Carbapenem resistance in Acinetobacter pittii isolates mediated by metallo-ß-lactamases.

OBJECTIVES: To characterize the genetic environment of metallo-ß-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.

An appeal for strengthening genomic pathogen surveillance to improve pandemic preparedness and infection prevention: the German perspective.

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.

[Establishment of genomic pathogen surveillance to strengthen pandemic preparedness and infection prevention in Germany]. / Etablierung der Genomischen Erreger-Surveillance zur Stärkung des Pandemie- und Infektionsschutzes in Deutschland.

The SARS-CoV­2 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.

Reduced Clostridioides difficile infections in hospitalised older people through multiple quality improvement strategies.

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.

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

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

The Washing Machine as a Reservoir for Transmission of Extended-Spectrum-Beta-Lactamase (CTX-M-15)-Producing Klebsiella oxytoca ST201 to Newborns.

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.

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

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

[Effective management of an outbreak with multiresistent Klebsiella pneumoniae in a neurorehabilitation unit]. / Effektives Management eines Ausbruchs mit multiresistenten Klebsiella pneumoniae in der Neurorehabilitation.

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.

Transmission Risk on a Neonatal Intensive Care Unit: Escherichia coli versus Klebsiella pneumoniae.

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.

Protracted Regional Dissemination of GIM-1-Producing Serratia marcescens in Western Germany.

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 blaGIM-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 blaGIM-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.

Comparison of Phenotypic Tests and an Immunochromatographic Assay and Development of a New Algorithm for Detection of OXA-48-like Carbapenemases.

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-ß-lactamases or Klebsiella pneumoniae carbapenemases (KPC), no specific inhibitor is available for the phenotypic detection of OXA-48. Molecular detection of blaOXA-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.

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

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

Dissemination of blaOXA-58 in Proteus mirabilis isolates from Germany.

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 .

Multicentre investigation of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in German hospitals.

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.

Description of IMP-31, a novel metallo-ß-lactamase found in an ST235 Pseudomonas aeruginosa strain in Western Germany.

OBJECTIVES: The objective of this study was to characterize a novel IMP-type metallo-ß-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 ß-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 ß-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 ß-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.

Development and evaluation of a novel universal ß-lactamase gene subtyping assay for blaSHV, blaTEM and blaCTX-M using clinical and livestock-associated Escherichia coli.

OBJECTIVES: Antibiotic resistance among Escherichia coli is globally an increasing problem in public healthcare. Understanding the spread of plasmid-mediated ESBL genes is of great importance in elucidating their molecular epidemiology. However, differentiation of subtypes and alleles is frequently hampered by the lack of comprehensive diagnostic tools. We therefore developed a novel universal blaSHV, blaTEM and blaCTX-M subtyping assay based on PCR and Sanger sequencing that results in large amplicons of >700 bp, enabling differentiation of bla alleles as precisely as possible. METHODS: The assay was established using 10 reference strains with known bla genotypes that represent all examined primer groups and 101 uncharacterized ESBL-producing E. coli of clinical and livestock-associated origins from different German regions. All isolates were tested in parallel with established blaSHV, blaTEM and blaCTX-M subtyping assays for the respective ß-lactamases and their alleles. RESULTS: The novel assay yielded equal (n = 92) or better (n = 47) subtyping results compared with established subtyping methods and reliably detected all expected enzymes in the reference strains. Overall, the occurring enzymes could be differentiated into groups representing one (n = 9), two (n = 5) or three (n = 4) highly similar alleles. Moreover, ESBL and non-ESBL allelic variants of blaSHV and blaTEM occurring in the same isolate were distinguished reliably. CONCLUSIONS: We established a highly discriminatory assay for the subtyping of clinically important ESBL genes that can easily be used in epidemiological analyses.

Molecular epidemiology of VIM-1 producing Escherichia coli from Germany referred to the National Reference Laboratory.

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-ß-lactams susceptibility rates of >90% were observed for amikacin, tigecycline, colistin, fosfomycin and nitrofurantoin.

Community-acquired adult Escherichia coli meningitis leading to diagnosis of unrecognized retropharyngeal abscess and cervical spondylodiscitis: a case report.

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 ß-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.

Fosfomycin susceptibility in carbapenem-resistant Enterobacteriaceae from Germany.

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.

Detection of carbapenemases by real-time PCR and melt curve analysis on the BD Max system.

We developed a multiplex SYBR green real-time PCR for the BD Max instrument (BD Diagnostics, Sparks, MD) to detect a panel of carbapenemases. The assay was evaluated with 152 consecutive isolates sent to the German National Reference Laboratory, and 65/65 of the carbapenemase-positive and 87/87 of the carbapenemase-negative strains were identified correctly.