Spread of carbapenemase-producing Morganella spp from 2013 to 2021: a comparative genomic study.

BACKGROUND: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp. METHODS: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A. FINDINGS: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-ß-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A. INTERPRETATION: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons. FUNDING: None.

Successful SARS-CoV-2 mRNA Vaccination Program in Allogeneic Hematopoietic Stem Cell Transplant Recipients-A Retrospective Single-Center Analysis.

(1) Background: mRNA COVID-19 vaccines are effective but show varied efficacy in immunocompromised patients, including allogeneic hematopoietic stem cell transplant (HSCT) recipients. (2) Methods: A retrospective study on 167 HSCT recipients assessed humoral response to two mRNA vaccine doses, using the manufacturer cut-off of ≥7.1 BAU/mL, and examined factors affecting non-response. (3) Results: Twenty-two percent of HSCT recipients failed humoral response. Non-responders received the first vaccine a median of 10.2 (2.5-88.9) months post-HSCT versus 35.3 (3.0-215.0) months for responders (p < 0.001). Higher CD19 (B cell) counts favored vaccination response (adjusted odds ratio (aOR) 3.3 per 100 B-cells/microliters, p < 0.001), while ongoing mycophenolate mofetil (MMF) immunosuppression hindered it (aOR 0.04, p < 0.001). By multivariable analysis, the time from transplant to first vaccine did not remain a significant risk factor. A total of 92% of non-responders received a third mRNA dose, achieving additional 77% seroconversion. Non-converters mostly received a fourth dose, with an additional 50% success. Overall, a cumulative seroconversion rate of 93% was achieved after up to four doses. (4) Conclusion: mRNA vaccines are promising for HSCT recipients as early as 3 months post-HSCT. A majority seroconverted after four doses. MMF usage and low B cell counts are risk factors for non-response.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Humoral response after a third and fourth dose of mRNA-based SARS-CoV-2 vaccine in previously seronegative kidney transplant recipients.

Growing evidence shows diminished response to mRNA-based SARS-CoV­2 vaccination in kidney transplant recipients. We aimed to investigate the seroconversion rate after a 3rd and 4th dose of mRNA vaccination in kidney transplant recipients without prior antibody response to two or three vaccination doses.This retrospective study included 324 prevalent kidney transplant recipients of a single tertiary transplantation center of which 157 remained seronegative, defined as anti-spike-RBD-IgG antibody titer < 7.1 BAU/ml, after two doses of mRNA-based SARS-CoV­2 vaccination. Maintenance immunosuppression was not changed. The median patient age was 60.6 years (IQR 51.4-68.1 years), 66.9% were male. Positivity for anti-spike-RBD-IgG (≥ 7.1 BAU/ml) was measured 4-5 weeks after administration of a 3rd and 4th vaccine dose.Seroconversion rates were 63.9% after a 3rd dose and 29.3% after a 4th dose of vaccine. Cumulative prevalence of seropositivity was 51.5% after 2 doses, 80.5% after 3 doses and 84.2% after 4 doses.In conclusion, seroconversion can be achieved in the majority of the kidney transplant recipients by administrating three or four doses of mRNA vaccine without changing maintenance immunosuppression.

Efficacy and safety of voriconazole as invasive fungal infection prophylaxis in patients with acute myeloid leukemia.

Invasive fungal infections (IFIs) are commonly observed in patients, who are at high risk of severe infections during the neutropenic phase. The aim of this retrospective single-center study was to evaluate the efficacy and safety of voriconazole as a fungal prophylaxis after induction chemotherapy for acute myeloid leukemia (AML) in adult patients. Six proven/probable IFIs were diagnosed in 213 patients with AML (median age 61 years, range 18-85), who received a total of 377 induction chemotherapies. This yielded an incidence rate of 1.6% based on all induction cycles administered. Voriconazole prophylaxis was administered as intended in 317 out of 377 (84%) induction cycles until the end of neutropenia with a median duration of 20 days (range: 2-101 days). In conclusion, voriconazole demonstrates efficacy and safety as a first-line IFI prophylaxis comparable to published data on posaconazole, which is the standard fungal prophylaxis recommendation for AML patients in international guidelines today.

Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Oxazolidinone Resistance Mediated by optrA in Clinical Enterococcus faecalis Isolates in Upper Austria: First Report and Characterization by Whole Genome Sequencing.

The genetic mechanisms associated with acquisition of linezolid (LZD) resistance are diverse, including point mutations in the V domain of the 23S rRNA and the 50S ribosomal proteins as well as cfr, optrA, and/or poxtA genes, which may be plasmid- or chromosomally encoded. The aim of this study was to investigate through Whole Genome Sequencing (WGS)-based typing the presence and location of genes and point mutations associated with LZD resistance in two Enterococcus faecalis isolates from Upper Austrian patients. The isolates were retrieved during screening by LZD disk diffusion test of a total of 911 clinical E. faecalis isolates in 2017. The two E. faecalis isolates had LZD minimum inhibitory concentrations of 8 and 32 mg/L and were optrA-positive (ST476 and ST585). Bioinformatic analysis revealed the presence of optrA located in the chromosome of both isolates. One isolate carried the optrA gene in the transposon 6674, previously reported as chromosomally encoded, and the second isolate in fragments originating from the integrative plasmid pEF10748. Additional mechanisms of LZD resistance on the 23S rRNA and the 50S ribosomal proteins were detected. None of the patients reported travels to geographical areas with high LZD resistance or previous LZD treatments. This is the first report of optrA carrying E. faecalis, including characterization by WGS from Austria. LZD resistance in a low-prevalence setting is of concern and should be further monitored.

Hospital outbreak caused by linezolid resistant Enterococcus faecium in Upper Austria.

Background: Enterococcus faecium is part of the human gastrointestinal flora but may act as opportunistic pathogen. Environmental persistence, high colonization capability and diverse intrinsic and acquired resistance mechanisms make it especially successful in nosocomial high-risk settings. In March 2014, an outbreak of Linezolid resistant Enterococcus faecium (LREfm) was observed at the hematooncology department of a tertiary care center in Upper Austria. Methods: We report on the outbreak investigation together with the whole genome sequencing (WGS)-based typing results including also non-outbreak LREfm and susceptible isolates. Results: The 54 investigated isolates could be divided in six clusters based on cgMLST. Cluster one comprised LREfm isolates of genotype ST117 and CT24, which was identified as the causative clone of the outbreak. In addition, the detection of four other clusters comprising isolates originating from hematooncology patients but also at other hospitals, pointed to LREfm transmission between local healthcare facilities. LREfm patients (n = 36) were typically at risk for acquisition of nosocomial pathogens because of immunosuppression, frequent hospitalization and antibiotic therapies. Seven of these 36 patients developed LREfm infection but were successfully treated. After termination of the initial outbreak, sporadic cases occurred despite a bundle of applied outbreak control interventions. Conclusions: WGS proved to be an effective tool to differentiate several LREfm clusters in an outbreak. Active screening for LREfm is important in a high-risk setting such as hematooncology, where multiple introductions are possible and occur despite intensified infection control measures.

Truncation of GdpP mediates ß-lactam resistance in clinical isolates of Staphylococcus aureus.

OBJECTIVES: High-level ß-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of ß-lactam resistance and sought to understand the molecular basis of the resistance observed. METHODS: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the ß-lactam resistance profiles for the isolates and the isolates were further investigated by WGS. RESULTS: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. CONCLUSIONS: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased ß-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.

Antifungal susceptibility of yeast bloodstream isolates collected during a 10-year period in Austria.

BACKGROUND: Candida-associated infections put a significant burden on western healthcare systems. Development of (multi-)resistant fungi can become untreatable and threaten especially vulnerable target groups, such as the immunocompromised. OBJECTIVES: We assessed antifungal susceptibility and explored possible influence factors of clinical Candida isolates collected from Austrian hospitals between 2007 and 2016. METHODS: Thousand three hundred and sixty clinical Candida spp. isolated from blood cultures were subjected to antifungal susceptibility testing (AFST) in a liquid-handling aided continuous microdilution assay. We tested against fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin and micafungin according to EUCAST with additional recording of growth curves. We performed rigid quality control on each assay via growth curve assessment and included two standard reference strains. Minimal inhibitory concentrations (MIC) were quantified according to EUCAST guideline E.DEF 7.3.1, and susceptibility was evaluated using EUCAST clinical breakpoints. RESULTS: The isolate collection consisted of Candida albicans (59%), C. glabrata (19%), C. parapsilosis (9%), C. tropicalis (5%) and C. krusei (3%) and few other Candida species and fungi (5%). During the observed time period, species abundance and antifungal resistance rates remained constant. Multi-resistance was rare and we found no single isolate which was resistant to both azoles and echinocandins. Within the antifungal resistance profile of our strain collection, we observed clusters along species boundaries. CONCLUSIONS: Over the last decade, the distribution of Candida species and its level of antifungal resistance remained constant in Austria. Our data compare well with other European countries. Principal component analysis of the susceptibility profile of this collection revealed species-specific clusters and substantial intra-species variation, especially for C. glabrata.

Whole-Genome Analysis of a Human Enterobacter mori Isolate Carrying a blaIMI-2 Carbapenemase in Austria.

Enterobacterales belonging to the genus Enterobacter are well-known human pathogens and blaIMI carrying strains have been described in several countries. From Austria, we report on the first human Enterobacter mori isolate, typically a plant pathogen, which showed an undescribed multilocus sequence type and carried a blaIMI-2 carbapenemase.

SeptiFast versus blood culture in clinical routine - A report on 3 years experience.

BACKGROUND: In recent years a multiplex real-time PCR (SeptiFast) has been introduced, allowing detection of 25 common blood pathogens considerably faster than conventional blood culture. METHODS: SeptiFast was applied routinely in addition to blood culture in cases of critically ill patients with fever and other signs of severe systemic infections. In this study data of 470 episodes were retrospectively analysed to assess the impact of various parameters, such as clinical indications, assigning ward and antimicrobial treatment on test outcome using a multivariate logistic model. RESULTS: After exclusion of microorganisms classified as contaminants, the concordance between SeptiFast and blood culture was 85.5%. SeptiFast detected 98 out of 120, while blood culture merely found 63 out of 120 potential pathogens. In comparison to blood culture, SeptiFast showed considerably higher positivity rates in sepsis, pneumonia and febrile immunosuppression and a lower rate in endocarditis. The highest positivity and concordance between tests was shown in patients from the emergency room (P = 0.007). CONCLUSIONS: The results obtained in this study are similar to those from prospective settings confirming the robustness of the SeptiFast assay in routine use. Our data suggest that SeptiFast is a valuable add-on to blood culture and may increase the diagnostic efficiency of a microbiological laboratory.

Detection of the mcr-1 Gene in a Multidrug-Resistant Escherichia coli Isolate from an Austrian Patient.

Since colistin resistance based on the plasmid-encoded mcr-1 gene was first described, this resistance gene in Enterobacteriaceae has been found worldwide. These organisms are typically of heterogeneous genetic background and show exceptional clonal diversity. We describe the first confirmation of mcr-1 in a human Escherichia coli strain cultured from a surveillance stool sample of an Austrian oncology patient.

Cilastatin does not affect Carba NP test performance for detection of carbapenemase production in Enterobacteriaceae.

The Carba NP test is a simple confirmation method for carbapenemase-producing Enterobacteriaceae (CPE) but reagents have to be freshly prepared as imipenem sodium salt is unstable. We evaluated the Carba NP test performance based on a commercially available 10-fold cheaper drug formulation containing cilastatin against 217 CPE and 78 non-CPE isolates with reduced meropenem susceptibility. Specificity and sensitivity were 100 % and 98.6 %, respectively and 3 false negative results of blaVIM-1-producing Proteus mirabilis were reproducible with the RAPIDEC® Carba NP test. Cilastatin does not disturb test performance provided that the imipenem drug quantity is doubled.

Multi-center and multi-method evaluation of in vitro activities of ceftaroline against S. aureus.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5µg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

Exposure to cigarette smoke and Chlamydia pneumoniae infection in mice: Effect on infectious burden, systemic dissemination and cytokine responses: A pilot study.

Cigarette smoke exposure has been considered a risk factor for infection with Chlamydia pneumoniae. C. pneumoniae infection is associated with respiratory tract infection and chronic respiratory disease, which is a serious public health concern. To determine whether prior exposure to cigarette smoke worsens C. pneumoniae infection (specifically, increases infectious burden and systemic dissemination) as well as alters cytokine responses in mice, adult female C57BL/6 mice were exposed to either filtered air (FA) or mainstream cigarette smoke (MCS) (15 mg/m(3), total suspended particulates) for 5 days/week for 2 weeks and then infected with C. pneumoniae (10(5) IFU) via intratracheal instillation. Mice were euthanized on Days 7, 14 or 26 post-infection (p.i.). Chlamydial burdens in the lungs and spleen were quantified by quantitative PCR (qPCR) and histologic analyses were performed; cytokine levels (TNFα, IL-4, IFNγ) in bronchoalveolar lavage fluid and serum were assayed by enzyme-linked immunosorbent assay (ELISA). The results indicated that: (1) mice exposed to either FA or MCS had similar chlamydial burdens in the lungs and spleen on Days 14 and 26 p.i.; (2) proximal and distal airway inflammation was observed on Day 14 p.i. in both FA and MCS mice, but persisted in MCS mice until Day 26 p.i.; FA exposed mice demonstrated resolution of distal airway inflammation; and (3) MCS mice displayed higher serum levels of IFNγ and IL-4 on Day 26 p.i. These findings indicate that exposure of mice to MCS (at a concentration equivalent to smoking < 1 pack cigarettes/day) led to greater C. pneumoniae-induced inflammation, as indicated by prolonged inflammatory changes.

Prevalence and resistance patterns of commensal S. aureus in community-dwelling GP patients and socio-demographic associations. A cross-sectional study in the framework of the APRES-project in Austria.

BACKGROUND: The aim of the present study was to assess the prevalence and resistance of commensal S. aureus in the nasal microbiota of community-dwelling persons in Austria, as well as to identify possible associations with socio-demographic factors. Multi-drug resistance in this population was additionally studied. METHOD: This cross-sectional study was conducted within the context of the European APRES project. In nine European countries, nasal swabs were collected from 32,206 general practice patients who received care for non-infectious reasons. In Austria, 20 GPs attempted to recruit 200 consecutive patients without infectious diseases, with each patient completing demographic questionnaires as well as providing a nose swab sample. Isolation, identification, and resistance testing of S. aureus were performed. Statistical analyses included subgroup analyses and logistic regression models. RESULTS: 3309 nose swabs and corresponding questionnaires from Austrian subjects were analyzed. S. aureus was identified in 16.6 % (n = 549) of nose swabs, of which 70.1 % were resistant against one or more antibiotics, mainly penicillin. S. aureus carrier status was significantly associated with male sex (OR 1.6; 1.3-2.0), younger age (OR 1.3; 1.0-1.8), living in a rural area (OR 1.4; 1.1-1.7) and working in the healthcare sector (OR 1.5; 1.0-2.1). Multi-drug resistances were identified in 13.7 % (n = 75) of the S. aureus carriers and 1.5 % (n = 8) tested positive for MRSA. The highest resistance rate was observed against penicillin (64.8 %), followed by azithromycin (13.5 %) and erythromycin with 13.3 %. CONCLUSION: This study describes the prevalence and resistance patterns of commensal S. aureus in community-dwelling persons in Austria and shows that differences exist between socio-demographic groups. Demographic associations have been found for S. aureus carriers but not for carriers of resistant S. aureus strains. Only two thirds of S. aureus strains were found to be resistant against small spectrum penicillin. As it is recognized that one of the corner stones for the containment of antibiotic resistance is the appropriate prescription of antibiotics in the outpatient sector, this finding lends support to the avoidance of prescription of broad-spectrum antibiotics to treat S. aureus infections in the community.

Evaluation of a multiplex ligation-dependent probe amplification assay for the detection of respiratory pathogens in oncological patients.

BACKGROUND: Respiratory tract infections are widespread and may cause significant morbidity and mortality in immunosuppressed populations such as oncological patients. OBJECTIVES: The RealAccurate Respiratory RT PCR Kit covering 14 respiratory viruses was compared to the RespiFinder Smart22, a broad-spectrum multiplex ligation-dependent probe amplification (MLPA) test, targeting 22 viral and bacterial respiratory pathogens. STUDY DESIGN: After verification of its analytical performance, the clinical performance of the RespiFinder Smart22 was evaluated by re-analysis of 96 respiratory samples from oncological patients. Additionally, the time to result (TTR) of both methods was compared. RESULTS: The analytical performance of the RespiFinder Smart22 fulfilled all previously specified criteria. Concordant results in both assays were achieved in 74.0% of all clinical specimens and in 91.2% when only positive results were taken into account. The RespiFinder Smart22 yielded additional results in a total of 22 (22.9% of 96) samples due to higher test sensitivity and a broader, highly multiplexed spectrum of pathogens. The TTR of a typical routine test consisting of three samples were 206 and 356 min for the RealAccurate Respiratory RT PCR Kit and the RespiFinder Smart22, respectively. However, hands-on time was reduced by 59.0% applying the MLPA method. CONCLUSIONS: In our hands, the RespiFinder Smart22 showed excellent analytical performance while hands-on time was halved in comparison to the RT PCR method. Regarding the clinical evaluation, the MLPA method provided additional results in 22.9% (22/96) of specimens due to its comprehensive format, higher test sensitivity and the capability to detect 22 pathogens compared to 14 with the RealAccurate Respiratory RT PCR Kit.