Emergence of OXA-48-like producing Citrobacter species, Germany, 2011 to 2022.

BackgroundCarbapenemase-producing Enterobacterales are a public health threat worldwide and OXA-48 is the most prevalent carbapenemase in Germany and western Europe. However, the molecular epidemiology of OXA-48 in species other than Escherichia coli and Klebsiella pneumoniae remains poorly understood.AimTo analyse the molecular epidemiology of OXA-48 and OXA-48-like carbapenemases in Citrobacter species (spp.) in Germany between 2011 and 2022.MethodsData of 26,822 Enterobacterales isolates sent to the National Reference Centre (NRC) for Gram-negative bacteria were evaluated. Ninety-one Citrobacter isolates from 40 German hospitals harbouring bla OXA-48/OXA-48­like were analysed by whole genome sequencing and conjugation experiments.ResultsThe frequency of OXA-48 in Citrobacter freundii (CF) has increased steadily since 2011 and is now the most prevalent carbapenemase in this species in Germany. Among 91 in-depth analysed Citrobacter spp. isolates, CF (n = 73) and C. koseri (n = 8) were the most common species and OXA-48 was the most common variant (n = 77), followed by OXA-162 (n = 11) and OXA­181 (n = 3). Forty percent of the isolates belonged to only two sequence types (ST19 and ST22), while most other STs were singletons. The plasmids harbouring bla OXA­48 and bla OXA-162 belonged to the plasmid types IncL (n = 85) or IncF (n = 3), and plasmids harbouring bla OXA­181 to IncX3 (n = 3). Three IncL plasmid clusters (57/85 IncL plasmids) were identified, which were highly transferable in contrast to sporadic plasmids.ConclusionIn CF in Germany, OXA-48 is the predominant carbapenemase. Dissemination is likely due to distinct highly transmissible plasmids harbouring bla OXA­48 or bla OXA-48-like and the spread of the high-risk clonal lineages ST19 and ST22.

Uncovering the spread of drug-resistant bacteria through next-generation sequencing based surveillance: transmission of extended-spectrum ß-lactamase-producing Enterobacterales by a contaminated duodenoscope.

Contamination of duodenoscopes is a significant concern due to the transmission of multidrug-resistant organisms (MDROs) among patients who undergo endoscopic retrograde cholangiopancreatography (ERCP), resulting in outbreaks worldwide. In July 2020, it was determined that three different patients, all had undergone ERCP with the same duodenoscope, were infected. Two patients were infected with blaCTX-M-15 encoding Citrobacter freundii, one experiencing a bloodstream infection and the other a urinary tract infection, while another patient had a bloodstream infection caused by blaSHV-12 encoding Klebsiella pneumoniae. Molecular characterization of isolates was available as every ESBL-producing isolate undergoes Next-Generation Sequencing (NGS) for comprehensive genomic analysis in our center. After withdrawing the suspected duodenoscope, we initiated comprehensive epidemiological research, encompassing case investigations, along with a thorough duodenoscope investigation. Screening of patients who had undergone ERCP with the implicated duodenoscope, as well as a selection of hospitalized patients who had ERCP with a different duodenoscope during the outbreak period, led to the discovery of three additional cases of colonization in addition to the three infections initially detected. No microorganisms were detected in eight routine culture samples retrieved from the suspected duodenoscope. Only after destructive dismantling of the duodenoscope, the forceps elevator was found to be positive for blaSHV-12 encoding K. pneumoniae which was identical to the isolates detected in three patients. This study highlights the importance of using NGS to monitor the transmission of MDROs and demonstrates that standard cultures may fail to detect contaminated medical equipment such as duodenoscopes.

Enterobacterales carrying chromosomal AmpC ß-lactamases in Europe (EuESCPM): Epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020-2022.

INTRODUCTION: The ESCPM group (Enterobacter species including Klebsiella aerogenes - formerly Enterobacter aerogenes, Serratia species, Citrobacter freundii complex, Providencia species and Morganella morganii) has not yet been incorporated into systematic surveillance programs. METHODS: We conducted a multicentre retrospective observational study analysing all ESCPM strains isolated from blood cultures in 27 European hospitals over a 3-year period (2020-2022). Diagnostic approach, epidemiology, and antimicrobial susceptibility were investigated. RESULTS: Our study comprised 6,774 ESCPM isolates. MALDI-TOF coupled to mass spectrometry was the predominant technique for bacterial identification. Susceptibility to new ß-lactam/ß-lactamase inhibitor combinations and confirmation of AmpC overproduction were routinely tested in 33.3% and 29.6% of the centres, respectively. The most prevalent species were E. cloacae complex (44.8%) and S. marcescens (22.7%). Overall, third-generation cephalosporins (3GC), combined third- and fourth-generation cephalosporins (3GC + 4GC) and carbapenems resistance phenotypes were observed in 15.7%, 4.6%, and 9.5% of the isolates, respectively. AmpC overproduction was the most prevalent resistance mechanism detected (15.8%). Among carbapenemase-producers, carbapenemase type was provided in 44.4% of the isolates, VIM- (22.9%) and OXA-48-enzyme (16%) being the most frequently detected. E. cloacae complex, K. aerogenes and Providencia species exhibited the most notable cumulative antimicrobial resistance profiles, with the former displaying 3GC, combined 3GC + 4GC and carbapenems resistance phenotypes in 15.2%, 7.4%, and 12.8% of the isolates, respectively. K. aerogenes showed the highest rate of both 3GC resistant phenotype (29.8%) and AmpC overproduction (32.1%), while Providencia species those of both carbapenems resistance phenotype (42.7%) and carbapenemase production (29.4%). ESCPM isolates exhibiting both 3GC and combined 3GC + 4GC resistance phenotypes displayed high susceptibility to ceftazidime/avibactam (98.2% and 95.7%, respectively) and colistin (90.3% and 90.7%, respectively). Colistin emerged as the most active drug against ESCPM species (except those intrinsically resistant) displaying both carbapenems resistance phenotype (85.8%) and carbapenemase production (97.8%). CONCLUSIONS: This study presented a current analysis of ESCPM species epidemiology in Europe, providing insights to inform current antibiotic treatments and guide strategies for antimicrobial stewardship and diagnostics.

Detection of rare carbapenemases in Enterobacterales-comparison of two colorimetric and three CIM-based carbapenemase assays.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases (n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes.

Investigating environmental transmission to resolve a Bacillus cereus group outbreak in a neonatal intensive care unit using core genome multilocus sequence typing.

BACKGROUND: We analyzed an outbreak of Bacillus cereus group (Bcg) at a single-center neonatal intensive care unit level IV by conducting comprehensive sampling of both patients and the environment. METHODS: Between 06/2020 and 10/2021, all Bcg isolates identified by both regular colonization screening and additional sampling of the environment were subjected to whole-genome sequencing, followed by in vitro extraction of MLST ST, resistance genes and virulence factors. Using publicly available genome sequences, we defined an ad hoc core genome multilocus sequence typing (cgMLST) scheme comprising 2759 target genes for Bcg typing, which we applied to the detected isolates. We have compared the results with a stable cgMLST that was published in the meantime and completed the investigation with a SNP analysis. RESULTS: We analyzed 28 Bcg isolates from patient and environmental samples using MLST and cgMLST. This revealed multiple sequence types, with ST127 being the most common (n = 13). Both cgMLST schemes grouped ten of the 13 ST127 isolates into a cluster, including two invasive isolates from two different patients and several environmental samples. SNP analysis postulated a screen from a ventilation machine as a possible reservoir. CONCLUSION: In sensitive settings such as neonatal intensive care units, considering the environment in outbreak analyses is crucial, especially when investigating potential transmission routes through shared devices. When dealing with widespread bacteria such as Bcg, high-resolution typing techniques are necessary. In this study, we successfully resolved an outbreak of Bcg infections using a custom cgMLST scheme combined with a SNP analysis.

A side-by-side comparison of the new VITEK MS PRIME and the MALDI Biotyper sirius in the clinical microbiology laboratory.

PURPOSE: This study aims to evaluate the performance of two latest generation matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems in routine laboratory settings, focusing on turnaround time (TAT), time to results (TTR), hands-on time, and identification rate. METHODS: We conducted a time and motion study on three workflow scenarios to simulate different laboratory settings. Overall, 618 bacterial isolates from a tertiary hospital's laboratory were processed using the VITEK MS PRIME (bioMérieux) and the MALDI Biotyper sirius (Bruker Daltonics) and their corresponding databases VITEK IVD Database 3.2 and MBT reference library 12. RESULTS: The target preparation process showed no significant difference in TAT, but the Biotyper workflow had a shorter hands-on time by 3 to 6 min. In the measurement process, TTR was three to five times shorter for the Biotyper sirius while hands-on time was significantly shorter for VITEK MS PRIME (approximately 1.5 min per target). The identification rate without retesting was 97.9% for VITEK MS PRIME and 98.9% for Biotyper sirius. Both systems achieved 100% agreement at genus and 96.2% at species level. CONCLUSION: Both systems exhibited excellent identification rates for routine bacterial isolates. Due to its high speed, the Biotyper sirius is suited for laboratories with high sample throughput and a workflow designed for processing larger batches. The VITEK MS PRIME, with its "load and go" system accommodating up to 16 targets, reduces hands-on time, making it a reasonable choice for laboratories with fewer identifications overall but a higher number of targets and a workflow designed for parallel processing on different workstations.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study.

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

Surface water in Lower Saxony: A reservoir for multidrug-resistant Enterobacterales.

The emergence of extended-spectrum ß-lactamase and carbapenemase-producing Enterobacterales (ESBL-E and CPE, respectively) is a threat to modern medicine, as infections become increasingly difficult to treat. These bacteria have been detected in aquatic environments, which raises concerns about the potential spread of antibiotic resistance through water. Therefore, we investigated the occurrence of ESBL-E and CPE in surface water in Lower Saxony, Germany, using phenotypic and genotypic methods. Water samples were collected from two rivers, five water canals near farms, and 18 swimming lakes. ESBL-E and CPE were isolated from these samples using filters and selective agars. All isolates were analyzed by whole genome sequencing. Multidrug-resistant Enterobacterales were detected in 4/25 (16%) water bodies, including 1/2 rivers, 2/5 water canals and 1/18 lakes. Among all samples, isolates belonging to five different species/species complexes were detected: Escherichia coli (n = 10), Enterobacter cloacae complex (n = 4), Citrobacter freundii (n = 3), Citrobacter braakii (n = 2), and Klebsiella pneumoniae (n = 2). Of the 21 isolates, 13 (62%) were resistant at least to 3rd generation cephalosporins and eight (38%) additionally to carbapenems. CPE isolates harbored blaKPC-2 (n = 5), blaKPC-2 and blaVIM-1 (n = 2), or blaOXA-181 (n = 1); additionally, mcr-9 was detected in one isolate. Two out of eight CPE isolates were resistant to cefiderocol and two to colistin. Resistance to 3rd generation cephalosporins was mediated by ESBL (n = 10) or AmpC (n = 3). The presence of AmpC-producing Enterobacterales, ESBL-E and CPE in northern German surface water samples is alarming and highlights the importance of aquatic environments as a potential source of MDR bacteria.

Vancomycin-resistant enterococci (VRE) in hospital settings across European borders: a scoping review comparing the epidemiology in the Netherlands and Germany.

The rising prevalence of vancomycin-resistant enterococci (VRE) is a matter of concern in hospital settings across Europe without a distinct geographical pattern. In this scoping review, we compared the epidemiology of vancomycin-resistant Enterococcus spp. in hospitals in the Netherlands and Germany, between 1991 and 2022. We searched PubMed and summarized the national antibiotic resistance surveillance data of the two countries. We included 46 studies and summarized national surveillance data from the NethMap in the Netherlands, the National Antimicrobial Resistance Surveillance database in Germany, and the EARS-Net data. In total, 12 studies were conducted in hospitals in the Netherlands, 32 were conducted in German hospitals, and an additional two studies were conducted in a cross-border setting. The most significant difference between the two countries was that studies in Germany showed an increasing trend in the prevalence of VRE in hospitals, and no such trend was observed in studies in the Netherlands. Furthermore, in both Dutch and German hospitals, it has been revealed that the molecular epidemiology of VREfm has shifted from a predominance of vanA towards vanB over the years. According to national surveillance reports, vancomycin resistance in Enterococcus faecium clinical isolates fluctuates below 1% in Dutch hospitals, whereas it follows an increasing trend in German hospitals (above 20%), as supported by individual studies. This review demonstrates that VRE is more frequently encountered in German than in Dutch hospitals and discusses the underlying factors for the difference in VRE occurrence in these two neighboring countries by comparing differences in healthcare systems, infection prevention control (IPC) guidelines, and antibiotic use in the Netherlands and Germany.

Janus kinase-inhibition modulates hepatitis E virus infection.

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.

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.

Proteus mirabilis - analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection.

OBJECTIVES: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays. METHODS: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing. RESULTS: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-ß-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-ß-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France. DISCUSSION: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.

Comparison of Five Different Selective Agar for the Detection of Vancomycin-Resistant Enterococcus faecium.

Five commercially available selective agar were evaluated regarding sensitivity and specificity to detect vancomycin-resistant Enterococcus (E.) faecium. Altogether 187 E. faecium strains were included, comprising 119 van-carrying strains (phenotypically vancomycin-resistant n = 105; phenotypically vancomycin-susceptible VVE-B n = 14) and 68 vancomycin-susceptible isolates. Limit of detection was calculated for each selective agar for pure cultures, stool suspensions and artificial rectal swabs. After 24-h incubation sensitivity ranged between 91.6% and 95.0%. It increased in 2 out of 5 agar after 48-h incubation. Specificity ranged between 94.1% and 100% and was highest after 24 h in 4 out of the 5 agar. Sensitivity of van-carrying phenotypically vancomycin-resistant strains was higher after 24 h (97.1-100%) and 48 h (99.1-100%) when compared to van-carrying strains that tested vancomycin-susceptible (50.0-57.1% after both incubation periods). Overall, chromID VRE, CHROMagar VRE and Brilliance VRE demonstrated the highest detection rates after 24 h. Detection rates of Chromatic VRE and VRESelect improved after 48 h. Adjustment of incubation time depending on the applied media may be advised. As detection of VVE-B was impeded with all selective agar, screening for vancomycin-resistant enterococci relying solely on selective media would not be recommended for critical clinical samples, but rather in combination with molecular methods to improve detection of these strains. Furthermore, stool samples were demonstrated to be superior to rectal swabs and should be favoured, if possible, in screening strategies.

Susceptibility of Aerococcus urinae and Aerococcus sanguinicola to Standard Antibiotics and to Nitroxoline.

Aerococcus urinae and Aerococcus sanguinicola have been increasingly recognized as causative agents of urinary tract infection (UTI) during the last decade. Nitroxoline achieves high urinary concentrations after oral administration and is recommended in uncomplicated UTI in Germany, but its activity against Aerococcus spp. is unknown. The aim of this study was to assess the in vitro susceptibility of clinical Aerococcus species isolates to standard antibiotics and to nitroxoline. Between December 2016 and June 2018, 166 A. urinae and 18 A. sanguinicola isolates were recovered from urine specimens sent to the microbiology laboratory of the University Hospital of Cologne, Germany. Susceptibility to standard antimicrobials was analyzed by disk diffusion (DD) according to EUCAST methodology, nitroxoline was tested by DD and agar dilution. Susceptibility of Aerococcus spp. to benzylpenicillin, ampicillin, meropenem, rifampicin, nitrofurantoin, and vancomycin was 100% and resistance was documented only against ciprofloxacin (20 of 184; 10.9%). MICs of nitroxoline in A. urinae isolates were low (MIC50/90 1/2 mg/L) while significantly higher MICs were observed in A. sanguinicola (MIC50/90 64/128 mg/L). If the EUCAST nitroxoline breakpoint for E. coli and uncomplicated UTI was applied (16 mg/L), 97.6% of A. urinae isolates would be interpreted as susceptible while all A. sanguinicola isolates would be considered resistant. Nitroxoline demonstrated high activity against clinical A. urinae isolates, but low activity against A. sanguinicola. Nitroxoline is an approved antimicrobial for UTI and could be an alternative oral drug to treat A. urinae urinary tract infection, yet clinical studies are needed to demonstrate this potential in vivo. IMPORTANCE A. urinae and A. sanguinicola have been increasingly recognized as causative agents in urinary tract infections. Currently, there are few data available on the activity of different antibiotics against these species and no data on nitroxoline. We demonstrate that clinical isolates in Germany are highly susceptible to ampicillin, while resistance to ciprofloxacin was common (10.9%). Additionally, we show that nitroxoline is highly active against A. urinae, but not against A. sanguinicola, which based on the presented data, should be considered intrinsically resistant. The presented data will help to improve the therapy of urinary tract infections by Aerococcus species.

Usefulness of screening for Candida auris colonisation in international patients admitted to a large university hospital.

INTRODUCTION: Candida auris is an emerging pathogen in health care-associated infections. In contrast to many other countries with rising numbers of C. auris, only seven cases have been reported in Germany from 2015 to 2017, mostly from patients who received prior medical treatment abroad. We therefore established a mandatory screening for C. auris colonisation at our tertiary care centre for all patients who were admitted as international patients or previously hospitalised in a foreign country within the past 6 months. METHODS: Colonisation of patients was assessed using a previously established screening protocol for multidrug resistant bacteria. Since 2017, all screening samples were additionally analysed for C. auris using CHROMagar Candida (CHROMagar, Paris, France). Yeast isolates were identified using matrix-assisted laser ionisation time-of-flight (MALDI TOF), except for C. albicans (identified by the typical green colour on chromogenic agar). Data were analysed retrospectively. RESULTS: Our study cohort included 655 patients and an overall number of 1399 samples. Fifty-three patients were colonised with Candida species (C. albicans, n = 37; C. glabrata, n = 14; others n = 9). No case of C. auris was detected. Candida spp. were mainly detected from respiratory samples (5.4% positive) and gastrointestinal specimen (5.2%). Laboratory costs were 14,689 € and analyses resulted in 98.7 h of additional technician's work. CONCLUSION: No colonisation with C. auris was detected among patients with previous hospitalisation abroad. Universal C. auris screening of patients with any contact to foreign health care does not seem to be cost-effective in our setting and more targeted screening strategies have to be developed.

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-ß-lactamase-producing Gram-negative bacteria.

Ceftazidime-avibactam is one of the last resort antimicrobial agents for the treatment of carbapenem-resistant, Gram-negative bacteria. Metallo-ß-lactamase-producing bacteria are considered to be ceftazidime-avibactam resistant. Here, we evaluated a semi-automated antimicrobial susceptibility testing system regarding its capability to detect phenotypic ceftazidime-avibactam resistance in 176 carbapenem-resistant, metallo-ß-lactamase-producing Enterobacterales and Pseudomonas aeruginosa isolates. Nine clinical isolates displayed ceftazidime-avibactam susceptibility in the semi-automated system and six of these isolates were susceptible by broth microdilution, too. In all nine isolates, metallo-ß-lactamase-mediated hydrolytic activity was demonstrated with the EDTA-modified carbapenemase inactivation method. As zinc is known to be an important co-factor for metallo-ß-lactamase activity, test media of the semi-automated antimicrobial susceptibility testing system and broth microdilution were supplemented with zinc. Thereby, the detection of phenotypic resistance was improved in the semi-automated system and in broth microdilution. Currently, ceftazidime-avibactam is not approved as treatment option for infections by metallo-ß-lactamase-producing, Gram-negative bacteria. In infections caused by carbapenem-resistant Gram-negatives, we therefore recommend to rule out the presence of metallo-ß-lactamases with additional methods before initiating ceftazidime-avibactam treatment.

Emergence of Tn1999.7, a New Transposon in blaOXA-48-Harboring Plasmids Associated with Increased Plasmid Stability.

OXA-48 is the most common carbapenemase in Enterobacterales in Germany and many other European countries. Depending on the genomic location of blaOXA-48, OXA-48-producing isolates vary in phenotype and intra- and interspecies transferability of blaOXA-48. In most bacterial isolates, blaOXA-48 is located on one of seven variants of Tn1999 (Tn1999.1 to Tn1999.6 and invTn1999.2). Here, a novel Tn1999 variant, Tn1999.7, is described, which was identified in 11 clinical isolates from 2016 to 2020. Tn1999.7 differs from Tn1999.1 by the insertion of the 8,349-bp Tn3 family transposon Tn7442 between the lysR gene and blaOXA-48 open reading frame. Tn7442 carries genes coding for a restriction endonuclease and a DNA methyltransferase as cargo, forming a type III restriction modification system. Tn1999.7 was carried on an ~71-kb IncL plasmid in 9/11 isolates. In one isolate, Tn1999.7 was situated on an ~76-kb plasmid, harboring an additional insertion sequence in the plasmid backbone. In one isolate, the plasmid size is only ~63 kb due to a deletion adjacent to Tn7442 that extends into the plasmid backbone. Mean conjugation rates of the Tn1999.7-harboring plasmids in J53 ranged from 4.47 × 10-5 to 2.03 × 10-2, similar to conjugation rates of other pOXA-48-type IncL plasmids. The stability of plasmids with Tn1999.7 was significantly higher than that of a Tn1999.2-harboring plasmid in vitro. This increase in stability could be related to the insertion of a restriction-modification system, which can promote postsegregational killing. The increased plasmid stability associated with Tn1999.7 could contribute to the further spread of OXA-48.

Patterns and trends of antibacterial treatment in patients with urinary tract infections, 2015-2019: an analysis of health insurance data.

BACKGROUND: Urinary tract infections are among the most common reason for encounter and subsequent antibiotic prescriptions. Due to the risk of collateral damage and increasing resistance rates, explicit recommendations against the use of fluoroquinolones like ciprofloxacin in uncomplicated urinary tract infections have been issued. However, to what extent these recommendations were followed and if there are relevant differences between the disciplines involved (general practitioners, urologists, paediatricians and gynaecologists) are unknown. METHODS: We used anonymized data from a local statutory health insurance (SHI) company, which covered about 38% of all SHI-insured persons in the federal state of Bremen, Germany between 2015-2019. Data included demographics, outpatient diagnoses and filled prescriptions on an individual level. RESULTS: One-year prevalence of urinary tract infections was 5.8% in 2015 (females: 9.2%, males: 2.5%). Of all 102,715 UTI cases, 78.6% referred to females and 21.4% to males, 6.0% of cases were younger than 18 years. In females, general practitioners were the most common diagnosing speciality (52.2%), followed by urologists (20.0%) and gynaecologists (16.1%). Overall, fluoroquinolones were most often prescribed (26.3%), followed by fosfomycin (16.1%) and the combination of sulfamethoxazole and trimethoprim (14.2%). Fluoroquinolones were most often prescribed by urologists and general practitioners, while gynaecologists preferred fosfomycin. During the study period, shares of fluoroquinolones decreased from 29.4% to 8.7% in females and from 45.9% to 22.3% in males. CONCLUSIONS: Despite a clear trend toward a more guideline adherent prescription pattern, there is still room for improvement regarding the use of second-line antibiotics especially fluoroquinolones. The choice of antibiotics prescribed differs between specialities with higher uptake of guideline-recommended antibiotics by gynaecologists, mainly because of higher prescription shares of fosfomycin.

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study.

Background: Ibrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. Objective: The aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. Methods: Ibrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. Results: Ibrexafungerp MICs ranged from 0.016 to ≥8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06-≥8 mg/L). Modal MICs/MIC50s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. Conclusion: Ibrexafungerp showed a potent in vitro activity against Candida.

In vitro activity of mecillinam, temocillin and nitroxoline against MDR Enterobacterales.

Background: With increasing resistance to common antibiotics the treatment of urinary tract infections has become challenging and alternative therapeutic options are needed. In the present study, we evaluate the activity of three older and less frequently used antibiotics against MDR Enterobacterales. Methods: Susceptibility of mecillinam, temocillin and nitroxoline was assessed in Enterobacterales isolated from urinary specimens with elevated MICs of third-generation cephalosporins. Susceptibility was determined by the recommended reference MIC methods and additionally by disc diffusion. All isolates were characterized for common ß-lactamases by phenotypic and molecular assays. Results: In total 394 Enterobacterales were included. The most common resistance mechanisms were ESBLs (n = 273), AmpC (n = 132), carbapenemases [n = 12, including OXA-48-like (n = 8), VIM (n = 2), KPC (n = 1) and NDM (n = 1)] or others (n = 2). Resistance was observed in 59% of isolates to ceftazidime, in 41% to piperacillin/tazobactam and in 54% to ciprofloxacin. In comparison, resistance was less frequent against mecillinam (15%), temocillin (13%) or nitroxoline (2%). Mecillinam showed higher activity in Enterobacter spp., Escherichia coli and in OXA-48-like-producing isolates compared with temocillin, which was more active in Proteus mirabilis and in ESBL-producing isolates. Activity of nitroxoline was high against all isolates, including carbapenemase-producing isolates. Correlation between disc diffusion and MIC methods was good for mecillinam and moderate for temocillin and nitroxoline. Conclusions: Mecillinam, temocillin and nitroxoline show good to excellent in vitro activity in MDR Enterobacterales. The activity of mecillinam and temocillin was higher in certain species and restricted depending on ß-lactamase production while nitroxoline showed universally high activity irrespective of species or ß-lactamase present.