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The SARS-CoV-2 pandemic has highlighted the importance of viable infection surveillance and the relevant infrastructure. From a German perspective, an integral part of this infrastructure, genomic pathogen sequencing, was at best fragmentary and stretched to its limits due to the lack or inefficient use of equipment, human resources, data management and coordination. The experience in other countries has shown that the rate of sequenced positive samples and linkage of genomic and epidemiological data (person, place, time) represent important factors for a successful application of genomic pathogen surveillance. Planning, establishing and consistently supporting adequate structures for genomic pathogen surveillance will be crucial to identify and combat future pandemics as well as other challenges in infectious diseases such as multi-drug resistant bacteria and healthcare-associated infections. Therefore, the authors propose a multifaceted and coordinated process for the definition of procedural, legal and technical standards for comprehensive genomic pathogen surveillance in Germany, covering the areas of genomic sequencing, data collection and data linkage, as well as target pathogens. A comparative analysis of the structures established in Germany and in other countries is applied. This proposal aims to better tackle epi- and pandemics to come and take action from the "lessons learned" from the SARS-CoV-2 pandemic. [ABSTRACT FROM AUTHOR]

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. bla GIM-1 and bla VIM-2 were plasmid-encoded, bla VIM-4 was chromosomally encoded, while bla NDM-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. bla NDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the bla VIM-2- and bla GIM-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. [ABSTRACT FROM AUTHOR]

Objectives: Characterization of Proteus mirabilis isolates harbouring bla OXA-58 with emphasis on the genetic environment of this resistance determinant.Methods: Strains of P. mirabilis ( n  =   37) isolated from different patients were tested for the presence of bla OXA-58 . The genetic context of bla OXA-58 was determined by WGS of two strains and Sanger sequencing. Clonality of the strains was assessed by PFGE. Susceptibility testing was performed by microdilution according to EUCAST.Results: Four strains isolated in different geographical regions of Germany were positive for bla OXA-58 , and WGS showed that this resistance gene was harboured on a plasmid. Sanger sequencing confirmed the presence of two nearly identical plasmids, 6219 and 6208 bp in size, in all four strains. Upstream of bla OXA-58 an IS Aba 3-like transposase gene was located. The P. mirabilis strains were not clonally related according to PFGE. MICs of meropenem for three of the strains were only just above the EUCAST breakpoint and the Carba NP test was positive for only two of the strains.Conclusions: To our knowledge, this is the first description of bla OXA-58 in the species P. mirabilis . The resistance gene is harboured by almost identical plasmids in strains not clonally related and from different geographical regions. Apart from an IS Aba 3-like transposase gene upstream of bla OXA-58 the genetic context is different from bla OXA-58 harboured on plasmids in the genus Acinetobacter . With MICs of meropenem well below the EUCAST breakpoint or only just above it and equivocal or false negative results from the Carba NP test, bla OXA-58 can be easily overlooked in P. mirabilis . [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

Aim of this study was to determine the incidence and molecular epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Germany. E. coli and K. pneumoniae isolates from clinical samples which were non-susceptible to carbapenems were collected in laboratories serving 20 hospitals throughout Germany from November 2013 to April 2014. The isolates were tested for the presence of carbapenemases by PCR and phenotypic methods and typed by multilocus sequence typing. Risk factors including a previous hospitalization abroad were analysed. Carbapenemases were detected in 24 isolates from 22 patients out of 464,514 admissions. Carbapenemases included OXA‐48 (n = 14), KPC‐2 (n = 8) and NDM‐1 (n = 2). Except for two K. pneumoniae isolates with ST101, all OXA‐48 producing strains belonged to different clones. In contrast, half of KPC‐2 producing K. pneumoniae were of ST258 and both NDM‐1 producing strains were of ST11. Compared to carbapenem-susceptible controls, patients with carbapenemase-producing strains differed by a significantly higher proportion of males, a higher proportion of isolates from wound samples and a more frequent previous stay abroad in univariate analysis. This multicentre study demonstrated an incidence of carbapenemase-producing E. coli and K. pneumoniae from clinical samples in Germany of 0.047 cases per 1000 admissions. OXA‐48 was more frequent than KPC‐2 and NDM‐1 and showed a multiclonal background. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli is a rare cause of community-acquired meningitis in adults unless predisposing factors are present (e.g., previous penetrating cranio-cerebral injury or neurosurgery, immunosuppression, chronic alcoholism, history of cancer, diabetes mellitus, advanced age).Case Presentation: We describe the case of a 53-year-old woman, resident in Germany, suffering from community-acquired bacterial meningitis caused by CTX-M-9 type extended spectrum β-lactamase producing Escherichia coli. Because typical predisposing factors were not apparent, pathogen identification resulted in expanded diagnostics to exclude a distant or contiguous primary focus. By magnetic resonance tomography, a previously unrecognized large retropharyngeal abscess with cervical spondylodiscitis was detected. In retrospect, the patient had complained about neck pain for a few weeks prior to meningitis onset, but the symptoms were interpreted as being related to a herniated disk. Meningitis and osteomyelitis resolved completely under surgical treatment and meropenem therapy.Conclusion: In case of adult Escherichia coli meningitis, underlying diseases should always be carefully excluded, especially if predisposing factors are not apparent. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Urinary tract infections (UTIs) are among the most common types of bacterial infection in outpatient medicine. Rising rates of antibiotic resistance and a better understanding of the ecological adverse effects (collateral damage) of antibiotics warrant a reevaluation of the treatment recommendations for uncomplicated UTI. The new S3 guideline contains updated recommendations. Methods: The new S3 guideline is based on a review of publications on un -complicated UTI retrieved by a systematic search of the Medline and Cochrane Library databases. Guidelines from abroad were also considered in the review. Results: Uncomplicated UTI is classified as either uncomplicated cystitis (UC) or uncomplicated pyelonephritis (UP). The choice of a suitable antibiotic is determined by the following main criteria: the patient's individual risk profile and prior antibiotic treatment, if any; the spectrum of pathogens and antibiotic susceptibility; the proven efficacy of the antibiotic; the ecological adverse effects (collateral damage) of antimicrobial therapy; the side effects for the patient under treatment. On the basis of these criteria, co-trimoxazole/trimethoprim and fluoroquinolones can no longer be recommended as first-line empirical treatment for UC. Rather, the new recommended treatment of first choice consists of fosfomycin-trometamol, nitrofurantoin, or pivmecillinam. High-dose flu-oroquinolones are still recommended, however, as first-line oral treatment for U P. Asymptomatic bacteriuria should only be treated in exceptional situations such as pregnancy or before urological procedures that will probably inure the mucosa of the urinary tract. Conclusion: The new S3 guideline on uncomplicated UTI incorporates a forward- looking approach to the use of antibiotics in treating this common type of infection. It is intended to bring about a sustained improvement in the quality of care. [ABSTRACT FROM AUTHOR]

During the time period from April 2012 to May 2013, 13 newborns (1 - 4 weeks old) and one child in a paediatric hospital ward in Germany were colonized with an extended spectrum betalactamase (ESBL) producing Klebsiella oxytoca (CTX-M-15). A microbiological source-tracking analysis, including 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. Isolates of the K. oxytoca strains were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine, used in the same ward to wash laundry of the newborns as well as in two sinks. These strains were typed using PFGE and multi-locus sequence typing (MLST). The environmental findings were compared with those of the human strains and the isolates detected on the clothing. The results showed that the strains were identical using both techniques, (ST201 and pulse-field-gel-electrophoresis [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 (MDR). After the washing machine had been taken out of use, no further colonizations were detected over a following 4-year period. [ABSTRACT FROM AUTHOR]

Objective: The number of hospitalized patients with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) does not differentiate between patients admitted due to coronavirus disease 2019 (COVID-19) (ie, primary cases) and incidental SARS-CoV-2 infection (ie, incidental cases). We developed an adaptable method to distinguish primary cases from incidental cases upon hospital admission., Design: Retrospective cohort study., Setting: Data were obtained from 3 German tertiary-care hospitals., Patients: The study included patients of all ages who tested positive for SARS-CoV-2 by a standard quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay upon admission between January and June 2022., Methods: We present 2 distinct models: (1) a point-of-care model that can be used shortly after admission based on a limited range of parameters and (2) a more extended point-of-care model based on parameters that are available within the first 24-48 hours after admission. We used regression and tree-based classification models with internal and external validation., Results: In total, 1,150 patients were included (mean age, 49.5±28.5 years; 46% female; 40% primary cases). Both point-of-care models showed good discrimination with area under the curve (AUC) values of 0.80 and 0.87, respectively. As main predictors, we used admission diagnosis codes (ICD-10-GM), ward of admission, and for the extended model, we included viral load, need for oxygen, leucocyte count, and C-reactive protein., Conclusions: We propose 2 predictive algorithms based on routine clinical data that differentiate primary COVID-19 from incidental SARS-CoV-2 infection. These algorithms can provide a precise surveillance tool that can contribute to pandemic preparedness. They can easily be modified to be used in future pandemic, epidemic, and endemic situations all over the world.

The SARS-CoV‑2 pandemic has shown a deficit of essential epidemiological infrastructure, especially with regard to genomic pathogen surveillance in Germany. In order to prepare for future pandemics, the authors consider it urgently necessary to remedy this existing deficit by establishing an efficient infrastructure for genomic pathogen surveillance. Such a network can build on structures, processes, and interactions that have already been initiated regionally and further optimize them. It will be able to respond to current and future challenges with a high degree of adaptability.The aim of this paper is to address the urgency and to outline proposed measures for establishing an efficient, adaptable, and responsive genomic pathogen surveillance network, taking into account external framework conditions and internal standards. The proposed measures are based on global and country-specific best practices and strategy papers. Specific next steps to achieve an integrated genomic pathogen surveillance include linking epidemiological data with pathogen genomic data; sharing and coordinating existing resources; making surveillance data available to relevant decision-makers, the public health service, and the scientific community; and engaging all stakeholders. The establishment of a genomic pathogen surveillance network is essential for the continuous, stable, active surveillance of the infection situation in Germany, both during pandemic phases and beyond., (© 2023. The Author(s).)

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 , IS Aba1-bla OXA-51 , bla IMI , bla FIM and bla DIM 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.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 , IS Aba1-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.

Background: In August 2015, 17 neonates with Enterobacter cloacae (E. cloacae) colonization were identified in a neonatal intensive care unit (NICU) in Germany. Two developed severe brain abscesses. Despite temporary NICU closure in September, another infant with E. cloacae colonization was detected in October 2015., Methods: We defined potential cases as inpatients treated in the NICU or any pediatric/maternity ward in 2015 with E. cloacae in any specimen before molecular typing. Cases were at first confirmed by arbitrarily-primed-polymerase-chain-reaction and later by XbaI-macrorestriction/pulsed-field gel electrophoresis and next-generation-sequencing. Enhanced barrier precautions and cohorting were implemented for all potential cases and microbiologic screening was extended from NICU to all pediatric/maternity wards., Results: Of 41 potential cases (occurring between 08/04/2015 and 15/11/2015 in 4 wards), the isolates of 23 shared identical arbitrarily-primed-polymerase-chain-reaction patterns; 3 without plausible epidemiologic link. Pulsed-field gel electrophoresis analyses verified only 10 cases (all in the NICU); next-generation-sequencing analysis confirmed these results. In addition 6 cases without isolates available for genotyping were closely linked in place and time., Conclusions: Forty-one suspected patients were cohorted and the NICU was temporarily closed. Further analyses revealed that only 16 cases belonged to the outbreak. Only close interdisciplinary collaboration and highly discriminatory genotyping methods allowed to clearly differentiate between cases and noncases in this E. cloacae outbreak.

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 bla OXA-48 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.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., Competing Interests: Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: In addition to acute care hospitals, rehabilitation centres are increasingly confronted with multi-resistant pathogens. Long durations of stay and intensive treatments impose special hygienic challenges., Material and Methods: We investigated an extended spectrum beta-lactamase-Klebsiella pneumoniae (ESBL-K. pneumoniae) outbreak in a neurorehabilitation centre. We defined confirmed cases as patients who stayed in the centre during the outbreak period and from whom ESBL-K. pneumoniae was isolated with the outbreak sequence type. Probable cases had an epidemiological link to at least one confirmed case but no isolate for typing. Next generation sequencing (NGS) was performed on 53 isolates from patients. Environmental sampling was performed. Systematic microbiological screening was implemented and ESBL-K. pneumoniae-positive patients were cohorted in a designated ward., Results: We identified 30 confirmed and 6 probable cases. NGS revealed three genetic clusters: Cluster 1 - the outbreak cluster - with isolates of 30 cases (sequence type ST15), Cluster 2 with 7 patients (ST405) and Cluster 3 with 8 patients (ST414). In two patients, the outbreak strain developed further antibiotic resistance, one with colistin resistance and the other carbapenem resistance. The outbreak ceased after strict isolation measures., Discussion: Epidemiology and NGS results paired with the effectiveness of cohorting suggest that transmission occurred mainly from person to person in this outbreak. There was an apparent association of the probability to acquire ESBL-K. pneumoniae and treatment intensity, whereas infection rate was related to morbidity. The identification of the outbreak clone and additional clusters plus the development of additional antibiotic resistance shows the relevance of NGS and highlights the need for timely and efficient outbreak management.

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., (Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: The objective of this study was to characterize a novel IMP-type metallo-β-lactamase (MBL) found in an MDR clinical isolate of Pseudomonas aeruginosa., Methods: The P. aeruginosa isolate NRZ-00156 was recovered from an inguinal swab from a patient hospitalized in Western Germany and showed high MICs of carbapenems. MBL production was analysed by Etest for MBLs, an EDTA combined disc test and an EDTA bioassay. Typing of the isolate was performed by MLST. Genetic characterization of the new blaIMP gene was performed by sequencing the PCR products. A phylogenetic tree was constructed. The novel blaIMP gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization., Results: The P. aeruginosa isolate NRZ-00156 expressed the ST235 allelic profile and was resistant to all the β-lactams tested except aztreonam. The isolate was positive for MBL production and harboured a new IMP allele, blaIMP-31, located on a disrupted class I integron [also carrying the blaOXA-35, aac(6')-Ib, aac(3)-Ic and aphA15 genes]. Its closest relative was IMP-35, with 96.7% amino acid identity. Expression of blaIMP-31 demonstrated that E. coli TOP10 producing IMP-31 had elevated resistance to all the β-lactams tested except aztreonam. Kinetic data were obtained for both IMP-31 and IMP-1. In comparison with IMP-1, IMP-31 showed weaker hydrolytic activity against all the β-lactams tested, which resulted from lower kcat values., Conclusions: The characterization of the new IMP-type gene blaIMP-31 from an ST235 P. aeruginosa isolate indicates an ongoing spread of highly divergent IMP-type carbapenemases in clinical P. aeruginosa strains and highlights the continuous need for the prevention of nosocomial infections caused by MDR Gram-negative bacteria., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: We aimed to retrospectively reconstruct the timing of transmission events and pathways in order to understand why extensive preventive measures and investigations were not sufficient to prevent new cases., Methods: We extracted available information from patient charts to describe cases and to compare them to the normal population of the ward. We conducted a cohort study to identify risk factors for pathogen acquisition. We sequenced the available isolates to determine the phylogenetic relatedness of Klebsiella pneumoniae isolates on the basis of their genome sequences., Results: The investigation comprises 37 cases and the 10 cases with ESBL (extended-spectrum beta-lactamase)-producing K. pneumoniae bloodstream infection. Descriptive epidemiology indicated that a continuous transmission from person to person was most likely. Results from the cohort study showed that 'frequent manipulation' (a proxy for increased exposure to medical procedures) was significantly associated with being a case (RR 1.44, 95% CI 1.02 to 2.19). Genome sequences revealed that all 48 bacterial isolates available for sequencing from 31 cases were closely related (maximum genetic distance, 12 single nucleotide polymorphisms). Based on our calculation of evolutionary rate and sequence diversity, we estimate that the outbreak strain was endemic since 2008., Conclusions: Epidemiological and phylogenetic analyses consistently indicated that there were additional, undiscovered cases prior to the onset of microbiological screening and that the spread of the pathogen remained undetected over several years, driven predominantly by person-to-person transmission. Whole-genome sequencing provided valuable information on the onset, course and size of the outbreak, and on possible ways of transmission., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: To establish the source and contamination routes resulting in positive clinical and surveillance microbiological cultures with carbapenem-resistant, GIM-1 metallo-β-lactamase-positive Acinetobacter pitii and Acinetobacter radioresistens from 21 patients in 8 departments., Design: 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-1 Acinetobacter species 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-positive Acinetobacter (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-resistant Acinetobacter species) 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 MIC50 and MIC90 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Objectives: To characterize the mechanisms involved in reduced susceptibility to carbapenems in two Enterobacter cloacae clinical isolates., Methods: Two E. cloacae isolates recovered from different regions in Germany and showing reduced susceptibility to carbapenems were analysed. Susceptibility testing, conjugation, transformation assays, plasmid analysis, sequencing and molecular typing using rep-PCR were performed., Results: The two clinical isolates carried the bla(GIM-1) gene and showed resistance to ertapenem, with variable MIC values of imipenem and meropenem. The isolates were clonally unrelated. The bla(GIM-1) gene was located on self-transferable and non-typeable plasmids. Both isolates harboured distinct plasmids and integron structures containing the bla(GIM-1) gene cassette. Interestingly, one of the two plasmids was able to replicate in Pseudomonas aeruginosa, demonstrating its broad host range., Conclusions: This is the first identification in E. cloacae of the bla(GIM-1) gene, which is responsible for reduced susceptibility to carbapenems. We showed that this gene, previously identified in P. aeruginosa, was located in a different genetic background in E. cloacae. The bla(GIM-1) gene might spread quite efficiently in Enterobacteriaceae and P. aeruginosa, as it is difficult to detect and in addition is located on conjugative plasmids.

Although previous studies investigating the MALDI Biotyper database (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification) have proven its high accuracy for bacterial identification, the studies differed in sample preparation, number of replicates, quantity of shots and target types used. In particular, the score cut-off values of special importance for reliable species identification varied. The aim of the present study was to identify species-specific differences in the mean score values for staphylococci. Cut-off values recommended by the manufacturer were adapted using the 20th percentile to rule out unknown score-modifying factors, even though the specificity was high and the lowest cut-off values would also yield an accurate result. Whilst correct species diagnosis was obtained in 97.32 % of samples (1382/1420), only 220 of all duplicates (15.49 %) revealed a score of ≥2.3, whilst 968 (68.17 %) had a score between 2.0 and 2.299, and 194 (13.66 %) had a score of <2.0. Ten of 21 species had a calculated 20th percentile of <2.0 and one species of <1.7. In conclusion, the use of species-specific cut-off values improves the relative sensitivity of species identification in staphylococci.

Objectives: To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital., Methods: PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate., Results: Sequencing of the PCR product obtained using primers for bla(NDM-1) revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and bla(NDM-2) was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103., Conclusions: This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1.