Clinical relevance of Staphylococcus saccharolyticus detection in human samples: a retrospective cohort study.

PURPOSE: To characterize the clinical relevance of S. saccharolyticus and to identify criteria to distinguish between infection and contamination. METHODS: We retrospectively investigated clinical features of patients with S. saccharolyticus detection between June 2009 and July 2021. Based on six criteria, infection was considered likely for patients with a score from 3 to 6 points, infection was considered unlikely for patients with a score from 0 to 2 points. We performed group comparison and logistic regression to identify factors than are associated with likely infection. In addition, whole genome sequencing (WGS) of 22 isolates was performed. RESULTS: Of 93 patients in total, 44 were assigned to the group "infection likely" and 49 to the group "infection unlikely". Multiple regression analysis revealed "maximum body temperature during hospital stay" to have the strongest predictive effect on likely infection (adjusted odds ratio 4.40, 95% confidence interval 2.07-9.23). WGS revealed two different clades. Compared to isolates from clade A, isolates from clade B were more frequently associated with implanted medical devices (3/10 vs. 9/12, p = 0.046) and a shorter time to positivity (TTP) (4.5 vs. 3, p = 0.016). Both clades did neither differ significantly in terms of causing a likely infection (clade A 7/10 vs. clade B 5/12, p = 0.23) nor in median length of hospital stay (28 vs. 15.5 days, p = 0.083) and length of stay at the ICU (21 vs. 3.5 days, p = 0.14). CONCLUSION: These findings indicate that S. saccharolyticus can cause clinically relevant infections. Differentiation between infection and contamination remains challenging.

Post-COVID-19-pandemic changes and clinical characteristics of invasive group a streptococcal infections from 2015 to 2023.

PURPOSE: Since winter 2022, invasive GAS (iGAS) infections have re-emerged in Europe, causing severe diseases in children and adults. We aimed to examine whether this reported post-pandemic increase was associated with an increased disease severity and/or a shift in clinical disease phenotypes. METHODS: We performed detailed clinical phenotyping of patients hospitalized with iGAS infections at a 1410-bed tertiary German Medical Center from 01/2015 to 09/2023. RESULTS: One hundred seventy-eight patients were included: 50 children (28.1%) and 128 adults (71.9%). IGAS infections of Q1/2023 exceeded the pre-pandemic average by 551% (1200% for children). The mean age of affected patients shifted significantly post-pandemically (49.5 ± 26.5 to 32.4 ± 28.2 years of age, p < 0.05), mainly due to the higher percentage of children affected with iGAS infections (15.2% pre-pandemic, 44.2% post-pandemic). CFR was significantly lower for children (2%) compared to adults (11.7%) (p < 0.05) and decreased from 13% to 6.5% post-pandemically (p = 0.148). Duration of antibiotic therapy (13.5 (10 to 21) to 10 (9 to 14) days), length of hospital (10 (4 to 25) to 7 (5 to 15) days), and ICU stay (7.0 (2.5 to 18.0) to 5.0 (3.0 to 8.5) days) were shorter post-pandemically. Despite the higher post-pandemic percentage of affected children, PICU admissions (57% before to 32% after), use of catecholamines (28.6% to 11.8%), invasive ventilation (35.7% to 17.6%) and CFR (7% to 0%) were all lower after the pandemic. CONCLUSION: Children were at higher risk for iGAS infections post-pandemically. The surge of post-pandemic iGAS infections was not accompanied by increased iGAS-associated morbidity and mortality.

Genomic Modification of TonB and Emergence of Small-Colony Phenotype in VIM- and NDM-Producing Escherichia coli following Cefiderocol Exposure In Vitro.

Knowledge on resistance mechanisms toward cefiderocol, a novel siderophore-conjugated cephalosporin antibiotic, is still limited. Although the presence of New-Delhi metallo-ß-lactamase has been demonstrated to facilitate the resistance development toward cefiderocol via siderophore receptor mutations in Enterobacter cloacae and Klebsiella pneumoniae, the impact of metallo-ß-lactamases on facilitating such mutations in Escherichia coli is not yet elucidated. Our study aimed to study the effect of the presence of various ß-lactamases, such as NDM-5, VIM-1, KPC-2, and OXA-48, on the development of cefiderocol resistance in E. coli. To this end, we performed liquid mating to transfer these ß-lactamases onto a defined K-12 E. coli background (J53) and exposed these transconjugants to increasing cefiderocol concentrations in a serial passage experiment. Cefiderocol-resistant isolates were genotyped by whole-genome sequencing to investigate the underlying resistance mechanism. Cefiderocol-resistant isolates emerged only in isolates producing VIM-1 and NDM-5 metallo-ß-lactamase, but not in those producing the serine ß-lactamases KPC-2 and OXA-48. We observed two distinct morphological changes of the J53 E. coli strain exhibiting reduced colony size after insertions of transposable elements in the tonB gene leading to alterations in the TonB binding site and morphological changes consistent with the small-colony variant (SCV) phenotype due to mutations in the hemB and hemH genes. Passaging experiments suggested that these phenotypes were highly plastic. The SCV phenotype is attributed to immune evasion and decreased susceptibility toward antibiotics. The emergence of SCV following cefiderocol exposure may have clinical implications for bacterial clearance and warrants further investigation.

Association Between Soluble Notch Ligand Delta-like Ligand 1 and Bleeding Complications in Patients With Dengue Fever Infection.

Bleeding associated with endothelial damage is a key feature of severe dengue fever. In the current study, we investigated whether Notch ligands were associated with bleeding in 115 patients with confirmed dengue infection in Vietnam. Soluble Notch ligands were determined by means of enzyme-linked immunosorbent assay. Seventeen of 115 patients (14.8%) experienced bleeding manifestations. High soluble delta-like ligand 1 (sDLL1) plasma levels was associated with bleeding (median, 15 674 vs 7117 pg/mL; P < .001). Receiver operating characteristic (ROC) curve analysis demonstrated that sDLL1 had the best test performance (area under the ROC curve, 0.852), with 88% sensitivity and 84% specificity. The combination with alanine aminotransferase and aspartate aminotransferase slightly increased sDLL1 performance. sDLL1 may be useful to guide clinical management of patients with patients in endemic settings.

Rapid Development of Cefiderocol Resistance in Carbapenem-resistant Enterobacter cloacae During Therapy Is Associated With Heterogeneous Mutations in the Catecholate Siderophore Receptor cirA.

We report a case of resistance development toward cefiderocol in a patient with intra-abdominal and bloodstream infections caused by carbapenemase-producing Enterobacter cloacae within 21 days of cefiderocol therapy. Whole genome sequencing revealed heterogeneous mutations in the cirA gene, encoding a catecholate siderophore receptor, conferring phenotypic resistance to cefiderocol.

New Delhi Metallo-Beta-Lactamase Facilitates the Emergence of Cefiderocol Resistance in Enterobacter cloacae.

Cefiderocol is a promising novel siderophore cephalosporin for the treatment of multidrug-resistant Gram-negative bacilli and with stability against degradation by metallo-ß-lactamases. Nonetheless, the emergence of cefiderocol in metallo-ß-lactamase-producing Enterobacterales during therapy has been reported on more than one occasion. To understand the underlying mechanisms and factors facilitating the resistance development, we conducted an in vitro evolution experiment using clinical E. cloacae isolates via serial passaging under cefiderocol pressure. In this study, we showed that the presence of the New Delhi metallo-ß-lactamase (NDM) facilitates the emergence of resistance via nonsynonymous mutations of the CirA catecholate siderophore receptor. Inhibition of metallo-ß-lactamase activity using dipicolinic acid prevented the emergence of cefiderocol-resistant mutants successfully. This finding implies that caution should be taken when using cefiderocol for the treatment of infections caused by metallo-ß-lactamase-producing bacteria.

The acquisition of transferable extrachromosomal fec operon is associated with a cefiderocol MIC increase in Enterobacterales.

BACKGROUND: Cefiderocol is a novel siderophore cephalosporin active against MDR Gram-negative bacilli, including MBL-harbouring Enterobacterales. The detection of multiple cefiderocol-resistant blaVIM-carrying Enterobacterales isolates (MIC = 4 mg/L) from a single patient suggested an additional, potentially transferable, resistance determinant as blaVIM typically does not elevate cefiderocol MIC above the resistance threshold. METHODS: Transfer of a mobile genetic element was performed in liquid mating experiments. All donor isolates and transconjugants were characterized by short-read WGS to identify potential resistance determinants. mRNA expression of siderophore receptors was determined by quantitative RT-PCR. Validation was performed by transformation. Antibiotic susceptibility was determined by broth microdilution. RESULTS: Liquid mating experiments indicated the presence of transferable resistance determinants. Comparative genomic analysis of the clinical isolates and their respective transconjugants revealed the transfer of an accessory fec operon (fecABCDEIR). Transformation of the fec operon-containing vector into a TOP10 Escherichia coli led to an elevation of the cefiderocol MIC by at least 16-fold. Higher expression of fecA as a proxy for the fec operon mRNA expression was associated with phenotypic cefiderocol resistance. Both VIM and the accessory fec operon contribute to the elevation of cefiderocol MIC beyond the resistance threshold. The acquisition of an accessory fec operon via liquid mating confers phenotypic cefiderocol resistance in both E. coli J53 and Pseudomonas aeruginosa PAO1, indicating a broad-host-range nature of this mobile resistance determinant. CONCLUSIONS: The emergence of a transferable cefiderocol resistance determinant without prior exposure to the substance is worrisome and should be monitored closely.

Staphylococcus massiliensis isolated from human blood cultures, Germany, 2017-2020.

Clinical and laboratory data on newly described staphylococcal species is rare, which hampers decision-making when such pathogens are detected in clinical specimens. Here, we describe Staphylococcus massiliensis detected in three patients at a university hospital in southwest Germany. We report the discrepancy of microbiological findings between matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 16S-rRNA polymerase chain reaction, and whole-genome sequencing for all three isolates. Our findings highlight the diagnostic pitfalls pertinent to novel and non-model organisms in daily microbiological practice, in whom the correct identification is dependent on database accuracy.

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

AIM: The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected meningitis in a resource-limited setting without extensive bioinformatics expertise. METHODS: DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the EPI2ME cloud platform. The Nanopore sequencing was done in parallel to routine microbiological diagnostics. RESULTS: Nanopore sequencing detected bacterial pathogens to species level in 13 of 30 (43%) samples. CSF culture showed 40% (12/30) positivity. In 21 of 30 patients (70%) with suspected bacterial meningitis, both methods yielded concordant results. About nine of 30 samples showed discordant results, of these five were false positive and four were false negative. In five of the culture negative results, nanopore sequencing was able to detect pathogen genome, due to the higher sensitivity of the molecular diagnostics. In two other samples, the CSF culture revealed Cryptococcus neoformans and Streptococcus pneumoniae, which were not detected by Nanopore sequencing. Overall, using both the cultures and 16S Nanopore sequencing, positivity rate increased from 40% (12/30) to 57% (17/30). CONCLUSION: Next-generation sequencing could detect pathogens within six hours and could become an important tool for both pathogen screening and surveillance in low- and middle-income countries (LMICs) that do not have direct access to extensive bioinformatics expertise.

Direct-PCR from rectal swabs and environmental reservoirs: A fast and efficient alternative to detect blaOXA-48 carbapenemase genes in an Enterobacter cloacae outbreak setting.

Carbapenemase-producing bacteria are a risk factor in clinical settings worldwide. The aim of the study was to accelerate the time to results during an outbreak situation with blaOXA-48-positive Enterobacter cloacae by using a real-time multiplex quantitative PCR (qPCR) directly on rectal swab specimens and on wastewater samples to detect carbapenemase-producing bacteria. Thus, we analyzed 681 rectal swabs and 947 environmental samples during a five-month period by qPCR and compared the results to culture screening. The qPCR showed a sensitivity of 100% by testing directly from rectal swabs and was in ten cases more sensitive than the culture-based methods. Environmental screening for blaOXA-48-carbapenemase genes by qPCR revealed reservoirs of different carbapenemase genes that are potential sources of transmission and might lead to new outbreaks. The rapid identification of patients colonized with those isolates and screening of the hospital environment is essential for earlier patient treatment and eliminating potential sources of nosocomial infections.

Host factors facilitating SARS-CoV-2 virus infection and replication in the lungs.

SARS-CoV-2 is the virus causing the major pandemic facing the world today. Although, SARS-CoV-2 primarily causes lung infection, a variety of symptoms have proven a systemic impact on the body. SARS-CoV-2 has spread in the community quickly infecting humans from all age, ethnicities and gender. However, fatal outcomes have been linked to specific host factors and co-morbidities such as age, hypertension, immuno-deficiencies, chronic lung diseases or metabolic disorders. A major shift in the microbiome of patients suffering of the coronavirus disease 2019 (COVID-19) have also been observed and is linked to a worst outcome of the disease. As many co-morbidities are already known to be associated with a dysbiosis of the microbiome such as hypertension, diabetes and metabolic disorders. Host factors and microbiome changes are believed to be involved as a network in the acquisition of the infection and the development of the diseases. We will review in detail in this manuscript, the immune response toward SARS-CoV-2 infection as well as the host factors involved in the facilitation and worsening of the infection. We will also address the impact of COVID-19 on the host's microbiome and secondary infection which also worsen the disease.

Acquisition and Transmission of Carbapenemase-Producing (blaKPC-2) Enterobacter cloacae in a Highly Frequented Outpatient Clinic.

The role of outpatient clinics as a potential transmission ground for multidrug-resistant organisms has not been adequately investigated. Here, we report a transmission cluster of blaKPC-2-positive Enterobacter cloacae among patients treated in a highly frequented outpatient department.

Molecular analysis of an increase in trimethoprim/sulfamethoxazole-resistant MRSA reveals multiple introductions into a tertiary care hospital, Germany 2012-19.

OBJECTIVES: Increasing spread of resistance could jeopardize the use of antifolates against MRSA infections. METHODS: We compared the prevalence of phenotypic trimethoprim/sulfamethoxazole resistance in 20 534 clinical Staphylococcus aureus isolates (19 096 MSSA and 1438 MRSA) of non-redundant patients at Heidelberg University Hospital over 8 years and performed WGS on trimethoprim/sulfamethoxazole-resistant MRSA. RESULTS: From 2012 to 2019, trimethoprim/sulfamethoxazole resistance in MSSA (674/19 096; 3.5%) ranged between 1.5% and 7.2% and in MRSA (135/1438; 9.4%) between 0.5% and 20.2%, reaching a peak in 2016 and 2018, respectively (Ptrend < 0.001). Trimethoprim/sulfamethoxazole resistance was more likely in outpatients than inpatients (P = 0.005), younger patients (P < 0.001), skin and soft tissue infections (SSTIs) (MRSA only, P = 0.05), submissions from pulmonology (MRSA only, P = 0.001), the upper respiratory tract (MSSA only, P < 0.001) and general surgery (MSSA only, P = 0.001). WGS of 76 trimethoprim/sulfamethoxazole-resistant MRSA revealed that 59% belonged to major pandemic CA-MRSA clones (ST22, ST8, ST398, ST772, ST30), 47% harboured Panton-Valentine leucocidin (PVL), 97% SCCmec IV/V, 71% dfrG and 28% dfrA. SNP-based phylogeny of trimethoprim/sulfamethoxazole-resistant MRSA core genomes favoured independent introduction over clonal expansion as the source, most prominently of dfrA+ trimethoprim/sulfamethoxazole-resistant ST22 MRSA from the Gaza Strip. CONCLUSIONS: The presented results support that trimethoprim/sulfamethoxazole-resistant S. aureus, formerly associated with SSTI from outpatients and S. aureus in the (sub)tropics, is on the rise in the temperate zone, potentially due to migration. Closer monitoring of trimethoprim/sulfamethoxazole resistance in S. aureus is recommended to safeguard the effectiveness of antifolate compounds.

Pitfalls in genotypic antimicrobial susceptibility testing caused by low expression of blaKPC in Escherichia coli.

BACKGROUND: There is a growing interest in the rapid genotypic identification of antimicrobial resistance (AMR). In routine diagnostics, we detected multiple KPC-positive Escherichia coli (KPC-Ec) with discordant phenotypic meropenem susceptibility from a single patient's blood cultures, which prompted a more thorough investigation. OBJECTIVES: We investigated the potential clinical relevance of, and the mechanism behind, discordant phenotypic and genotypic meropenem susceptibility in KPC-Ec. METHODS: WGS was used to perform a comparative analysis of the isolates' genetic characteristics and their blaKPC-2 locus. Expression of blaKPC-2 was determined by quantitative PCR and the potency of meropenem hydrolysis was determined using a semi-quantitative carbapenem inactivation method. An in vivo infection assay using Galleria mellonella was performed to assess the potential clinical relevance of KPC expression in E. coli. RESULTS: Despite the presence of blaKPC-2, three of five isolates were susceptible to meropenem (MICVITEK2 ≤ 0.25 mg/L), while two isolates were resistant (MICVITEK2 ≥ 16 mg/L). The isolates with high MICs had significantly higher blaKPC-2 expression, which corresponds to phenotypic meropenem inactivation. The genetic environment of blaKPC-2, which may impact KPC production, was identical in all isolates. In vivo infection assay with G. mellonella suggested that meropenem was effective in reducing mortality following infection with low-expressing KPC-Ec. CONCLUSIONS: Our findings clearly highlight a limitation of genotypic AMR prediction for blaKPC. For the time being, genotypic AMR prediction requires additional analysis for accurate antibiotic therapy decision-making.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam.

BACKGROUND: Blood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI. METHODS: A total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay. RESULTS: 58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76-93% 95% CI) and 54% (39-48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07). CONCLUSION: A high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

Challenges in interpretation of WGS and epidemiological data to investigate nosocomial transmission of vancomycin-resistant Enterococcus faecium in an endemic region: incorporation of patient movement network and admission screening.

OBJECTIVES: VRE are listed, by the WHO, among the leading resistant pathogens causing greatest public concern; hence the spread and transmission of VRE, especially in hospitalized patients, need to be monitored. Despite the advancements in typing methods since the implementation of WGS for outbreak investigations, data interpretation, especially for vancomycin-resistant Enterococcus faecium (VREfm) in an endemic setting, remains challenging. In this study we explored the potential added benefit of incorporating patient movement data and admission screening to accurately estimate the magnitude of an outbreak. METHODS: We sequenced 73 VREfm isolates from patients with bacteraemia (n = 43) and rectal colonization (n = 30/32). Genetic relatedness was determined by SNP distance (≤10) between isolates. Patient movements were visualized in a movement network, along with contact intensity and rectal colonization status prior to infection onset. RESULTS: ST117, ST80 and ST203 were the predominant STs in our study population. Forty-four percent (18/41) of VREfm bacteraemia cases were of endogenous origin. SNP analysis of infection and colonization isolates revealed nine clonal groups. Eighty-six percent (37/43) of the patients were visualized in a transmission network due to spatiotemporal overlap. Nineteen out of 43 (44%) belonged to five transmission clusters. Incorporation of prior colonization status revealed that transmission was very likely in only 63% (12/19) of patients in these transmission clusters. DISCUSSION: Although interpretation of WGS data is challenging, incorporation of patient movement data and colonization status by admission screening of high-risk patients may provide additional resolution when interpreting the magnitude of an outbreak in an endemic setting.

A new sequential animal model for infection-related non-unions with segmental bone defect.

BACKGROUND: The treatment of fracture-related infections (FRI) is still a challenge for orthopedic surgeons. The prevalence of FRI is particularly high in open fractures with extensive soft-tissue damage. This study aimed to develop a new two-step animal model for non-unions with segmental bone defects, which could be used to evaluate new innovative bone substitutes to improve the therapeutic options in humans with FRI and bone defects. METHODS: After randomization to infected or non-infected groups, 30 Sprague-Dawley rats underwent a transverse osteotomy of the mid-shaft femur with a 5 mm defect. Additionally, the periosteum at the fracture zone was cauterized at both sides. After intramedullary inoculation with 103 CFU Staphylococcus aureus (infected group) or PBS (non-infected group), a fracture stabilization was done by intramedullary K-wires. After 5 weeks, the bone healing process was evaluated, and revision surgery was performed in order to obtain increased bone healing. The initial K-wires were removed, and debridement of the osteotomy-gap was done followed by a more stable re-osteosynthesis with an angle-stable plate. After further 8 weeks all rats were euthanized and the bone consolidation was tested biomechanically and the callus formation quantitatively by micro-CT analysis. RESULTS: We developed and presented a new two-stage non-union animal model through a targeted S. aureus infection. After 5 weeks, all animals showed a non-union irrespective of assignment to the infected and non-infected group. Lane and Sandhu score showed a higher callus formation in the infected group. In all infected animals, the inoculated S. aureus strain was detected in the revision surgery. The second surgery did not improve bone healing, as shown by the Lane Sandhu score and in the µ-CT analysis. Similarly, biomechanical testing showed in both groups a significantly lower maximum torque as compared to the contralateral side (p < 0.0001). CONCLUSIONS: We were able to successfully develop a new two-stage non-union animal model, which reflects a genuine clinical situation of an infection-related non-union model with segmental bone defects. This model could be used to evaluate various therapeutic anti-infectious and osteoinductive strategies in FRIs.

Emergence of carbapenem-resistant ST131 Escherichia coli carrying blaOXA-244 in Germany, 2019 to 2020.

The dissemination of carbapenem-producing Gram-negative bacteria is a major public health concern. We report the first detection of OXA-244-producing ST131 O16:H5 Escherichia coli in three patients from two tertiary hospitals in the south-west of Germany. OXA-244 is emerging in Europe. Because of detection challenges, OXA-244-producing E. coli may be under-reported. The emergence of carbapenem resistance in a globally circulating high-risk clone, such as ST131 E. coli is of clinical relevance and should be monitored closely.

Molecular characterization of carbapenem-resistant Acinetobacter baumannii using WGS revealed missed transmission events in Germany from 2012-15.

BACKGROUND: Infection and colonization with multi-resistant Acinetobacter baumannii causes therapeutic and economic problems in the nosocomial setting. Due to the sensitivity issue of screening schemes for A. baumannii, it is difficult to implement adequate transmission prevention measures. The high discriminatory power of WGS for transmission-chain analysis provides us with the necessary tool to study and identify transmission events. We retrospectively sequenced and analysed 39 A. baumannii isolates from 2012-15 to search for possible missed transmission events. METHODS: Molecular typing by WGS was performed for non-repetitive (n=39) carbapenem-resistant A. baumannii. Retrospective assessment of patient records was performed to investigate and confirm possible transmission events. RESULTS: Between July 2012 and September 2015, A. baumannii was isolated from 268 patients, of which 16% (42/268) were carbapenem resistant. Thirty-nine of these isolates were recoverable and sequenced. Fifteen percent (6/39) of these were resistant to all antibiotics tested. Most isolates belong to the circulating IC2 clonal type. SNP analysis revealed four potential outbreak clusters. Two of these clusters showed high concordance with the local spatio-temporal epidemiology, suggesting that transmission events were very likely. CONCLUSIONS: Our data suggest that there were two independent transmission events, which would have been missed by conventional MLST owing to high clonality. The routine implementation of WGS can optimize surveillance and initiation of suitable containment measures. In addition, emerging resistance to salvage therapy is a major therapeutic problem and should be monitored closely.

Significant increase in cultivation of Gardnerella vaginalis, Alloscardovia omnicolens, Actinotignum schaalii, and Actinomyces spp. in urine samples with total laboratory automation.

While total laboratory automation (TLA) is well established in laboratory medicine, only a few microbiological laboratories are using TLA systems. Especially in terms of speed and accuracy, working with TLA is expected to be superior to conventional microbiology. We compared in total 35,564 microbiological urine cultures with and without incubation and processing with BD Kiestra TLA for a 6-month period each retrospectively. Sixteen thousand three hundred thirty-eight urine samples were analyzed in the pre-TLA period and 19,226 with TLA. Sixty-two percent (n = 10,101/16338) of the cultures processed without TLA and 68% (n = 13,102/19226) of the cultures processed with TLA showed growth. There were significantly more samples with two or more species per sample and with low numbers of colony forming units (CFU) after incubation with TLA. Regarding the type of bacteria, there were comparable amounts of Enterobacteriaceae in the samples, slightly less non-fermenting Gram-negative bacteria, but significantly more Gram-positive cocci, and Gram-positive rods. Especially Alloscardivia omnicolens, Gardnerella vaginalis, Actinomyces spp., and Actinotignum schaalii were significantly more abundant in the samples incubated and processed with TLA. The time to report was significantly lower in the TLA processed samples by 1.5 h. We provide the first report in Europe of a large number of urine samples processed with TLA. TLA showed enhanced growth of non-classical and rarely cultured bacteria from urine samples. Our findings suggest that previously underestimated bacteria may be relevant pathogens for urinary tract infections. Further studies are needed to confirm our findings.