High Genetic Diversity in Third-Generation Cephalosporin-Resistant Escherichia coli in Wastewater Systems of Schleswig-Holstein.

The spread of multidrug-resistant bacteria from humans or livestock is a critical issue. However, the epidemiology of resistant pathogens across wastewater pathways is poorly understood. Therefore, we performed a detailed comparison of third-generation cephalosporin-resistant Escherichia coli (3GCREC) from wastewater treatment plants (WWTPs) to analyze dissemination pathways. A total of 172 3GCREC isolated from four WWTPs were characterized via whole genome sequencing. Clonal relatedness was determined using multi-locus sequence typing (MLST) and core genome MLST. Resistance genotypes and plasmid replicons were determined. A total of 68 MLST sequence types were observed with 28 closely related clusters. Resistance genes to eight antibiotic classes were detected. In fluoroquinolone-resistant isolates, resistance was associated with three-or-more point mutations in target genes. Typing revealed high genetic diversity with only a few clonal lineages present in all WWTPs. The distribution paths of individual lines could only be traced in exceptional cases with a lack of enrichment of certain lineages. Varying resistance genes and plasmids, as well as fluoroquinolone resistance-associated point mutations in individual isolates, further corroborated the high diversity of 3GCREC in WWTPs. In total, we observed high diversity of 3GCREC inside the tested WWTPs with proof of resistant strains being released into the environment even after treatment processes.

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.

Long time persistence and evolution of carbapenemase-producing Enterobacterales in the wastewater of a tertiary care hospital in Germany.

BACKGROUND: Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. METHODS: In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). RESULTS: Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum ß-lactamases decreased over time. CONCLUSIONS: This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future.

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.

Reading the Score of the Air-Change in Airborne Microbial Load in Contrast to Particulate Matter during Music Making.

The potential impact of music-making on air quality around musicians was inferred at the outset of the SARS-CoV-2 pandemic from measurements on individual musical instruments and from theoretical considerations. However, it is unclear to what extent playing together in an orchestra under optimal ventilation conditions really increases infection risks for individual musicians. In this study, changes in indoor air quality were assessed by measuring common parameters, i.e., temperature, relative humidity, and carbon dioxide, along with particle counting and determining the presence of airborne pharyngeal bacteria under different seating arrangements. The study was conducted in cooperation with a professional orchestra on a stage ventilated by high volume displacement ventilation. Even with a full line-up, the particle load was only slightly influenced by the presence of the musicians on stage. At the same time, however, a clear increase in pharyngeal flora could be measured in front of individual instrument groups, but independent of seat spacing. Simultaneous measurement of various air parameters and, above all, the determination of relevant indicator bacteria in the air, enables site-specific risk assessment and safe music-making even during a pandemic.

Evidence of surface contamination in hospital rooms occupied by patients infected with monkeypox, Germany, June 2022.

The extent of monkeypox virus environmental contamination of surfaces is unclear. We examined surfaces in rooms occupied by two monkeypox patients on their fourth hospitalisation day. Contamination with up to 105 viral copies/cm2 on inanimate surfaces was estimated by PCR and the virus was successfully isolated from surfaces with more than 106 copies. These data highlight the importance of strict adherence of hospital staff to recommended protective measures. If appropriate, pre-exposure or early post-exposure vaccination should be considered for individuals at risk.

High burden and diversity of carbapenemase-producing Enterobacterales observed in wastewater of a tertiary care hospital in Germany.

Hospitals are one of the main reservoirs of multi-resistant Enterobacterales (MRE). As MRE are resistant to the most frequently used antibiotics, therapy for patients with MRE infections is challenging. It has been previously described that MRE from hospital wastewater can pass into municipal wastewater and even surface water. In this study, we investigated the diversity and epidemiology of MRE in the wastewater of a large tertiary care hospital. Wastewater samples were collected for a four-day period and tested for the presence of Enterobacterales resistant to 3rd gen. cephalosporins. Representative isolates were further characterized by whole genome sequencing. In 120 ß-glucuronidase-producing isolates, 68 Escherichia coli and, interestingly, also 52 Citrobacter freundii were identified. In 120 ß-glucosidase-producing isolates 45 Serratia marcescens, 34 Klebsiella oxytoca, 32 Enterobacter cloacae and 9 Klebsiella pneumoniae were observed. For all species various MLST sequence types and different clusters of resistance genes were determined, showing a great diversity within the different Enterobacterales, further corroborated by clonal analysis performed by cgMLST. The most prominent clone was wastewater associated E. coli ST635, which accounted for 47.1% of all E. coli isolates. Interestingly, 45.6% of E. coli, 88.5% of C. freundii, 95.6% of S. marcescens, 91.2% of K. oxytoca, 96.9% of E. cloacae and 88.9% of K. pneumoniae isolates carried a carbapenemase gene, indicating a high burden with carbapenemase-producing Enterobacterales. Comparison with clinical isolates from the same hospital displayed few clonal matches. One wastewater isolate of K. pneumoniae was identified to be closely related compared to a clone that had been introduced into the hospital during an outbreak four years earlier. One E. coli isolate was identified as identical to an isolate from a patient, with inpatient stay during the sampling period. The data obtained in this study highlight the problem of antibiotic resistance of Enterobacterales in hospital wastewater. In particular, the clustered occurrence of carbapenemase genes is of great concern and underscores the problem of increasingly scarce antibiotic options against these bacteria.

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.