A global view on carbapenem-resistant Acinetobacter baumannii.

To give an update on the molecular epidemiology and global distribution of carbapenemase encoding genes, we subjected 313 carbapenem-resistant Acinetobacter baumannii isolated from 114 study centers in 47 countries in five world regions, Africa, Asia, Europe, Latin America, and North America, to whole genome sequencing. Numbers of isolates investigated were proportional to the population size of the contributing countries. Molecular epidemiology was investigated using seven-loci and core genome multilocus sequence typing, whole-genome single nucleotide polymorphism phylogenies, and the intrinsic blaOXA-51-like variant. Carbapenemase encoding genes were identified by multiplex PCR and ResFinder. Among the total of 313 isolates, 289 (92.3%) were assigned to A. baumannii international clones (IC) IC1-IC8. IC2 predominated with 196 isolates (62.6%) and was spread worldwide, followed by IC5 with 44 isolates (14.1%) mainly confined to Latin America. Six isolates (1.9%) originating from Belgium, Egypt, Italy, and Pakistan represent the novel IC9. Acquired OXA-type carbapenemase genes were found in 300 (96%) isolates with blaOXA-23-like and blaOXA-40-like predominating, which constitutes a significant increase compared to our findings from 2010. Metallo-beta-lactamases were rare with seven isolates (2.2%). The distribution of ICs and carbapenemase determinants can vary widely among different geographical regions. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii are of increasing public health importance, as they are resistant to last-line antibiotics. International clones with well-characterized resistance genes dominate globally; however, locally, other lineages with different properties may be of importance to consider. This study investigated isolates from a broad geographic origin from 114 hospitals in 47 countries and from five world regions ensuring the greatest possible diversity in an organism known for its propensity for clonal epidemic spread and reflecting the current global epidemiology of carbapenem-resistant A. baumannii. In Latin America, a lineage different from other geographic regions circulates, with a different resistance gene profile. This knowledge is important to adjust local infection prevention measures. In a global world with migration and increasing use of antimicrobials, multidrug-resistant bacteria will continue to adapt and challenge our healthcare systems worldwide.

Three Klebsiella pneumoniae lineages causing bloodstream infections variably dominated within a Greek hospital over a 15 year period.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a major clinical and public health threat. The rapid dissemination of this pathogen is driven by several successful clones worldwide. We aimed to investigate the CRKP clonal lineages, their antibiotic resistance determinants and their potential transmissions in a tertiary care hospital located in Athens, Greece. Between 2003 and 2018, 392 CRKP isolates from bloodstream infections were recovered from hospitalized patients. Whole genome sequencing (WGS) was performed on the Illumina platform to characterize 209 of these isolates. In total, 74 % (n=155) of 209 isolates belonged to three major clonal lineages: ST258 (n=108), ST147 (n=29) and ST11 (n=18). Acquired carbapenemase genes were the mechanisms of resistance in 205 isolates (bla KPC, n=123; bla VIM, n=56; bla NDM, n=20; bla OXA-48, n=6). Strong associations (P=0.0004) were observed between carbapenemase genes and clonal lineages. We first isolated bla VIM-1-carrying ST147 strains during the early sampling period in 2003, followed by the emergence of bla KPC-2-carrying ST258 in 2006 and bla NDM-1-carrying ST11 in 2013. Analysis of genetic distances between the isolates revealed six potential transmission events. When contextualizing the current collection with published data, ST147 reflected the global diversity, ST258 clustered with isolates representing the first introduction into Europe and ST11 formed a distinct geographically restricted lineage indicative of local spread. This study demonstrates the changing profile of bloodstream CRKP in a tertiary care hospital over a 15 year period and underlines the need for continued genomic surveys to develop strategies to contain further dissemination. This article contains data hosted by Microreact.

Clinical and molecular features of NDM-producing Acinetobacter baumannii in a multicenter study in Israel.

BACKGROUND: NDM-producing Acinetobacter baumannii (NDMAb) were reported sporadically worldwide but little is known about the transmission, epidemiology and clinical features of NDMAb-infected patients. The goals of this study were to characterize (1) the epidemiology and clinical features of NDMAb-infected patients; (2) the microbiological and molecular features of NDMAb isolates and (3) the transmission networks of NDMAb within healthcare facilities. METHODS: The study was conducted at the Tel-Aviv Sourasky, Rambam and Sha'are-Zedek Medical centers (TASMC, RMC and SZMC, respectively) in Israel. All cases detected between January 2018 and July 2019 were included. Phylogenetic analysis was based on core genome SNP distances. Clonal transmission was defined according to molecular (≤ 5 SNP) and epidemiological criteria (overlapping hospital stay). NDMAb cases were compared at a ratio of 1:2 with non-NDM carbapenem-resistant A. baumannii (CRAb) cases. RESULTS: The study included 54 NDMAb-positive out of 857 CRAb patients, including 6/179 (3.3%) in TASMC, 18/441 (4.0%) in SZMC and 30/237 (12.6%) in RMC. Patients infected by NDMAb had similar clinical features and risk factors as patients with non-NDM CRAb. The length-of-stay was higher in NDMAb cases (48.5 days vs. 36 days, respectively, p = 0.097) and the in-hospital mortality was similarly high in both groups. Most isolates (41/54, 76%) were first detected from surveillance culture. The majority of isolates harbored the blaNDM-2 gene allele (n = 33), followed by the blaNDM-1 (n = 20) allele and the blaNDM-4 allele (n = 1). The majority of isolates were related within the ST level to other isolates in SZMC and RMC: 17/18 and 27/30 isolates, respectfully. The common ST's were the blaNDM-1 harboring ST-2 (n = 3) and ST-107 (n = 8) in SZMC and the blaNDM-2 harboring ST-103 in SZMC (n = 6) and in RMC (n = 27). All blaNDM alleles were located within a conserved mobile genetic environment flanked by the ISAb125 and IS91 family transposon. Clonal transmission was identified in most hospital-acquired cases in RMC and SZMC. CONCLUSION: NDMAb constitutes a minor part of CRAb cases and are clinically similar to non-NDM CRAb. Transmission of NDMAb occurs mostly by clonal spread.

Total escape of SARS-CoV-2 from dual monoclonal antibody therapy in an immunocompromised patient.

Monoclonal antibodies (mAbs) directed against the spike of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective therapeutic options to combat infections in high-risk patients. Here, we report the adaptation of SARS-CoV-2 to the mAb cocktail REGN-COV in a kidney transplant patient with hypogammaglobulinemia. Following mAb treatment, the patient did not clear the infection. During viral persistence, SARS-CoV-2 acquired three novel spike mutations. Neutralization and mouse protection analyses demonstrate a complete viral escape from REGN-COV at the expense of ACE-2 binding. Final clearance of the virus occurred upon reduction of the immunosuppressive regimen and total IgG substitution. Serology suggests that the development of highly neutralizing IgM rather than IgG substitution aids clearance. Our findings emphasise that selection pressure by mAbs on SARS-CoV-2 can lead to development of escape variants in immunocompromised patients. Thus, modification of immunosuppressive therapy, if possible, might be preferable to control and clearance of the viral infection.

Taking hospital pathogen surveillance to the next level.

High-throughput bacterial genomic sequencing and subsequent analyses can produce large volumes of high-quality data rapidly. Advances in sequencing technology, with commensurate developments in bioinformatics, have increased the speed and efficiency with which it is possible to apply genomics to outbreak analysis and broader public health surveillance. This approach has been focused on targeted pathogenic taxa, such as Mycobacteria, and diseases corresponding to different modes of transmission, including food-and-water-borne diseases (FWDs) and sexually transmitted infections (STIs). In addition, major healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and carbapenemase-producing Klebsiella pneumoniae are the focus of research projects and initiatives to understand transmission dynamics and temporal trends on both local and global scales. Here, we discuss current and future public health priorities relating to genome-based surveillance of major healthcare-associated pathogens. We highlight the specific challenges for the surveillance of healthcare-associated infections (HAIs), and how recent technical advances might be deployed most effectively to mitigate the increasing public health burden they cause.

Molecular features and transmission of NDM-producing Enterobacterales in Israeli hospitals.

OBJECTIVES: NDM-producing Enterobacterales (NDME) account for 34.9% of new carbapenemase-producing Enterobacterales cases in Israeli hospitals. The goals of this study were to characterize the genomic composition of NDME isolates and mobile genetic elements (MGEs) and to identify NDME transmission events (TEs). METHODS: The study was conducted at the Tel-Aviv Sourasky, Rambam and Sha'are-Zedek Medical Centers (TASMC, RMC and SZMC, respectively). All NDME isolates detected between January 2018 and July 2019 were included.Phylogenetic analysis was based on core-genome SNP distances. Core-genome distance of ≤5 SNPs between isolates from patients with overlapping hospitalization periods was suggestive of a potential TE. MGEs were classified by comparison of the blaNDM gene flanking regions. RESULTS: The study included 212 NDME isolates from 203 patients, including 104 isolates from TASMC, 30 isolates from RMC and 78 isolates from SZMC. The majority of isolates (n = 157; 74%) harboured the blaNDM-1 gene, followed by the blaNDM-5 (n = 48) and blaNDM-15 genes (n = 7). The most common NDME species were Klebsiella pneumoniae (n = 67), Escherichia coli (n = 65) and Enterobacter cloacae (n = 45), all showing a highly diverse clonal structure. Most blaNDM-1-harbouring isolates (134/157; 85%) were divided into nine different MGE modules, variably distributed across species and hospitals.The numbers of post-admission acquisition cases (n = 118) that could be linked to other cases by both molecular and epidemiological criteria were 13/58 (24.2%), 3/48 (6.3%) and 4/12 (33.3%) in TASMC, SZMC and RMC, respectively. CONCLUSIONS: The study depicted a complex and diverse population structure, suggesting that NDME had not spread via clonal expansion.

Mortality associated with third-generation cephalosporin resistance in Enterobacteriaceae bloodstream infections at one South African hospital.

OBJECTIVES: Enterobacteriaceae are common pathogens causing bloodstream infection (BSI) in sub-Saharan Africa and frequently express third-generation cephalosporin (3GC) resistance; however, the impact of 3GC resistance on clinical outcomes is rarely studied. METHODS: We conducted a single-site prospective cohort study at Tygerberg Hospital, Cape Town, South Africa to examine the feasibility of measuring impacts of 3GC resistance in Enterobacteriaceae BSI. We included patients with 3GC-susceptible and 3GC-resistant BSIs and matched each BSI patient to two uninfected patients. We determined the concordance of initial antibiotic treatment with the corresponding isolate's susceptibility profile. We performed exploratory impact analysis using multivariable regression models. RESULTS: Between 1 June 2017 and 31 January 2018, we matched 177 Enterobacteriaceae BSI patients to 347 uninfected patients. Among these BSIs, 35% were phenotypically 3GC resistant. Parameters describing clinical comorbidity showed strong associations with mortality. We found that 18% of 3GC-R and 3% of 3GC-S BSI patient received non-concordant initial therapy. In multivariable Cox regression, we found a mortality impact over their matched patients for both 3GC-R (cause-specific hazard ratio 23.77; 95% CI 5.12-110.3) and 3GC-S (HR 7.49; 95%CI 3.08-18.19) BSI. There was a nonsignificant ratio of these ratios (HR 3.18; 95% CI 0.54-18.70), limited by the small sample size. CONCLUSION: This form of impact estimation was feasible in one hospital in South Africa where 3GC-R status was associated with non-concordant initial antibiotic treatment. There was a possible increase in mortality among individuals with 3GC-resistant Enterobacteriaceae, but with broad confidence intervals. These analytical approaches could be applied to larger datasets to improve precision of estimates.

Evaluation of the BD Phoenix CPO detect panel for prediction of Ambler class carbapenemases.

Rapid detection of carbapenemases as a cause of resistance is beneficial for infection control and antimicrobial therapy. The BD Phoenix NMIC-502 panel and CPO detect test identifies presence of carbapenemases in Enterobacterales such as Klebsiella pneumoniae and assigns them to Ambler classes. To evaluate the performance of the CPO detect panel, we employed a European collection of 1222 K. pneumoniae including carbapenem non-susceptible and susceptible clinical isolates from 26 countries, for which draft genomes were available after Illumina sequencing and the presence of carbapenemase genes had been identified by ARIBA gene calling. The CPO panel detected 488 out of 494 carbapenemase-encoding isolates as positive and six as negative. One-hundred and two isolates were tested positive for carbapenemase in the absence of any carbapenemase gene. The CPO panel identified 229 out of 230 KPC-positive isolates as carbapenemase producing and classified 62 of these as class A enzyme. Similarly, the CPO panel correctly specified 167 of 182 as class D. Regarding metallo-beta-lactamases, the CPO panel assigned 78 of 90 MBL positive isolates to class B enzymes. The sensitivity of the CPO panel in detecting carbapenemase activity was 99.5%, 97.7% and 98.3% for class A, B and D enzymes, respectively. The sensitivity in assignation to Ambler class A, B and D was 27%, 86% and 91%, respectively. An overall sensitivity of 98.8% and specificity of 86% in unclassified detection of carbapenemases was observed, with frequent false positive detection of carbapenemase producing organisms, thus rendering further confirmatory tests necessary.

Characterisation of mcr-4.3 in a colistin-resistant Acinetobacter nosocomialis clinical isolate from Cape Town, South Africa.

OBJECTIVES: Colistin resistance in Acinetobacter spp. is increasing, resulting in potentially untreatable nosocomial infections. Plasmid-mediated colistin resistance is of particular concern due to its low fitness cost and potential transferability to other bacterial strains and species. This study investigated the colistin resistance mechanism in a clinical Acinetobacter nosocomialis isolate from Cape Town, South Africa. METHODS: A colistin-resistant A. nosocomialis isolate was identified from a blood culture in 2017. PCR and Illumina whole-genome sequencing (WGS) were performed to identify genes and mutations conferring resistance to colistin. Plasmid sequencing was performed on an Oxford Nanopore platform. mcr functionality was assessed by broth microdilution after cloning the mcr gene into pET-48b(+) and expressing it in SHuffle® T7 Escherichia coli and after curing the plasmid using 62.5 mg/L acridine orange. RESULTS: The colistin minimum inhibitory concentration (MIC) of the A. nosocomialis isolate was 16 mg/L. The mcr-4.3 gene was detected by PCR and WGS. No other previously described colistin resistance mechanism was found by WGS. The mcr-4.3 gene was identified on a 24 024-bp RepB plasmid (pCAC13a). Functionality studies showed that recombinant mcr-4.3 did not confer colistin resistance in E. coli. However, plasmid curing of pCAC13a restored colistin susceptibility in A. nosocomialis. CONCLUSION: We describe the first detection of a plasmid-mediated mcr-4.3 gene encoding colistin resistance in A. nosocomialis and the first detection of mcr-4.3 in a clinical isolate in Africa. Recombinant expression of mcr-4.3 did not confer colistin resistance in E. coli, suggesting that its functionality may be RepB plasmid-dependent or species-specific.

Colistin Resistance Mechanisms in Clinical Escherichia coli and Klebsiella spp. Isolates from the Western Cape of South Africa.

Objectives: Colistin is a last-resort antibiotic for the treatment of carbapenem-resistant Gram-negative infections. Colistin resistance thus poses a threat to human health. Colistin resistance is most commonly encoded by mutations in chromosomal pmrA, pmrB, phoP, phoQ, ccrB, and mgrB genes, and the presence of plasmid-mediated mcr genes. This study describes colistin resistance mechanisms in clinical Enterobacterales isolates from the Western Cape, South Africa. Results: Escherichia coli (n = 22) and Klebsiella spp. (n = 7) isolates, from nine health care facilities, were confirmed to be colistin resistant during 2016 and 2017. mcr-1 was present in 55% (12/22) of E. coli and 71% (5/7) of Klebsiella spp. isolates. Colistin resistance mutations in pmrB were identified in 8/10 mcr-negative E. coli isolates using whole-genome sequencing, with pmrB Pro-94→Gln being the most frequent with presence in 4 isolates. One mcr-negative Klebsiella spp. isolate had a complete deletion of the mgrB and one contained an insertion sequence (IS1) in mgrB. Conclusion: A reduction in the proportion of colistin-resistant isolates harboring mcr-1 from 2016 to 2017 was observed. Colistin-resistant E. coli attributed by chromosomal mutations in pmrB in 2017 were mostly clonal related, which contrasts with the 2016 unrelated mcr-1-positive isolates. The diverse strains, hospitals, and resistance mechanisms may suggest that selective pressure is the main driver of colistin resistance.

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131.

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years, Escherichia coli sequence type 131 (ST131) has emerged as a major cause of extraintestinal infection frequently exhibiting a multidrug resistance (MDR) phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones, however, is not well studied. We characterized strains from an international collection of clinical MDR E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by principal-component analysis (PCA). PCA revealed a group of 17 MDR E. coli isolates (n = 34) exhibiting increased susceptibility to treatment with NMP, suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed enhanced efflux phenotype [EEP] strains). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino acid substitutions occurring in AcrR, MarR, and SoxR. Quantitative reverse transcription-PCR (qRT-PCR) of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC system in EEP strains, whereas in the remaining strains, we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli strains exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC efflux pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

Antiphospholipid antibodies may be associated with uveitis.

PURPOSE: To evaluate the prevalence of a spectrum of autoantibodies in adult patients with non-infectious uveitis compared to healthy controls. METHODS: This is a case-control study conducted in a tertiary referral center. Serum positivity to auto-antibodies directed at membranous phospholipids (aPL), nuclear antigens, and cytoplasmic (ANCA) antigens were assessed in sera from 63 non-infectious uveitis patients, and 78 healthy controls. Uveitis patients' demographic and clinical data were collected retrospectively from their medical charts. RESULTS: Of the spectrum of antibodies evaluated only aPL were linked with uveitis (OR 11.2, CI 1.4-92.1), as 13 (20.6%) uveitis patients were positive to at least one of the screened aPL, namely either anti-cardiolipin (aCL), anti-ß2-glycoprotein (aß2GPI), or anti-phosphatidylserine/prothrombin (aPS/PT). aCL antibodies were detected in 5/63 (7.9%) of uveitis patients and in none of controls (p = 0.016). Positivity to either aCL or aß2GPI was noted in 8/63 (12.7%) of uveitis patients and in 1 (1.3%) of the controls (p = 0.011). Of the 13 uveitis patients positive to any of the aPL antibodies, 8 (62%) had exclusively anterior uveitis, 9 (69%) were idiopathic, and none had evidence of posterior vaso-occlusive involvement or systemic thrombotic manifestations. CONCLUSION: An association between aPL and uveitis among an unselected population of patients with no evidence of thrombosis or presence of the antiphospholipid syndrome was documented in this study. This link was observed, alike the general population of uveitis patients, mainly in patients with anterior eye inflammation. A possible interaction between aPL and uveitis, mediated by non-thrombotic mechanisms, requires further studies.

Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among Klebsiella pneumoniae.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

Protocol for a prospective cohort study: Prevention of Transmissions by Effective Colonisation Tracking in Neonates (PROTECT-Neo).

INTRODUCTION: Transmissions of opportunistic bacterial pathogens between neonates increase the risk of infections with negative repercussions, including higher mortality, morbidity and permanent disabilities. The probability of transmissions between patients is contingent on a set of intrinsic (patient-related) and extrinsic (ward-related) risk factors that are not clearly quantified. It is the dual objective of the Prevention of Transmissions by Effective Colonisation Tracking-Neo study to determine the density of transmission events in a level III neonatal intensive care unit (NICU) and to identify risk factors that may be causally associated with transmission events. METHODS AND ANALYSIS: A full cohort of patients treated in a 17-bed level III NICU will be prospectively followed and transmission events between two or more patients will be documented. A transmission event occurs when isogenic isolates from two different patients can be identified. Isolates will be obtained by routine weekly screening. Isogenicity will be determined by whole-genome sequencing. During the study, relevant intrinsic and extrinsic risk factors will be recorded. Specimen and data will be collected for 1 year. We postulate that transmission density increases during episodes when demand for intensive care cannot be met by existing staff, and that threshold dynamics have a bearing on cohorting and hand hygiene performance. Poisson logistic regression, proportional hazard and multilevel competing risk models will be used to estimate the effect of explanatory variables. ETHICS AND DISSEMINATION: This study has been approved by the local ethics committee (study ID 287/18). The results will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders. TRIAL REGISTRATION NUMBER: The German Clinical Trials Registry (DRKS00017733); Pre-results.

Clonal expansion of colistin-resistant Acinetobacter baumannii isolates in Cape Town, South Africa.

OBJECTIVES: To describe colistin-resistant Acinetobacter baumannii isolates in Cape Town, South Africa. METHODS: A. baumannii isolates identified on Vitek 2 Advanced Expert System were collected from Tygerberg Hospital referral laboratory between 2016 and 2017. Colistin resistance was confirmed using broth microdilution and SensiTest. mcr-1-5 were detected using PCR and strain typing was performed by rep-PCR. Whole genome sequencing (WGS) was performed on a subset of isolates to identify chromosomal colistin resistance mechanisms and strain diversity using multilocus sequence typing (MLST) and pairwise single nucleotide polymorphism analyses. RESULTS: Twenty-six colistin-resistant and six colistin-susceptible A. baumannii were collected separately based on Vitek susceptibility; 20/26 (77%) were confirmed colistin-resistant by broth microdilution. Four colistin-resistant isolates were isolated in 2016 and 16 in 2017, from five healthcare facilities. Thirteen colistin-resistant isolates and eight colistin-susceptible isolates were identical by rep-PCR and MLST (ST1), all from patients admitted to a tertiary hospital during 2017. The remaining colistin-resistant isolates were unrelated. CONCLUSIONS: An increase in colistin-resistant A. baumannii isolates from a tertiary hospital in 2017 appears to be clonal expansion of an emerging colistin-resistant strain. This strain was not detected in 2016 or from other hospitals. Identical colistin-susceptible isolates were also isolated, suggesting relatively recent acquisition of colistin resistance.

Molecular characterization of carbapenem-resistant Acinetobacter baumannii using WGS revealed missed transmission events in Germany from 2012-15.

BACKGROUND: Infection and colonization with multi-resistant Acinetobacter baumannii causes therapeutic and economic problems in the nosocomial setting. Due to the sensitivity issue of screening schemes for A. baumannii, it is difficult to implement adequate transmission prevention measures. The high discriminatory power of WGS for transmission-chain analysis provides us with the necessary tool to study and identify transmission events. We retrospectively sequenced and analysed 39 A. baumannii isolates from 2012-15 to search for possible missed transmission events. METHODS: Molecular typing by WGS was performed for non-repetitive (n=39) carbapenem-resistant A. baumannii. Retrospective assessment of patient records was performed to investigate and confirm possible transmission events. RESULTS: Between July 2012 and September 2015, A. baumannii was isolated from 268 patients, of which 16% (42/268) were carbapenem resistant. Thirty-nine of these isolates were recoverable and sequenced. Fifteen percent (6/39) of these were resistant to all antibiotics tested. Most isolates belong to the circulating IC2 clonal type. SNP analysis revealed four potential outbreak clusters. Two of these clusters showed high concordance with the local spatio-temporal epidemiology, suggesting that transmission events were very likely. CONCLUSIONS: Our data suggest that there were two independent transmission events, which would have been missed by conventional MLST owing to high clonality. The routine implementation of WGS can optimize surveillance and initiation of suitable containment measures. In addition, emerging resistance to salvage therapy is a major therapeutic problem and should be monitored closely.

Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread.

Public health interventions to control the current epidemic of carbapenem-resistant Klebsiella pneumoniae rely on a comprehensive understanding of its emergence and spread over a wide range of geographical scales. We analysed the genome sequences and epidemiological data of >1,700 K. pneumoniae samples isolated from patients in 244 hospitals in 32 countries during the European Survey of Carbapenemase-Producing Enterobacteriaceae. We demonstrate that carbapenemase acquisition is the main cause of carbapenem resistance and that it occurred across diverse phylogenetic backgrounds. However, 477 of 682 (69.9%) carbapenemase-positive isolates are concentrated in four clonal lineages, sequence types 11, 15, 101, 258/512 and their derivatives. Combined analysis of the genetic and geographic distances between isolates with different ß-lactam resistance determinants suggests that the propensity of K. pneumoniae to spread in hospital environments correlates with the degree of resistance and that carbapenemase-positive isolates have the highest transmissibility. Indeed, we found that over half of the hospitals that contributed carbapenemase-positive isolates probably experienced within-hospital transmission, and interhospital spread is far more frequent within, rather than between, countries. Finally, we propose a value of 21 for the number of single nucleotide polymorphisms that optimizes the discrimination of hospital clusters and detail the international spread of the successful epidemic lineage, ST258/512.

Genomic identification of cryptic susceptibility to penicillins and ß-lactamase inhibitors in methicillin-resistant Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all ß-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with ß-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/ß-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for 'susceptible' MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/ß-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections.

Epidemiological Typing of Serratia marcescens Isolates by Whole-Genome Multilocus Sequence Typing.

Serratia marcescens is an opportunistic bacterial pathogen. It is notorious for its increasing antimicrobial resistance and its potential to cause outbreaks of colonization and infections, predominantly in neonatal intensive care units (NICUs). There, its spread requires rapid infection control response. To understand its spread, detailed molecular typing is key. We present a whole-genome multilocus sequence typing (wgMLST) method for S. marcescens Using a set of 299 publicly available whole-genome sequences (WGS), we developed an initial wgMLST system consisting of 9,377 gene loci. This included 1,455 loci occurring in all reference genomes and 7,922 accessory loci. This closed system was validated using three geographically diverse collections of S. marcescens consisting of 111 clinical isolates implicated in nosocomial dissemination events in three hospitals. The validation procedure showed a full match between epidemiological data and the wgMLST analyses. We set the cutoff value for epidemiological (non)relatedness at 20 different alleles, though for the majority of outbreak-clustered isolates, this difference was limited to 4 alleles. This shows that the wgMLST system for S. marcescens provides prospects for successful future monitoring for the epidemiological containment of this opportunistic pathogen.

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.