Emergence of linezolid-resistance in vancomycin-resistant Enterococcus faecium ST117 associated with increased linezolid-consumption.

OBJECTIVE: We aim to describe the epidemiological, clinical and microbiological characteristics of the linezolid- and vancomycin- resistant Enterococcus faecium (LVRE) in a tertiary care hospital in Germany. METHODS: We conducted a retrospective analysis of 196 LVRE cases observed from 1st January 2012 to 31th December 2018. Patients' medical charts were reviewed and available LVRE (n = 102) were subjected to whole-genome-sequencing. Antibiotic consumption was measured in defined daily dose (DDD)/100 bed-days (BD). RESULTS: The prevalence of LVRE isolates among VRE was 6.3 % in 2018. Most patients had an onco-hematological disease (134/196, 68.4 %). From 2012-2018 an increase of +356.7 % of linezolid defined daily dose/100 bed-days was observed. In 71.4 % (90/126, 70 missing values) of the patients, linezolid was prescribed in the previous 6 months. The median exposure to linezolid was 15 days (Interquartile, IQR 9-23). 42/196 (21.4 %) patients had an LVRE-related infection with an overall 30-day mortality rate of 33 %. In 121/196 (61.7 %) patients, linezolid-susceptible VREfm were isolated before LVRE, suggesting secondary acquisition of linezolid resistance. Genetic analysis revealed that most isolates belonged to ST117 (64/102 available isolates, 62.7 %). The G2576T 23S rDNA mutation was identified as the most common resistance mechanism (96/102, 94.1 %). poxtA was identified in two isolates, while cfr, and optrA were not detected. CONCLUSIONS: Incidence of LVRE related to 23S rDNA mutations is rising and probably associated with antibiotic consumption. Restrictions in the use of linezolid may be needed in order to retain therapeutic options in VRE.

Monocenter study on epidemiology, outcomes, and risk factors of infections in recipients of 166 allogeneic stem cell transplantations during 1 year.

OBJECTIVES: During allogeneic hematopoietic stem cell transplantation (allo-SCT), infections significantly contribute to morbidity and mortality. A monocentric prospective analysis was performed to assess epidemiology, risk factors, and outcomes of infections during the peri-transplant period. METHODS: Data were recorded prospectively using a predefined questionnaire. RESULTS: In 2015, 163 consecutive patients, 37.4% female, median age 59 (range 18-79) years received 166 allo-SCT. Median duration of leukopenia <109 /L was 14.5 days (range 4-43 days). Fever of unknown origin (FUO) occurred in 118/166 patients (71.1%). Severe sepsis developed in 95, and septic shock developed in 26 patients. Intensive diagnostic workup helped to identify causative microorganisms only in a small number of infectious courses. All but 13 patients needed antibiotic therapy, each according to the standard operating procedures of the department. Cumulative incidence of death by infection after 1 year was 16.6% (95% CI: 11.3-22.7). The only risk factor for FUO in neutropenia was duration of neutropenia

Efficacy of introducing a checklist to reduce central venous line associated bloodstream infections in the ICU caring for adult patients.

BACKGROUND: Central line-associated bloodstream infections (CLABSI) are a major source of sepsis in modern intensive care medicine. Some years ago bundle interventions have been introduced to reduce CLABSI. The use of checklists may be an additional tool to improve the effect of these bundles even in highly specialized institutions. In this study we investigate if the introduction of a checklist reduces the frequency of CLABSI. METHODS: During the study period from October 2011 to September 2012, we investigated the effect of implementing a checklist for the placement of central venous lines (CVL). Patients were allocated either to the checklist group or to the control group, roughly in a 1:2 ratio. The frequency of CLABSI was compared between the two groups. RESULTS: During the study period 4416 CVL were inserted; 1518 in the checklist group and 2898 in the control group. The use of the checklist during CVL placement resulted in a lower CLABSI frequency. The incidence in the checklist group was 3.8 per 1000 catheter days as compared to 5.9 per 1000 catheter days in the control group (IRR = 0.57; p = 0.001). The use of the checklist also reduced the frequency of catheter colonisation significantly, 36.3 per 1000 catheter days in the checklist group vs 21.2 per 1000 catheter days in the control group, respectively (IRR = 0.58; p < 0.001). CONCLUSION: The introduction of a checklist to improve the adherence to hygiene standards while placement of central venous lines reduced the frequency of infections significantly.

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 .

Emergence of ceftazidime/avibactam non-susceptibility in an MDR Klebsiella pneumoniae isolate.

Background: Avibactam is a novel broad-range ß-lactamase inhibitor active against Ambler class A (including ESBL and KPC) and some Ambler class C and D (e.g. OXA-48) enzymes. We here report on the emergence of ceftazidime/avibactam resistance in clinical, multiresistant, OXA-48 and CTX-M-14-producing Klebsiella pneumoniae isolate DT12 during ceftazidime/avibactam treatment. Methods and results: Comparative whole-genome sequence analysis identified two SNPs in the CTX-M-14-encoding gene leading to two amino acid changes (P170S and T264I). Compared with WT CTX-M-14, expression of the CTX-M-14Δ170Δ264 isoform in Escherichia coli led to a >64- and 16-fold increase in ceftazidime and ceftazidime/avibactam MICs, respectively, functionally linking the observed SNPs and elevated MICs. The mutated CTX-M-14 isoform exhibited augmented ceftazidime hydrolytic activity, which was a reasonable cause for impaired susceptibility to avibactam inhibition. The P170S exchange in CTX-M-14 was found in association with elevated ceftazidime/avibactam MICs for independent K. pneumoniae isolates, but was not sufficient for full resistance. Apparently, additional CTX-M-independent mechanisms contribute to ceftazidime/avibactam resistance in K. pneumoniae DT12. Conclusions: This study on the molecular basis of ceftazidime/avibactam resistance in clinical K. pneumoniae emerging in vivo underscores the need for continuous monitoring of ceftazidime/avibactam susceptibility during therapy. Despite sustained inhibition of OXA-48, rapid development of CTX-M-14 isoforms exhibiting augmented ceftazidime hydrolytic activity may limit the usefulness of ceftazidime/avibactam monotherapies in infections caused by isolates carrying blaCTX-M-14 and blaOXA-48.

Extended spectrum beta-lactamase producing Enterobacteriaceae causing bloodstream infections in rural Ghana, 2007-2012.

BACKGROUND: High prevalence of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae threatens treatment options for invasive bloodstream infections in sub-Saharan Africa. OBJECTIVES: To explore the frequency and genotype distribution of ESBL producing Enterobacteriaceae causing bloodstream infections in a primary health care setting in rural Ghana. METHODS: Blood cultures from all patients with fever ≥38°C within 24h after admission (community-acquired) and from all neonates with suspected neonatal sepsis (hospital-acquired) were obtained. ESBL-producing isolates were characterized by combined disc test and by amplifying the blaCTX-M, blaTEM and blaSHV genes. Multilocus sequence typing (MLST) was performed for all ESBL-producing Klebsiella pneumoniae and Escherichia coli isolates, and all K. pneumoniae isolates were differentiated by pulsed-field gel electrophoresis (PFGE). RESULTS: Among 426 Enterobacteriaceae isolated from blood cultures, non-typhoid Salmonella (n=215, 50.8%), S. Typhi (n=110, 26.0%), E. coli (n=50, 11.8%) and K. pneumoniae (n=41, 9.7%) were the most frequent. ESBL-producing isolates were restricted to the CTX-M-15 genotype and the species K. pneumoniae (n=34, 82.9%), Enterobacter cloacae complex (n=2, 66.7%) and E. coli (n=5, 10.0%). The rates of ESBL-producers in K. pneumoniae were 55.6% and 90.6% in community-acquired and neonatal bloodstream infections, respectively. MLST and PFGE analysis identified four outbreak clusters among neonates. CONCLUSIONS: Considering the rural primary health care study setting, the high proportion of ESBL-producing Klebsiella pneumoniae is worrisome and might be devastating in the absence of second line antibiotics. Therefore, enhanced diagnostic laboratories for surveillance purposes and sustainable hospital hygiene measures must be considered to prevent further spread of multidrug resistant bacteria within rural communities.

Prevalence and genotypes of extended spectrum beta-lactamases in Enterobacteriaceae isolated from human stool and chicken meat in Hamburg, Germany.

Chicken meat has been proposed to constitute a source for extended spectrum beta-lactamase (ESBL)-carrying Enterobacteriaceae that colonize and infect humans. In this study the prevalence of ESBL-producing Enterobacteriaceae in stool samples from ambulatory patients who presented in the emergency department of the University Medical Centre Hamburg-Eppendorf with gastrointestinal complains and in chicken meat samples from the Hamburg region were analysed and compared with respect to ESBL-genotypes, sequence types and antibiotic resistance profiles. Twenty-nine (4.1%) of 707 stool samples and 72 (60%) of 120 chicken meat samples were positive for ESBL-producing Enterobacteriaceae. The distribution of ESBL genes in the stool vs. chicken meat isolates (given as % of total isolates from stool vs. chicken meat) was as follows: CTX-M-15 (38% vs. 0%), CTX-M-14 (17% vs. 6%), CTX-M-1 (17% vs. 69%), SHV-12 (3% vs. 18%) and TEM-52 (3% each). Comparison of ESBL- and multilocus sequence type revealed no correlation between isolates of human and chicken. Furthermore, ESBL-producing E. coli from stool samples were significantly more resistant to fluoroquinolones, aminoglycosides and/or trimethoprim-sulfamethoxazole than chicken isolates. The differences in ESBL-genotypes, sequence types and antibiotic resistance patterns indicate that in our clinical setting chicken meat is not a major contributor to human colonization with ESBL-carrying Enterobacteriaceae.

Duration of fecal shedding of Shiga toxin-producing Escherichia coli O104:H4 in patients infected during the 2011 outbreak in Germany: a multicenter study.

BACKGROUND: In May-July 2011, Germany experienced a large food-borne outbreak of Shiga toxin 2-producing Escherichia coli (STEC O104:H4) with 3842 cases, including 855 cases with hemolytic uremic syndrome (HUS) and 53 deaths. METHODS: A multicenter study was initiated in 5 university hospitals to determine pathogen shedding duration. Diagnostics comprised culture on selective media, toxin enzyme-linked immunosorbent assay, and polymerase chain reaction. Results were correlated with clinical and epidemiologic findings. Testing for pathogen excretion was continued after discharge of the patient. RESULTS: A total of 321 patients (104 male, 217 female) were included (median age, 40 years [range, 1-89 days]). Median delay from onset of symptoms to hospitalization was 4 days (range, 0-17 days). Two hundred nine patients presented with HUS. The estimate for the median duration of shedding was 17-18 days. Some patients remained STEC O104:H4 positive until the end of the observation time (maximum observed shedding duration: 157 days). There was no significant influence of sex on shedding duration. Patients presenting with HUS had a significantly shortened shedding duration (median, 13-14 days) compared to non-HUS patients (median, 33-34 days). Antimicrobial treatment was also significantly associated with reduced shedding duration. Children (age≤15 years) had longer shedding durations than adults (median, 35-41 vs 14-15 days). CONCLUSIONS: STEC O104:H4 is usually eliminated from the human gut after 1 month, but may sometimes be excreted for several months. Proper follow-up of infected patients is important to avoid further pathogen spread.

Activity of antimicrobial examination gloves under realistic conditions: challenge not fulfilled.

BACKGROUND: Antimicrobial materials or surfaces are advertised as part of infection prevention bundles. However, the efficacy of such antimicrobial surfaces has not been sufficiently investigated in hospitals. In this study, the antimicrobial activity of examination gloves with light-activated antimicrobial properties against Gram-positive microorganisms was investigated modelling real live conditions. METHOD: In a standardized experimental set-up with dry and realistic contamination, the antimicrobial properties of gloves claiming light dependent antimicrobial activity against Gram-positive organisms were tested in comparison with conventional examination gloves. All gloves were contaminated through a standardized activity of the test persons for construction with contaminated building blocks. For contamination suspensions of Enterococcus faecium ATCC 6057, Acinetobacter baumannii (outbreak strain), methicillin resistant Staphylococcus aureus ATCC 43300 or E. faecium (VRE) patient isolate were dried on the surfaces. After the standardized activity, the gloves were held for 10 min in the light present in the room (bright conditions) and the grade of contamination was determined subsequently by quantitative culture. In one experimental series gloves were held in a dark box after contamination as a control (dark conditions). RESULTS: The light intensity in all experiments under bright conditions was significantly above the limit value specified by the manufacturer for the activation of antimicrobial properties (> 500 lx). The mean values for experiments with antimicrobial active and non-active gloves were 955 and 935 lx, respectively. As claimed by the manufacture, the gloves showed no sufficient efficacy against A. baumannii under bright conditions. Against Gram-positive microorganisms such as E. faecium, E. faecium (VRE) and methicillin resistant S. aureus the gloves showed only very low antimicrobial activity with a reduction factor < 1 log10 even after 10 min in bright conditions. Interestingly, comparable results for experiments with A. baumannii and E. faecium were shown under dark conditions. CONCLUSION: The lack of activity of the active principle against Gram-negative microorganisms could be confirmed. The reduction factors of > 4 log10 within 5 min for Gram-positive microorganisms claimed for the product using a standard test procedure (ASTM D7907) could not be confirmed in a realistic experimental test set-up even after 10 min of light exposure. The effectiveness against Gram-positive microorganisms should be further investigated under realistic (dry) conditions, including patient care. At this stage, the use of supposedly antimicrobial gloves should not be recommended, as the belief in their efficacy may encourage the misuse of gloves.

Phenotypic Variation in Clinical S. aureus Isolates Did Not Affect Disinfection Efficacy Using Short-Term UV-C Radiation.

Pigmentation, catalase activity and biofilm formation are virulence factors that cause resistance of Staphylococcus aureus to environmental stress factors including disinfectants. In recent years, automatic UV-C room disinfection gained greater importance in enhanced disinfection procedures to improve disinfection success in hospitals. In this study, we evaluated the effect of naturally occurring variations in the expression of virulence factors in clinical S. aureus isolates on tolerance against UV-C radiation. Quantification of staphyloxanthin expression, catalase activity and biofilm formation for nine genetically different clinical S. aureus isolates as well as reference strain S. aureus ATCC 6538 were performed using methanol extraction, a visual approach assay and a biofilm assay, respectively. Log10 reduction values (LRV) were determined after irradiation of artificially contaminated ceramic tiles with 50 and 22 mJ/cm2 UV-C using a commercial UV-C disinfection robot. A wide variety of virulence factor expression was observed, indicating differential regulation of global regulatory networks. However, no direct correlation with the strength of expression with UV-C tolerance was observed for either staphyloxanthin expression, catalase activity or biofilm formation. All isolates were effectively reduced with LRVs of 4.75 to 5.94. UV-C disinfection seems therefore effective against a wide spectrum of S. aureus strains independent of occurring variations in the expression of the investigated virulence factors. Due to only minor differences, the results of frequently used reference strains seem to be representative also for clinical isolates in S. aureus.

Infectivity of SARS-CoV-2 on Inanimate Surfaces: Don't Trust Ct Value.

SARS-CoV-2 RNA is frequently identified in patient rooms and it was speculated that the viral load quantified by PCR might correlate with infectivity of surfaces. To evaluate Ct values for the prediction of infectivity, we investigated contaminated surfaces and Ct-value changes after disinfection. Viral RNA was detected on 37 of 143 investigated surfaces of an ICU. However, virus isolation failed for surfaces with a high viral RNA load. Also, SARS-CoV-2 could not be cultivated from surfaces artificially contaminated with patient specimens. In order to evaluate the significance of Ct values more precisely, we used surrogate enveloped bacteriophage Φ6. A strong reduction in Φ6 was achieved by three different disinfection methods. Despite a strong reduction in viability almost no change in the Ct values was observed for UV-C and alcoholic surface disinfectant. Disinfection using ozone resulted in a lack of Φ6 recovery as well as a detectable shift in Ct values indicating strong degradation of the viral RNA. The observed lack of significant effects on the detectable viral RNA after effective disinfection suggest that quantitative PCR is not suitable for predicting the infectivity of SARS-CoV-2 on inanimate surfaces. Ct values should therefore not be considered as markers for infectivity in this context.

MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages.

Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m/z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.

Evaluation of the Effectiveness of Two Automated Room Decontamination Devices Under Real-Life Conditions.

To evaluate the effectiveness of automated room decontamination devices, a common aerosolized hydrogen peroxide (aHP) as well as a recent gaseous ozone-based device, which produces the disinfectant reagent without the need of consumables, were tested under real-life conditions. Twenty-two contaminated surfaces were positioned in different areas in a patient room with adjacent bathroom and anteroom. Following the decontamination process bacteria were recovered and reduction factors were calculated after performing quantitative culture. Following the manufactures instructions, the ozone-based device displayed a bactericidal effect (log10 > 5), whereas the aHP system failed for a high bacterial burden and achieves only a complete elimination of a realistic bioburden (log10 2). After increasing the exposure time to 30 min, the aHP device also reached a bactericidal effect. Nevertheless, our results indicate, that further research and development is necessary, to get knowledge about toxicity, efficacy and safety by using in complex hospital conditions and achieve meaningful integration in cleaning procedures, to reach positive effects on disinfection performance.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in local and imported poultry meat in Ghana.

Antibiotic use in animal husbandry has raised concerns on the spread of resistant bacteria. Currently animal products are traded globally with unprecedented ease, which has been challenging the control of antimicrobial resistance. This study aims to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from imported and locally produced poultry products sold in Ghana. Local and imported chicken meat was collected from 94 stores and markets throughout Kumasi (Ghana) and cultured on selective ESBL screening agar. Phenotypic ESBL-producing E. coli and K. pneumoniae isolates were confirmed by combined disc test and further characterized by antibiotic susceptibility testing, amplification of the blaCTX-M, blaTEM and blaSHV genes as well as multilocus sequence typing (MLST) and linked to the country of origin. Out of 200 meat samples, 71 (36%) samples revealed 81 ESBL-producing isolates (46 E. coli and 35 K. pneumoniae), with 44% (30/68) of local poultry and 31% (41/132) of imported products being contaminated. Most ESBL-producing isolates harboured the blaCTX-M-15 gene (61/81, 75%) and the dominant Sequence Types (ST) were ST2570 (7/35, 20%) among K. pneumoniae and ST10 (5/46, 11%) among E. coli. High numbers of ESBL-producing bacteria, particularly on local but also imported poultry meat, represent a potential source for human colonization and infection as well as spread within the community. Surveillance along the poultry production-food-consumer chain would be a valuable tool to identify sources of emerging multidrug resistant pathogens in Ghana.

Two-tier approach combining molecular and culture-based techniques for optimized detection of vancomycin-resistant enterococci.

Given constantly high or even rising incidences of both colonization and infection with vancomycin-resistant enterococci (VRE), timely and accurate identification of carriers in high-risk patient populations is of evident clinical importance. In this study, a two-tier approach consisting of PCR-based screening and cultural confirmation of positive results is compared to the conventional approach solely based on culture on selective media. The 2-tier strategy was highly consistent with the conventional approach, and was found to possess high sensitivity and specificity (93.1% and 100%, respectively). The introduction of the PCR-based combined VRE screening approach significantly (P<0.0001) reduced median overall time to result by 44.3hours. The effect was found to be most pronounced in VRE negative samples. Positive vanA PCR was highly consistent with culture (PPV: 92.0%, 95% CI: 72.5-98.6%, NPV: 99.6%, 95% CI: 98.9-99.6%), thus allowing for preliminary reporting of VRE detection. In contrast, a vanB positive PCR does not allow for preliminary reporting (PPV: 58.5%, 95% CI: 44.2-71.6%, NPV: 99.8%, 95% CI: 99.2-100%). The introduction of a molecular assay for rapid detection of VRE from rectal swabs combined with cultural confirmation proved to be reliable and time saving, especially in a setting of low VRE prevalence and predominance of vanA positive strains.

Yersinia virulence factor YopM induces sustained RSK activation by interfering with dephosphorylation.

BACKGROUND: Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells. METHODOLOGY/PRINCIPAL FINDINGS: In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221. CONCLUSIONS/SIGNIFICANCE: Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.