemases, including VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), and IMP (n = 9). Six isolates produced two different carbapenemases, and 43 carbapenemase-negative isolates were included as negative controls. The overall sensitivity for carbapenemase detection was 96.4% (95% confidence interval [CI], 91.9% to 98.5%) for the Xpert Carba-R assay and 100% (95% CI, 97.3% to 100%) for the GenePOC assay. The four most common carbapenemases (NDM, KPC, OXA-48-like, and VIM) were detected with a sensitivity of 100% (95% CI, 97.1% to 100%) by the two tests, with all double carbapenemase producers being correctly detected by both assays. The sensitivity of the Xpert Carba-R assay for IMP was 44.4% (95% CI, 18.9% to 73.3%), while that of the GenePOC assay was 100% (95% CI, 70.1% to 100%). The specificity of both assays was 100% (95% CI, 91.8% to 100%). The GenePOC Carba assay showed excellent sensitivity and specificity for the five most common carbapenemases, including IMP variants. Its simplicity and short turnaround time make it suitable for use in the routine microbiology laboratory for CPE detection.

Genetic and biochemical characterization of FRI-3, a novel variant of the Ambler class A carbapenemase FRI-1.

OBJECTIVES: To characterize a new variant of the FRI class A carbapenemase isolated from an MDR clinical Enterobacter cloacae isolate. METHODS: A carbapenem-resistant E. cloacae was isolated from a rectal swab from a patient in an ICU in Southern Germany. Various phenotypic tests confirmed production of a putative class A carbapenemase. The new bla gene variant, blaFRI-3, and its genetic environment were characterized by WGS. Biochemical characterization was performed by heterologous expression in Escherichia coli TOP10 and by purification of the enzyme with subsequent determination of its kinetic parameters. RESULTS: PCR and sequencing carried out for different class A carbapenemase genes confirmed the presence of a novel variant of blaFRI-1. The novel variant was named FRI-3 and exhibited 91%, 96% and 92% amino acid identity to FRI-1, FRI-2 and FRI-4, respectively. E. coli TOP10 expressing blaFRI-3 showed increased resistance to almost all ß-lactams. Comparing the catalytic behaviour of FRI-3 and FRI-1, it was shown that FRI-3 had the same substrate spectrum, but basically hydrolysed ß-lactams less efficiently than FRI-1. WGS data revealed that blaFRI-3 was located on a 111 kb plasmid. CONCLUSIONS: The biochemical characterization of FRI-3 illustrates that even a few differences in the amino acid sequence can lead to altered catalytic activities of ß-lactamases belonging to the same family.

Characterization of mutations in Escherichia coli PBP2 leading to increased carbapenem MICs.

OBJECTIVES: To examine the impact on carbapenem resistance of mutations in Escherichia coli PBP2 detected in clinical isolates showing increased MICs of imipenem, but not of meropenem. METHODS: The mutations in the PBP2-encoding gene mrdA were introduced into E. coli DH5α using the helper plasmid pTKRED. ß-Lactam MICs were determined by broth microdilution and interpreted according to EUCAST. Mutants were screened for secondary mutations by WGS. To detect a possible fitness cost related to these mutations, the generation times of the mutants and E. coli DH5α were measured and their cell morphology was examined. RESULTS: The 10 mutation patterns introduced into mrdA increased the MICs of imipenem and doripenem in all cases and of meropenem and mecillinam in some cases, but had no effect on ertapenem resistance. While no significant alteration of the generation time of the mutants could be detected, several mutants showed increased transverse diameters and thus altered cell morphology. CONCLUSIONS: Here we describe mutation patterns in PBP2 that contribute to increased MICs of carbapenems for clinical isolates of E. coli. We show that different mutations affect carbapenem MICs differently and that the mutations do not impact generation time, although some mutations affect cell morphology.

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

From June to October 2019, 17 patients (six infected, 11 colonised) with an extensively drug-resistant (XDR) Klebsiella pneumoniae strain were notified from four Western Pomerania medical facilities. The XDR K. pneumoniae 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 K. pneumoniae sequence type 307. Control measures and further investigations are ongoing.

LMB-1, a novel family of class B3 MBLs from an isolate of Enterobacter cloacae.

Objectives: To identify and characterize a novel MBL gene conferring carbapenem resistance to an isolate of Enterobacter cloacae from Austria. Methods: The novel MBL gene was heterologously expressed in Escherichia coli TOP10 to conduct comparative MIC studies and biochemical assays. Furthermore, WGS was performed using Illumina MiSeq and Oxford Nanopore MinION instruments to analyse the genetic environment of the novel MBL gene. Results: The novel MBL showed highest sequence homology to a predicted MBL precursor from the marine bacterium Rheinheimera pacifica and hence belongs to Ambler subgroup B3. The comparative MIC studies and biochemical assays showed activity of the novel enzyme against penicillins, cephalosporins and carbapenems, but not against aztreonam. It was named Linz MBL (LMB-1). The blaLMB-1 gene was shown to be located on a 108 kb plasmid of Inc type IncFIB(K). Of note, a gene adjacent to blaLMB-1 coded for a glycerophosphoryl diester phosphodiesterase that was also previously detected in R. pacifica. Conclusions: Homologies of the MBL gene itself and another gene located on the same plasmid to genes detected in marine bacterial species strongly suggest that this novel MBL was transferred to E. cloacae from a marine bacterium. This underlines the importance of natural reservoirs supplying hitherto unknown resistance genes to clinically relevant bacterial species and the importance of ongoing surveillance and research.

KPC-2 carbapenemase-producing Pseudomonas aeruginosa reaching Germany.

Background: Antimicrobial resistance due to carbapenemase expression poses a worldwide threat in healthcare. Inter-genus exchange of genetic information is of utmost importance in this context. Objectives: Here, to the best of our knowledge, we describe the first detection and characterization of a KPC-2-producing Pseudomonas aeruginosa in Germany. Methods: Characterization of the isolate was performed using MALDI-TOF MS, automated microdilution and MLST. Carbapenemase detection was performed using phenotypic and genotypic assays. The blaKPC-2-carrying plasmid was transformed into Escherichia coli NEB® 10-beta. The purified plasmid DNA was sequenced using the Illumina technique. Results: The isolate expressed ST235 and was resistant to carbapenems. Antimicrobial susceptibility testing revealed colistin to be the only antimicrobial agent active in vitro. The blaKPC-2 gene was located on a replicon type lncHI1 plasmid as part of Tn4401. Conclusions: The first detection (to the best of our knowledge) of plasmid-encoded KPC-2 in P. aeruginosa in Germany may point to a currently underestimated spread of carbapenemases among clinically relevant Gram-negative bacteria. Here, to the best of our knowledge, we also provide the first report of blaKPC-2 associated with the IncHI1 plasmid.

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-ß-lactamase found in a Pseudomonas aeruginosa clinical isolate.

Objectives: To characterize a novel subclass B1 metallo-ß-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. Methods: The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . Results: P. aeruginosa NRZ-03096 was resistant to all tested ß-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to ß-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. Conclusions: The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .