Search

Drinking water in hospitals is often tested for Pseudomonas aeruginosa because of its virulence potential. We here characterize a high-grade putative P. aeruginosa contamination of a hospital drinking water system suggesting a relevant risk for patients. According to EN ISO 16266, 7/11 (64%) simultaneously taken samples of one water system were reported as positive for P. aeruginosa. This resulted in extensive investigations and interventions and required a number of information and protection measures. However, supplemental analyses with more discrimination power (MALDI-TOF MS, 16S-rRNA sequencing) completely ruled out P. aeruginosa. We wish to raise awareness of this problem and propose to indicate the diagnostic uncertainty of results obtained by EN ISO 16266 on laboratory reports. Wrongly assuming the presence of P. aeruginosa in hospital water supply systems can lead to unnecessary control measures, since the described analytical uncertainty massively influences the health risk assessment and the remediation measures to be initiated, namely in medical environment.

ZusammenfassungDie SARS-CoV-2-Pandemie hat ein Defizit an essentieller infektionsepidemiologischer Infrastruktur, insbesondere in Bezug auf die Genomische Erreger-Surveillance (GES) in Deutschland, gezeigt. Zur Vorbereitung auf zukünftige pandemische Notlagen sehen es die Autor*innen als dringend erforderlich an, dieses bestehende Defizit durch den Aufbau einer leistungsfähigen Infrastruktur für GES zu beheben. Ein derartiges Netzwerk kann auf bereits regional initiierten Strukturen, Prozessen und Interaktionen aufbauen und diese weiter optimieren. Es kann zukünftig mit einer hohen Anpassungsfähigkeit auf aktuelle und kommende Herausforderungen reagieren.Ziele der vorliegenden Arbeit sind die Verdeutlichung der Dringlichkeit und Skizzierung von Vorschlägen zur Etablierung eines effizienten, anpassungsfähigen und reaktionsbereiten GES-Netzwerkes unter Berücksichtigung von externen Rahmenbedingungen und internen Standards. Die erarbeiteten Vorschläge basieren auf der Grundlage globaler und länderspezifischer Best Practices und Strategiepapiere. Zu den konkreten nächsten Schritten zur Realisierung einer integrierten GES zählen die Ermöglichung der Verknüpfung epidemiologischer Daten mit Genomdaten der Erreger, die gemeinsame und koordinierte Nutzung von vorhandenen Ressourcen, die Nutzbarmachung der so gewonnenen Surveillance-Daten für relevante Entscheidungstragende, den Öffentlichen Gesundheitsdienst und die wissenschaftliche Gemeinschaft sowie die Einbindung aller Stakeholder. Der Aufbau eines GES-Netzwerkes ist essentiell für die kontinuierliche, stabile, aktive Überwachung des Infektionsgeschehens in Deutschland sowohl während pandemischer Phasen als auch außerhalb dieser.

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant Escherichia coli isolated between 2007 and 2016 from German soldiers after deployments, with a particular focus on the African Sahel region. A total of 51 third-generation cephalosporine-resistant E. coli isolated from 51 military returnees from deployment collected during the assessment period between 2007 and 2016 were subjected to short-read next-generation sequencing analysis. Returnees from the Sahel region (Djibouti, Mali, South Sudan, Sudan, Sudan, and Uganda) comprised a proportion of 52.9% (27/51). Repeatedly isolated sequence types according to the Warwick University scheme from returnees from the Sahel region were ST38, ST131, and ST648, confirming previous epidemiological assessments from various sub-Saharan African regions. Locally prevalent resistance genes in isolates from returnees from the Sahel region associated with third-generation resistance were blaCTX-M-15, blaCTX-M-27, blaCTX-M-1, blaTEM-169, blaCTX-M-14, blaCTX-M-99-like, blaCTX-M-125, blaSHV-12, and blaDHA-1, while virulence genes were east1, sat, and tsh in declining order of frequency of occurrence each. In line with phenotypically observed high resistance rates for aminoglycosides and trimethoprim/sulfamethoxazole, multiple associated resistance genes were observed. A similar, slightly more diverse situation was recorded for the other deployment sites. In summary, this assessment provides first next-generation sequencing-based epidemiological data on third-generation cephalosporine-resistant E. coli imported by deployed German soldiers with a particular focus on deployments to the Sahel region, thus serving as a small sentinel. The detected sequence types are well in line with the results from previous epidemiological assessments in sub-Saharan Africa.

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant

Objectives To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Methods Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. Results In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described. Conclusions These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes.

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants. Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available. Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany. Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI. Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected. Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.

Objectives To reduce infections with Clostridioides difficile (CDI) in geriatric patients by interventions easily implementable in standard clinical care. Methods Prevalence and incidence of CDI between January 2015 and February 2020 were analysed (n = 25,311 patients). Pre-intervention status was assessed from April 2016 to March 2017 (n = 4,922). Between May 2017 and August 2019, a monocentric interventional crossover study (n = 4,655) was conducted including standard care and three interventions: (A) sporicidal cleaning of hospital wards, (B) probiotics and (C) improvement in personal hygiene for CDI patients. This was followed by a multicentric comparison of the interventional bundle (A + B + C) between September 2019 and February 2020 (n = 2,593) with the pre-intervention phase. In 98 CDI cases and matched controls individual risk factors for the development of CDI were compared. Results Time series analyses of CDI cases revealed a reduction in the prevalence of CDI in all three participating centres prior to the multicentric intervention phase. In the monocentric phase, no effect of individual interventions on CDI prevalence was identified. However, an aggregated analysis of CDI cases comparing the pre-intervention and the multicentric phase revealed a significant reduction in CDI prevalence. Risk factors for the development of CDI included use of antibiotics, anticoagulants, previous stay in long-term care facilities, prior hospital admissions, cardiac and renal failure, malnutrition and anaemia. Conclusions The observed reduction in CDI may be attributed to heightened awareness of the study objectives and specific staff training. Individual interventions did not appear to reduce CDI prevalence. A further randomised trial would be necessary to confirm whether the bundle of interventions is truly effective.

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. β-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.

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.

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.

Patients colonized or infected by multidrug-resistant bacteria (MDR) are entitled to the same medical treatment as other patients and infection control measures based on the current evidence are not in conflict with this aim. Recent studies indicate that the correct sampling technique is of utmost importance when screening for MRSA and usage of certain swabs might reduce the sensitivity considerably. Despite improvements in diagnostic strategies for direct MRSA detection by PCR, false positive results cannot be totally excluded. With regard to multidrug-resistant gramnegative bacteria differences between species should be emphasized to tailor infection control measures. E. g. easier transmissibility has been shown for K. pneumoniae compared to E. coli. Hospitals in Germany should strictly employ screening for carbapenemase-producing Enterobacterales as well as A. baumannii in all patients hospitalized abroad within the previous year. To avoid unnecessary social exclusion of MDR patients it must be emphasized that contact precautions are recommended for hospitals and not for long-time care facilities.

Neben Akutkliniken sind auch Rehabilitationskliniken zunehmend von der Problematik der multiresistenten Erreger betroffen. Lange Verweildauern und hohe Behandlungsintensitaten stellen dabei besondere Herausforderungen an die Hygiene dar. Systematische Untersuchung eines Ausbruchs mit „Extended Spectrum Beta-Lactamasen“-Klebsiella-pneumoniae (ESBL-K. pneumoniae) in einer Neurorehabilitationsklinik. Gesicherter Fall: Patient mit einem ESBL-K.-pneumoniae-Nachweis im Ausbruchszeitraum und Zugehorigkeit zum Ausbruchsklon, wahrscheinlicher Fall: kein typisiertes Isolat, aber epidemiologischer Link zum Ausbruchsgeschehen. Insgesamt 53 ESBL-K.-pneumoniae-Isolate wurden mittels Next Generation Sequencing (NGS) typisiert. Umgebungsuntersuchungen wurden durchgefuhrt und ein systematisches Screening wurde implementiert. Neben einer Starkung der Standardhygiene wurde eine Kohortierung von Patienten mit ESBL-K.-pneumoniae veranlasst. Insgesamt wurden 30 gesicherte und 6 wahrscheinliche Falle dem Ausbruchsgeschehen zugeordnet (Cluster 1, ST15). In der Typisierung konnten 2 weitere Cluster identifiziert werden: Cluster 2 (ST405) bei 7 Patienten und Cluster 3 (ST414) bei 8 Patienten. Bei 2 Patienten entwickelte der Ausbruchsstamm ST15 weitere Antibiotikaresistenzen (Colistin bzw. Carbapenem). ESBL-K.-pneumoniae wurde in Umgebungsuntersuchungen nicht identifiziert. Der Ausbruch konnte nach strikter Kohortierung beendet werden. Die Erregerausbreitung erfolgte vermutlich von Mensch zu Mensch. Die Ubertragungswahrscheinlichkeit stieg mit dem Grad der Interventionsintensitat, wahrend die Infektionsrate durch die Morbiditat der Patienten bedingt war. Die Identifikation eines Ausbruchsklons mit Entwicklung zusatzlicher Antibiotikaresistenzen und die Entdeckung weiterer Cluster demonstrieren die Relevanz einer Erregertypisierung und eines effektiven Ausbruchsmanagements.

Als prinzipiell gefahrdetes Kollektiv brauchen Fluchtlinge eine rasche, adaquate medizinische Versorgung. Dabei mussen vielfaltige Aspekte berucksichtigt werden, so beispielsweise Aufenthalte in Regionen mit in Westeuropa selten vorkommenden Infektionskrankheiten, oftmals andere Resistenzen sowie hohe Risiken fur psychische Belastungen durch traumatische Erlebnisse, Trennungssituation, Rechtsunsicherheit und/oder problematische Wohnverhaltnisse. Ein besonderer Fokus wird auf die Bedurfnisse schwangerer Fluchtlinge und Neugeborener gelegt. Essenziell fur eine angemessene und verlassliche Versorgung sind klar definierte Algorithmen. Exemplarisch wird die interne Regelung an einem Universitatsklinikum dargestellt.

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 Enterobacterales 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 blaNDM–1 was found to be present in 35 isolates of which seven additionally carried the carbapenemase gene blaKPC–2. Core genome MLST (cgMLST) revealed different clusters of closely related isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Morganella morganii or Enterobacter cloacae indicating clonal spread. The detailed reconstruction of the plasmid sequences revealed that in all outbreak-associated isolates blaNDM–1 was located on similar composite transposons, which were also very similar to Tn125 previously described for Acinetobacter baumannii. In contrast to Tn125, these structures were flanked by IS26 elements, which could facilitate horizontal gene transfer. Moreover, the identical plasmid was found to be shared by E. coli and M. morganii 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.

Washing machines should be further investigated as possible sites for horizontal gene transfer (ESBL genes) and cross-contamination with clinically important Gram-negative strains. Particularly in the health care sector, the knowledge of possible (re-)contamination of laundry (patients’ clothes and staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and to control nosocomial infections. This report describes an outbreak with a single strain of a multidrug-resistant bacterium (Klebsiella oxytoca sequence type 201) in a neonatal intensive care unit that was terminated only when the washing machine was removed. In addition, the study implies that changes in washing machine design and processing are required to prevent accumulation of residual water where microbial growth can occur and contaminate clothes., During the period from April 2012 to May 2013, 13 newborns (1 to 4 weeks of age) and 1 child in a pediatric hospital ward in Germany were colonized with Klebsiella oxytoca producing an extended-spectrum beta-lactamase (ESBL) (CTX-M-15). A microbiological source-tracking analysis with human and environmental samples was carried out to identify the source and transmission pathways of the K. oxytoca clone. In addition, different hygienic intervention methods were evaluated. K. oxytoca isolates were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine that was used in the same ward to wash laundry for the newborns, as well as in two sinks. These strains were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The environmental findings were compared with those for the human strains and the isolates detected on clothing. The results from both techniques showed that the strains were identical (sequence type 201 and PFGE type 00531, a clone specific to this hospital and not previously isolated in Germany), emphasizing the washing machine as a reservoir and fomite for the transmission of these multidrug-resistant bacteria. After the washing machine was taken out of use, no further colonizations were detected during the subsequent 4-year period. IMPORTANCE Washing machines should be further investigated as possible sites for horizontal gene transfer (ESBL genes) and cross-contamination with clinically important Gram-negative strains. Particularly in the health care sector, the knowledge of possible (re-)contamination of laundry (patients’ clothes and staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and to control nosocomial infections. This report describes an outbreak with a single strain of a multidrug-resistant bacterium (Klebsiella oxytoca sequence type 201) in a neonatal intensive care unit that was terminated only when the washing machine was removed. In addition, the study implies that changes in washing machine design and processing are required to prevent accumulation of residual water where microbial growth can occur and contaminate clothes.

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 Enterobacterales 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 blaNDM–1 was found to be present in 35 isolates of which seven additionally carried the carbapenemase gene blaKPC–2. Core genome MLST (cgMLST) revealed different clusters of closely related isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Morganella morganii or Enterobacter cloacae indicating clonal spread. The detailed reconstruction of the plasmid sequences revealed that in all outbreak-associated isolates blaNDM–1 was located on similar composite transposons, which were also very similar to Tn125 previously described for Acinetobacter baumannii. In contrast to Tn125, these structures were flanked by IS26 elements, which could facilitate horizontal gene transfer. Moreover, the identical plasmid was found to be shared by E. coli and M. morganii 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.

Patients colonized or infected by multidrug-resistant bacteria (MDR) are entitled to the same medical treatment as other patients and infection control measures based on the current evidence are not in conflict with this aim. Recent studies indicate that the correct sampling technique is of utmost importance when screening for MRSA and usage of certain swabs might reduce the sensitivity considerably. Despite improvements in diagnostic strategies for direct MRSA detection by PCR, false positive results cannot be totally excluded. With regard to multidrug-resistant gramnegative bacteria differences between species should be emphasized to tailor infection control measures. E. g. easier transmissibility has been shown for K. pneumoniae compared to E. coli. Hospitals in Germany should strictly employ screening for carbapenemase-producing Enterobacterales as well as A. baumannii in all patients hospitalized abroad within the previous year. To avoid unnecessary social exclusion of MDR patients it must be emphasized that contact precautions are recommended for hospitals and not for long-time care facilities.

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.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.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.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.

The distribution of carbapenemase genes in Escherichia coli strains isolated between September 2009 and May 2013 in Germany was investigated. Out of 192 isolates with carbapenemase production OXA-48 was found in 44.8%, VIM-1 in 18.8%, NDM-1 in 11.5% and KPC-2 in 6.8%. Patients with VIM-1 producing E. coli (n=36) differed from patients with OXA-48 by an older age, less frequent mention of travel history and an increased proportion of clinical over screening specimens. These data might indicate that introduction from abroad is of minor importance for VIM-1 producing E. coli compared to other carbapenemases. Multilocus sequence typing revealed that E. coli with VIM-1 were mostly multiclonal, emphasizing the role of horizontal gene transfer in its spread. Susceptibility testing of VIM-1 producing E. coli demonstrated aztreonam susceptibility in 55.6%. Among non-β-lactams susceptibility rates of >90% were observed for amikacin, tigecycline, colistin, fosfomycin and nitrofurantoin.

We characterized the molecular basis of carbapenemase production in carbapenem-resistant Gram-negative bacteria isolated from hospitalized patients from Saudi Arabia in the year 2012. Isolates were collected from across the Kingdom and phenotypically tested for carbapenemase production. Polymerase chain reaction detection of carbapenemase genes was also performed. Our results indicate that in Saudi Arabia, OXA-48 and NDM-1 are the dominant carbapenemases among Enterobacteriaceae with low prevalence of VIM. The latter is the most prevalent metallo-beta-lactamase in Pseudomonas aeruginosa, whereas oxacillinases, OXA-23 in particular, are the dominant carbapenemases in Acinetobacter baumannii. No KPC or IMP genes were detected. Our study is the first report of OXA-48, NDM-1, and VIM-4 enzymes in Enterobacter from the Kingdom. Also it is the first report of OXA-72 and NDM-1 in A. baumannii in Saudi Arabia, and the coexistence of blaOXA-23 and blaNDM-1 genes in this species in the country. Awareness of the role of international travel in the spread of carbapenem-resistant determinants in the Kingdom, as well as effective infection control interventions in hospitals and strict antimicrobial stewardship in healthcare facilities and the community are keys to combat the rise of carbapenemase producers in the Kingdom.

Isolation precautions required for neonatal intensive care units are part of a bundle with the aim to prevent transmission, colonization, and infection with multidrug-resistant gram-negative pathogens as neonates face an increased risk of mortality and morbidity in case of infection. The following short report describes a transmission of 3MDRGN Klebsiella pneumoniae on a neonatal intensive care unit in a university hospital in Germany. This transmission occurred even though intensified infection control measures were in place, which impressively shows the importance of surveillance, outbreak management, and awareness of contributing factors regarding outbreak situations.

Extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant K. pneumoniae 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 K. pneumoniae clones (ST15, n = 31; ST405, n = 7) and one CTX-M-15 producing Klebsiella quasipneumoniae strain (ST414, n = 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 K. pneumoniae outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and K. pneumoniae genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (refRank; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (SNPfilter; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.

Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Background From July 2010-April 2013, Leipzig University Hospital experienced the largest outbreak of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae (KPC-2- Kp ) strain observed in Germany to date. After termination of the outbreak, we aimed to reconstruct transmission pathways by phylogenetics based on whole-genome sequencing (WGS). Methods One hundred seventeen KPC-2- Kp isolates from 89 outbreak patients, 5 environmental KPC-2- Kp isolates, and 24 K pneumoniae strains not linked to the outbreak underwent WGS. Phylogenetic analysis was performed blinded to clinical data and based on the genomic reads. Results A patient from Greece was confirmed as the source of the outbreak. Transmission pathways for 11 out of 89 patients (12.4%) were plausibly explained by descriptive epidemiology, applying strict definitions. Five of these and an additional 15 (ie, 20 out of 89 patients [22.5%]) were confirmed by phylogenetics. The rate of phylogenetically confirmed transmissions increased significantly from 8 out of 66 (12.1% for the time period before) to 12 out of 23 patients (52.2% for the time period after; P .001) after implementation of systematic screening for KPC-2- Kp (33,623 screening investigations within 11 months). Using descriptive epidemiology, systematic screening showed no significant effect (7 out of 66 [10.6%] vs 4 out of 23 [17.4%] patients; P = .465). The phylogenetic analysis supported the assumption that a contaminated positioning pillow served as a reservoir for the persistence of KPC-2- Kp . Conclusions Effective phylogenetic identification of transmissions requires systematic microbiologic screening. Extensive screening and phylogenetic analysis based on WGS should be started as soon as possible in a bacterial outbreak situation.

Colistin is used as a last-resort antibiotic for treating infections caused by multidrug-resistant members of the family Enterobacteriaceae, in particular for carbapenem-resistant isolates. Resistance to colistin in Enterobacteriaceae species has been attributed to chromosomal mutations ([1][1])

OXA-48 is the most prevalent carbapenemase in Enterobacteriaceae in Europe and the Middle East, but it is frequently missed because many isolates display low MICs for carbapenems. Furthermore, in contrast to metallo-β-lactamases or Klebsiella pneumoniae carbapenemases (KPC), no specific inhibitor is available for the phenotypic detection of OXA-48. Molecular detection of bla OXA-48 is the “gold standard” but is not available in many laboratories. A few phenotypic assays have been described but have not been independently evaluated. The aim of this study was the systematic comparison of phenotypic tests and an immunochromatographic assay (ICT) for the detection of OXA-48/OXA-48-like carbapenemases and the development of an algorithm for reliable phenotypic detection of OXA-48. Four phenotypic tests (temocillin disk test, faropenem disk test, OXA-48 disk test, and high-inoculum [HI] OXA-48 disk test) and a new ICT (OXA-48 K -SeT) were compared by using a set of 166 Enterobacteriaceae isolates, including isolates producing OXA-48/OXA-48-like carbapenemases ( n = 84) or Ambler class A and B carbapenemases ( n = 41) and carbapenemase-negative isolates ( n = 41). The sensitivity and specificity for the different assays were 100% and 43.9% for temocillin, 57.1% and 98.8% for faropenem, 53.6% and 100% for the OXA-48 disk test, 98.8% and 97.6% for the HI OXA-48 disk test, and 100% and 100% for the ICT, respectively. The ICT displayed the highest sensitivity and specificity and was the most rapid assay, but it is more costly than phenotypic assays. Based on these results, a new algorithm incorporating temocillin, faropenem, and ICT which allows cost-effective detection of OXA-48 with 100% sensitivity and specificity was developed.

The members of the European Survey on Carbapenemase-Producing Enterobacteriaceae, (EuSCAPE) Working Group are: Portugal—Manuela Caniça and Vera Manageiro BACKGROUND: Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it difficult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the first structured survey on the occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in European hospitals. METHODS: National expert laboratories recruited hospitals with diagnostic capacities, who collected the first ten carbapenem non-susceptible clinical isolates of K pneumoniae or E coli and ten susceptible same-species comparator isolates and pertinent patient and hospital information. Isolates and data were relayed back to national expert laboratories, which made laboratory-substantiated information available for central analysis. FINDINGS: Between Nov 1, 2013, and April 30, 2014, 455 sentinel hospitals in 36 countries submitted 2703 clinical isolates (2301 [85%] K pneumoniae and 402 (15%) E coli). 850 (37%) of 2301 K pneumoniae samples and 77 (19%) of 402 E coli samples were carbapenemase (KPC, NDM, OXA-48-like, or VIM) producers. The ratio of K pneumoniae to E coli was 11:1. 1·3 patients per 10 000 hospital admissions had positive clinical specimens. Prevalence differed greatly, with the highest rates in Mediterranean and Balkan countries. Carbapenemase-producing K pneumoniae isolates showed high resistance to last-line antibiotics. INTERPRETATION: This initiative shows an encouraging commitment by all participants, and suggests that challenges in the establishment of a continent-wide enhanced sentinel surveillance for carbapenemase-producing Enterobacteriaeceae can be overcome. Strengthening infection control efforts in hospitals is crucial for controlling spread through local and national health care networks. European Centre for Disease Prevention and Control info:eu-repo/semantics/publishedVersion

Objective.To establish the source and contamination routes resulting in positive clinical and surveillance microbiological cultures with carbapenem-resistant, GIM-1 metallo-β-lactamase–positiveAcinetobacter pitiiandAcinetobacter radioresistensfrom 21 patients in 8 departmentsDesign.Retrospective, descriptive study.Setting.A 1,300-bed tertiary care academic medical facility consisting of 90 buildings linked by a pneumatic transport system (PTS).Methods.Microbiological workup of the cluster strains included matrix-assisted laser desorption/ionization time-of-flight species identification, phenotypic carbapenemase tests, polymerase chain reaction–based genotyping of carbapenemase, and pulsed-field gel electrophoresis. Outbreak management procedures were employed according to institutional regulations.Results.The rarity of GIM-1Acinetobacterspecies in the hospital and region, the lack of epidemiological links between patients, and the fact that in some patients the apparent colonization was clearly nonnosocomial prompted the suspicion of a pseudo-outbreak. Numerous environmental cultures were positive for GIM-1-positiveAcinetobacter(including archived sample requisition forms, PTS capsules, cultures from line-diverter and dispenser stations, and sterilized transport capsules following PTS delivery). Moreover, it was observed that condensation fluid from subterranean PTS tubing resulted in water entry in PTS capsules, possibly conferring specimen contamination. After extensive system disinfection, environmental surveys of the PTS were negative, and no further positive patient specimens were encountered.Conclusions.This is the first report of a PTS-associated pseudo-outbreak. The large number of falsely positive patient-related specimens in conjunction with the potential hazard of airborne and contact spread of multidrug-resistant microorganisms (in this case, GIM-1 carbapenem-resistantAcinetobacterspecies) underscores the need for implementation of infection control–based monitoring and operating procedures in a hospital PTS.

Due to the increase in multidrug-resistant Enterobacteriaceae , the interest in older antimicrobial agents, like fosfomycin, has increased. In this study, we used agar dilution for testing susceptibilities to fosfomycin in a collection of 107 carbapenem-nonsusceptible Enterobacteriaceae isolates, of which 80 produced various types of carbapenemases, including KPC, VIM, NDM, and OXA-48. Overall, 78% of the strains had fosfomycin MICs of ≤32 mg/liter and were thus considered to be susceptible according to the current EUCAST breakpoint. The MIC 50 and MIC 90 were 8 mg/liter and 512 mg/liter, respectively. Escherichia coli strains had significantly lower fosfomycin MICs than the Klebsiella pneumoniae and Enterobacter cloacae strains. Furthermore, comparisons of the susceptibility testing methods, like Etest and disk diffusion, were performed against agar dilution as the reference method. Essential agreement between Etest and agar dilution was 78.9%, and categorical agreement between the two methods was 92.5%, with 20% very major errors and 2.6% major errors. Disk diffusion was studied with 50-μg and 200-μg fosfomycin disks, but no inhibition zone breakpoint that reduced very major and major errors to an acceptable level was found. Etest and disk diffusion showed poor agreement with fosfomycin agar dilution.

Das Auftreten Carbapenem-resistenter Krankheitserreger stellt ein zunehmendes infektiologisches und krankenhaushygienisches Problem dar. Infolge eines Ausbruchs mit Klebsiella pneumoniae mit der Carbapenemase KPC-2 an einem sachsischen Klinikum initiierte das Sachsische Staatsministerium fur Soziales und Verbraucherschutz (SMS) eine Punktpravalenzerhebung zu Carbapenemase-bildenden Bakterien in Sachsen. Es wurden vorrangig Intensivstationen von 53 sachsischen Kliniken nacheinander von Oktober 2012 bis Februar 2013 in die Untersuchung einbezogen und dort Stuhlproben oder Rektalabstriche von 1037 Patienten auf das Vorhandensein von 4-fach multiresistenten gramnegativen Erregern (4MRGN) analysiert. Bei 3 Patienten [0,3 % CI95 (0,0596; 0,843)] wurden Carbapenemase-Bildner und bei 9 Patienten [0,9 % CI95 (0,397; 1,64)] Carbapenem-resistente Bakterien ohne Carbapenemasen detektiert. Daruber hinaus ergab die Resistenztestung bei 166 Patienten [16,0 % CI95 (13,82; 18,38)] ESBL (Extended-Spectrum-β-Lactamase)-Bildner. Zum Zeitpunkt der Untersuchung wurde K. pneumoniae mit der Carbapenemase KPC-2 in einer Klinik bei 2 Patienten nachgewiesen. Wachsamkeit ist aber auch im Hinblick auf andere Carbapenemase-Bildner erforderlich, wie der Nachweis von Pseudomonas aeruginosa mit der Carbapenemase VIM-1 in einer anderen Klinik zeigt.

Metallo-β-lactamases (MBLs) in Enterobacteriaceae are an increasing problem worldwide. This report describes the isolation of Citrobacter freundii carrying IMP-8 MBL from three patients during the period from March 2012 until March 2013 in Germany. The blaIMP-8 enzyme is predominantly found in Asia, where IMP-8 has spread to various enterobacterial species causing serious infections. To our best knowledge, this is the first report of blaIMP-8 habouring Enterobacteriaceae in Europe.

Zusammenfassung Im Zusammenhang mit den neuen KRINKO-Empfehlungen über Hygienemaßnahmen bei Infektionen oder Besiedlung mit multiresistenten gramnegativen Stäbchen wurde eine neue Multiresistenz-Definition, die MRGN-Klassifikation, eingeführt. Dies war nötig geworden, da bisherige Definitionen für krankenhaushygienische Zwecke wenig geeignet erschienen. Die MRGN-Klassifikation unterscheidet zwei Ausprägungsgrade der Multiresistenz, die als 3MRGN und 4MRGN bezeichnet werden und unterschiedliche hygienische Konsequenzen nach sich ziehen können. Sie beruht ferner darauf, dass Antibiotika unterschiedlich gewichtet werden und vor allem Resistenzen bei therapeutisch besonders bedeutsamen Gruppen wie Cephalosporinen, Carbapenemen und Fluorochinolonen in die Definition einfließen. Die MRGN-Klassifikation basiert weitestgehend auf Resistenzkategorien, nicht auf Resistenzmechanismen. Aufgrund der hohen epidemiologischen Bedeutung werden jedoch Carbapenemase-produzierende Enterobacteriaceae unabhängig vom restlichen Antibiogramm durchweg als 4MRGN klassifiziert. Die MRGN-Klassifikation erfordert Expertise und eine zuverlässige Carbapenemase-Detektion und ist daher ausdrücklich Aufgabe des mikrobiologischen Labors.

Objectives To characterize the mechanisms involved in the reduced carbapenem susceptibility of five Acinetobacter pittii strains isolated from different regions of Germany. Methods The strains were analysed by susceptibility testing, phenotypic tests for metallo-β-lactamase production, sequencing of the integron structure and strain typing by PFGE, as well as multilocus sequence typing (MLST) and plasmid analysis by S1 restriction and hybridization. Results Despite GIM-1 production, the MICs of imipenem were only 4 mg/L for four strains and some methods of phenotypic MBL detection failed. According to PFGE and MLST, the strains belonged to four different clones, but blaGIM-1 was present in identical integron structures in all strains and carried on plasmids of ∼60 kb. Conclusions For the first time, GIM-1 has been demonstrated in A. pittii. This resistance mechanism has previously been reported only in Enterobacteriaceae and Pseudomonas aeruginosa. As GIM-1 was found in strains with diverse clonal backgrounds, but encoded on plasmids of a similar size, further spread among Acinetobacter spp. seems possible. The detection of GIM-1 production might be challenging in some strains due to the low MICs of carbapenems.

In recent decades, Acinetobacter baumannii has emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants in A. baumannii in terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains of A. baumannii in a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants of A. baumannii in parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance.

New recommendations for infection control in the case of infection or colonization by multidrug-resistant Gram-negative bacteria were published by the Commission for Hospital Hygiene and Infection Prevention (KRINKO) at the Robert Koch Institute, Germany. In this context, a new definition for multidrug-resistance, the so-called MRGN classification, was introduced. This was necessary because previous definitions did not seem suitable for the purpose of hospital hygiene. The MRGN classification grades multidrug-resistance by severity distinguishing 3MRGN and 4MRGN, resulting in different recommendations for infection control. In addition, not all antibiotics are regarded as equally important in the classification, but only the most relevant classes, such as ureidopenicillins, extended-spectrum cephalosporins, carbapenems, and fluoroquinolones, are taken into account. The MRGN classification is mainly based on resistance categories, not on resistance mechanisms. An exception has been made for carbapenemase-producing Enterobacteriaceae, which are always classified as 4MRGN irrespective of their antibiogram due to their importance. The correct MRGN classification should only be performed by experienced microbiological laboratories and requires a reliable detection of carbapenemases.

The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens . Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the bla GIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the bla GIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control.

Objectives To characterize a novel subclass B1 metallo-β-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 β-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 β-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 .

Recently Gram-negative bacteria co-producing multiple carbapenemases have emerged in different parts of the world. We report the first isolation of an Escherichia coli strain co-producing the carbapenemases NDM-1 and OXA-232.

Background Antibiotic resistance in bacteria leads to massive health problems. Incidence of carbapenem and multidrug resistance in Gram-negative bacteria are increasing globally and turn out to be a very urgent challenge in health care. Resistant bacteria play an important clinical role during hospital outbreaks as well as in sepsis. Rapid diagnostic tests are necessary to provide immediate information for antimicrobial treatment and infection control measures. Methods Our mass spectrometry-based assay was validated with 63 carbapenemase-producing Gram-negative bacterial isolates, and 35 carbapenem-resistant Gram-negative species with no carbapenemase production. These were analyzed from solid culture media and positive blood culture vials. After 4 h of incubation the carbapenemase products were analyzed with the MALDI-TOF MS. All the isolates were genotyped for carbapenemase genes by PCR and sequencing. Results For culture isolates the concordance of hydrolysis assay to genetic results was 98 % for OXA variants, KPC, VIM, IMP, GIM, and NDM. In contrast, only 14 of 29 Acinetobacter baumannii isolates carrying the OXA and NDM genes could be identified from blood culture. However, from blood culture vials our method allowed the detection of carbapenemases in 98 % of Pseudomonas and Enterobacteriaceae isolates harboring different genes. Conclusions This MALDI-TOF MS–based assay permitted the detection of carbapenemases either from solid culture media (98–100 %) or blood culture vials (96 %) for all non-A. baumannii isolates within 4 h. In case of A. baumannii isolates the assay was highly sensitive for the detection of carbapenemases directly from solid culture media.

Summary MALDI-TOF MS-based peak differences in oxacillin-resistant Staphylococcus aureus and oxacillin-susceptible S. aureus isolates have been described previously. Unfortunately, these isolates were not isogenic with respect to their mec A gene. Ours is the first to use a SCC mec -harboring parent and a SCC mec -lacking daughter strain, with the same genetic background, to unequivocally rule out strain-specific protein peaks. We could not show differences in the peak profiles within the preset Biotyper settings used for MALDI-TOF-based identification in this pair of SCC mec -harboring parent and SCC mec -lacking daughter strains.