Linezolid Resistance Genes and Mutations among Linezolid-Susceptible Enterococcus spp.-A Loose Cannon?

The National Reference Centre for Enterococci receives an increasing number of linezolid-resistant Enterococcus isolates. Linezolid (LIN) resistance is mediated by G2576T 23S rDNA gene mutations and/or acquisition of resistance genes (cfr, optrA, poxtA). There are anecdotal reports that those resistance traits may be present in phenotypically linezolid-susceptible isolates. We aimed to determine the prevalence of LIN resistance genes and mutations in enterococci with a LIN MIC of 4 mg/L in broth microdilution (EUCAST = susceptible) isolated from German hospital patients 2019-2021. LIN MICs were additionally determined by ETEST® and VITEK2. Selected strains were subjected to LIN selective pressure and growth was monitored with increasing antibiotic concentrations. We received 195 isolates (LIN MIC = 4 mg/L). In total, 78/195 (40%) isolates contained either a putative resistance gene, the G2576T mutation, or a combination thereof. Very major error was high for broth microdilution. The ability to predict phenotypic resistance from genotypic profile was highest for G2576T-mediated resistance. Selection experiments revealed that, in particular, E. faecium isolates with resistance gene mutations or poxtA rapidly adapt to MICs above the clinical breakpoint. In conclusion, LIN resistance genes and mutations can be observed in phenotypically linezolid-susceptible enterococci. Those isolates may rapidly develop resistance under LIN selective pressure potentially leading to treatment failure.

CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

BNT162b2 vaccination reduced infections and transmission in a COVID-19 outbreak in a nursing home in Germany, 2021.

BACKGROUND: A SARS-CoV-2 outbreak was detected in a nursing home in February 2021 after residents and staff had received two doses of BNT162b2 vaccine in January 2021. METHODS: Nursing home staff, long-term residents and day-care receivers were included in a retrospective cohort study. We calculated attack rates (AR), secondary AR (SAR) and their 95% binomial confidence interval (CI), and we compared them using Fisher's exact test or chi-squared test, depending on the sample size. We used Poisson regression with robust error estimates to calculate vaccine effectiveness against SARS-COV-2 infections. We selected variables based on directed acyclic graphs. As a proxy for viral load at diagnosis, we compared the mean Ct values at diagnosis using t tests or Mann-Whitney U tests. RESULTS: The adjusted vaccine effectiveness against infection was 56% (95% CI: 15-77%, p = 0.04). Ct values at diagnosis were higher when intervals after receiving the second vaccination were longer (>21 vs. ≤21 days: 4.48 cycles, p = 0.08). The SAR was 67% lower in households of vaccinated (2/9 [22.2%]) than of unvaccinated infected staff (12/18 [66.7%]; p = 0.046). Vaccination rates were lowest among staff with close physical contact to care-receivers (46%). The highest AR in vaccinated staff had those working on wards (14%). CONCLUSIONS: Vaccination reduced the risk for SARS-CoV-2 infection, viral load and transmission; however, non-pharmaceutical interventions remain essential to reduce transmission of SARS-CoV-2 infections, even for vaccinated individuals. Vaccination coverage of staff ought to increase reduction of infections among themselves, their household members and residents.

Controlling an Unprecedented Outbreak with Vancomycin-Resistant Enterococcus faecium in Germany, October 2015 to November 2019.

Hospital outbreaks with vancomycin-resistant enterococci (VRE) pose a serious health threat and a challenge to infection prevention and control (IPC). We herein report on a VRE outbreak of unprecedented extent in Southern Germany (October 2015-November 2019). We used descriptive epidemiology and whole-genome sequencing (WGS) for a detailed outbreak investigation. Of the 2905 cases, 2776 (95.3%) were colonized, whereas from 127 (3.7%), VRE could be isolated from otherwise sterile body fluids or sites unlikely for enterococci colonization. Cases had a median age of 78 years (IQR 68-84) and 1339/2905 (46%) were female. The majority of isolates sequenced belonged to the clonal lineage ST80/CT1013 (212/397, 53%). Nosocomial transmission was observed as well as the constant import of VRE into the hospital. Extensive IPC measures were implemented and terminated the outbreak in late 2019, eventually. Our study shows that the combination of epidemiological and genomic analyses is indispensable for comprehensive outbreak investigations. The adaptation of IPC measures to these findings, their timely implementation, and strict execution also allow containment of large VRE outbreaks in hospital settings.

Combined Clinical, Epidemiological, and Genome-Based Analysis Identified a Nationwide Outbreak of Burkholderia cepacia Complex Infections Caused by Contaminated Mouthwash Solutions.

Background: In September 2018, Burkholderia cepacia complex (BCC) infections in 3 patients associated with exposure to a mouthwash solution (MWS) were reported to the Robert Koch Institute (RKI). As the product was still on the market and the scale of the outbreak was unclear, a nation-wide investigation was initiated. Methods: We aimed to investigate BCC infections/colonizations associated with MWS. Hospitals, laboratories, and public health services were informed that BCC isolates should be sent to the RKI. These isolates were typed by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) including development of an ad hoc core genome MLST (cgMLST) scheme. Results: In total, 36 patients from 6 hospitals met the case definition, the last patient in November 2018. Twenty-nine isolates from 26 of these patients were available for typing. WGS analysis revealed 2 distinct cgMLST clusters. Cluster 1 (Burkholderia arboris) contained isolates from patients and MWS obtained from 4 hospitals and isolates provided by the manufacturer. Patient and MWS isolates from another hospital were assigned to cluster 2 (B. cepacia). Conclusions: The combined clinical, epidemiological, and microbiological investigation, including whole-genome analysis, allowed for uncovering a supraregional BCC outbreak in health care settings. Strains of B. arboris and B. cepacia were identified as contaminating species of MWS bottles and subsequent colonization and putative infection of patients in several hospitals. Despite a recall of the product by the manufacturer in August 2018, the outbreak lasted until December 2018. Reporting of contaminated medical products and recalls should be optimized to protect patients.

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients.

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.

Factors preventing SARS-CoV-2 transmission during unintentional exposure in a GP practice: a cohort study of patient contacts; Germany, 2020.

Two general practitioners (GPs) with SARS-CoV-2 infection provided in-person patient care to patients of their joint medical practice before and after symptom onset, up until SARS-CoV-2 laboratory confirmation. Through active contact tracing, the local public health authorities recruited the cohort of patients that had contact with either GP in their putative infectious period. In this cohort of patient contacts, we assess the frequency and determinants of SARS-CoV-2-transmission from GPs to patients. We calculated incidence rate ratios (IRR) to explore the type of contact as an explanatory variable for COVID-19 cases. Among the cohort of 83 patient contacts, we identified 22 (27%) COVID-19 cases including 17 (21%) possible, three (4%) probable and two (2%) confirmed cases. All 22 cases had contact with a GP when the GP did not wear a mask, and/or when contact was ≥10 min. Importantly, patients who had contact <10 min with a GP wearing a facemask were at reduced risk (IRR 0.21; 95% CI 0.01-0.99) of COVID-19. This outbreak investigation adds to the body of evidence in supporting current guidelines on measures at preventing the transmission of SARS-CoV-2 in an outpatient setting.

Corrigendum: Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in Staphylococcus aureus.

[This corrects the article DOI: 10.3389/fmicb.2021.639660.].

Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in Staphylococcus aureus.

BACKGROUND: As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For S. aureus this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical S. aureus isolates. MATERIALS/METHODS: To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using in silico analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes. RESULTS: GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within mprF and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of mprF. Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome mec (SCCmec) revealed various mecA SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mecA mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in S. aureus. CONCLUSION: We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in S. aureus. However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.

Acinetobacter stercoris sp. nov. isolated from output source of a mesophilic german biogas plant with anaerobic operating conditions.

The Gram-stain-negative, oxidase negative, catalase positive strain KPC-SM-21T, isolated from a digestate of a storage tank of a mesophilic German biogas plant, was investigated by a polyphasic taxonomic approach. Phylogenetic identification based on the nearly full-length 16S rRNA gene revealed highest gene sequence similarity to Acinetobacter baumannii ATCC 19606T (97.0%). Phylogenetic trees calculated based on partial rpoB and gyrB gene sequences showed a distinct clustering of strain KPC-SM-21T with Acinetobacter gerneri DSM 14967T = CIP 107464T and not with A. baumannii, which was also supported in the five housekeeping genes multilocus sequence analysis based phylogeny. Average nucleotide identity values between whole genome sequences of strain KPC-SM-21T and next related type strains supported the novel species status. The DNA G + C content of strain KPC-SM-21T was 37.7 mol%. Whole-cell MALDI-TOF MS analysis supported the distinctness of the strain to type strains of next related Acinetobacter species. Predominant fatty acids were C18:1 ω9c (44.2%), C16:0 (21.7%) and a summed feature comprising C16:1 ω7c and/or iso-C15:0 2-OH (15.3%). Based on the obtained genotypic, phenotypic and chemotaxonomic data we concluded that strain KPC-SM-21T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter stercoris sp. nov. is proposed. The type strain is KPC-SM-21T (= DSM 102168T = LMG 29413T).

Analysis of Asymptomatic and Presymptomatic Transmission in SARS-CoV-2 Outbreak, Germany, 2020.

We determined secondary attack rates (SAR) among close contacts of 59 asymptomatic and symptomatic coronavirus disease case-patients by presymptomatic and symptomatic exposure. We observed no transmission from asymptomatic case-patients and highest SAR through presymptomatic exposure. Rapid quarantine of close contacts with or without symptoms is needed to prevent presymptomatic transmission.

The Mosaic mtr Locus as Major Genetic Determinant of Azithromycin Resistance of Neisseria gonorrhoeae-Germany, 2018.

Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.

Risk factors and outcomes associated with the carriage of tigecycline- and vancomycin-resistant Enterococcus faecium.

OBJECTIVES: Vancomycin-resistant E. faecium (VRE) is a common cause of healthcare-associated infections. The emergence of VRE with tigecycline resistance (TVRE) is increasing but its impact on patient outcome is still not well defined. This study aimed to assess risk factors for the acquisition of TVRE and of patient outcomes associated with TVRE carriage/infection. METHODS: At the University Hospital Frankfurt, we conducted a matched pair TVRE-VRE analysis to identify risk factors for TVRE carriage. Bed-to-bed contacts and potential transmission routes were reconstructed. TVRE were whole-genome sequenced to confirm suspected transmission events and to identify tigecycline resistance mechanisms. RESULTS: 76 TVRE cases were identified between 02/2014-04/2017 and compared to VRE colonized or infected controls. TVRE carriage was associated with exposure to tigecycline, an increased rate of bloodstream infections (BSI) with VRE or Candida spp., and higher mortality. Whole-genome sequencing-based analysis of 24 TVRE provided evidence for transmissions of TVRE, also across different wards. CONCLUSIONS: Tigecycline exposure is the main risk factor for TVRE carriage. VRE/TVRE- and Candida-BSI are associated with worse clinical outcome. Hospital transmission of TVRE may occur despite strict contact precautions, whereas both antimicrobial stewardship and infection control interventions are of high importance to prevent emergence and spread of TVRE.

Thirty years of VRE in Germany - "expect the unexpected": The view from the National Reference Centre for Staphylococci and Enterococci.

Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

The status of the genus Prolinoborus (Pot et al. 1992) and the species Prolinoborus fasciculus (Pot et al. 1992).

On the basis of two other publications (Yarza et al. 2013; Nemec et al. 2019) and on the basis of resequencing of the 16S rRNA gene of Prolinoborus fasciculus CIP 103579T it is concluded that Prolinoborus fasciculus CIP 103579T, which is the only available strain of the species from culture collections, does not conform to the original description given by Pot et al. (1992). The strain investigated is a member of the genus Acinetobacter within the Moraxellaceae, a family of the Gammaproteobacteria and not a member of the Betaproteobacteria as originally proposed. Prolinoborus fasciculus CIP 103579T shared 99.8 % 16S rRNA gene sequence similarity with Acinetobacter lwoffii DSM 2403T. The two strains clustered together by rpoB- and core genome-based phylogenetic analyses and shared an average nucleotide identity of 96.47% (reciprocal, 96.56 %) and a digital genome distance calculation (GGDC) value of 66.9 %. Furthermore, the two strains shared matrix-assisted laser desorption/ionization time of flight MS profiles to a high extent and showed highly similar cellular fatty acid profiles and physiological substrate utilization patterns. It is proposed that the Judicial commission consider (1) that the strain currently deposited as CIP 103579 be recognized as a member of Acinetobacter lwoffii; (2) placing Prolinoborus fasciculus (Pot et al. 1992) on the list of rejected names if a suitable replacement strain, or a neotype strain cannot be found within 2 years of publication of this request; and (3) place the genus name Prolinoborus (Pot et al. 1992) on the list of rejected names [Recommendation 20D (3) of the Code].

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Comprehensive integrated NGS-based surveillance and contact-network modeling unravels transmission dynamics of vancomycin-resistant enterococci in a high-risk population within a tertiary care hospital.

Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.

Comparative Target Analysis of Chlorinated Biphenyl Antimicrobials Highlights MenG as a Molecular Target of Triclocarban.

Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif, which is also present in other antibiotics, including the recently reported small molecule PK150. We show here that, like PK150, TCC exhibits an inhibitory effect on Staphylococcus aureus menaquinone metabolism via inhibition of the biosynthesis protein demethylmenaquinone methyltransferase (MenG). However, the activity spectrum (MIC90) of TCC across a broad range of multidrug-resistant staphylococcus and enterococcus strains was much narrower than that of PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 mode of action. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profiles of TCC and TCS were further investigated by proteomic analysis, showing complex but rather distinct changes in the protein expression profile of S. aureus Downregulation of the arginine deiminase pathway provided additional evidence for an effect on bacterial energy metabolism by TCC.IMPORTANCE TCC's widespread use as an antimicrobial agent has made it a ubiquitous environmental pollutant despite its withdrawal due to ecological and toxicological concerns. With its antibacterial mechanism of action still being unknown, we undertook a comparative target analysis between TCC, PK150 (a recently discovered antibacterial compound with structural resemblance to TCC), and TCS (another widely employed chlorinated biphenyl antimicrobial) in the bacterium Staphylococcus aureus We show that there are distinct differences in each compound's mode of action, but also identify a shared target between TCC and PK150, the interference with menaquinone metabolism by inhibition of MenG. The prevailing differences, however, which also manifest in a remarkably better broad-spectrum activity of PK150, suggest that even high levels of TCC or TCS resistance observed by continuous environmental exposure may not affect the potential of PK150 or related N,N'-diaryl urea compounds as new antibiotic drug candidates against multidrug-resistant infections.

A Nosocomial Cluster of Tigecycline- and Vancomycin-Resistant Enterococcus faecium Isolates and the Impact of rpsJ and tet(M) Mutations on Tigecycline Resistance.

Tigecycline-resistant enterococci are only rarely detected worldwide. In 2017, the National Reference Centre for Staphylococci and Enterococci noticed a nosocomial cluster of tigecycline- and vancomycin-resistant Enterococcus faecium (TVRE) in a hospital of tertiary care in Northern Germany. Nineteen E. faecium isolates were analyzed by means of antimicrobial susceptibility testing and pulsed-field gel electrophoresis. A subset of isolates was subjected to whole-genome sequencing. The genetic basis of tigecycline resistance was assessed by ResFinder and by comparative analyses to known tetracycline and tigecycline resistance genes. Phylogenetic investigations revealed the clustering of 11 TVRE that exhibited genotype ST117/CT1489. Two tigecycline-susceptible isolates were unrelated. Characterization of the genetic determinant putatively responsible for tigecycline resistance revealed two chromosomal changes in the TVRE population: (1) a deletion within the ribosomal protein gene rpsJ and (2) a serine insertion in and removal of transcriptional regulation of the ribosomal protection protein Tet(M). We here report the first nosocomial cluster of TVRE in a German hospital and disclosed the resistance mechanism that was most likely causative for tigecycline insusceptibility. Clonal spread of TVRE isolates can be assumed because all isolates were highly related and harbored identical chromosomal alterations associated with tigecycline resistance.