Nosocomial transmission of tet(x3), bla NDM-1 and bla OXA-97-carrying Acinetobacter baumannii conferring resistance to eravacycline and omadacycline, the Netherlands, March to August 2021.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

The agreement between bronchoalveolar lavage, bronchial wash and sputum culture: a retrospective study.

PURPOSE: Bronchoalveolar lavage is commonly used in clinical practice for unresolved pneumonia. However, bronchoalveolar lavage is not suitable for all patients as it is an invasive procedure and can worsen oxygenation. The diagnostic value of bronchial wash and sputum has been debated extensively over the years. In this study, we aim to compare the diagnostic value in several pathogens of bronchoalveolar lavage and bronchial wash, and secondarily bronchoalveolar lavage and sputum. METHODS: We retrospectively included all adult patients in our hospital who underwent bronchoalveolar lavage, bronchial wash, and where sputum sampling was done between January 1st of 2018 and December 31st of 2021. The intraclass correlation coefficient was computed for the three tests. RESULTS: In total, 308 patients were included. We found a level of correlation of 0.819 and 0.865, respectively, between bronchoalveolar lavage and bronchial wash for two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. For Stenotrophomonas maltophilia and Aspergillus fumigatus, we found an intraclass correlation coefficient of 0.568 and 0.624, respectively. Between bronchoalveolar lavage and sputum, we found varying levels of agreement. CONCLUSION: Our study shows reasonably well agreement levels between bronchoalveolar lavage and bronchial wash, suggesting that bronchial wash could potentially be an alternative to bronchoalveolar lavage.

Neonatal Elizabethkingia anophelis meningitis originating from the water reservoir of an automated infant milk dispenser, the Netherlands, February 2024.

Elizabethkingia anophelis is a multidrug-resistant pathogen causing high mortality and morbidity in adults with comorbidities and neonates. We report a Dutch case of E. anophelis meningitis in a neonate, clonally related to samples taken from an automated infant milk dispenser located at the family's residence. We inform about the emergence of E. anophelis and suggest molecular surveillance in hospitals and other health settings. This is the first case connecting an automated formula dispenser to an invasive infection in a neonate.

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.

Genetic Determinants in MRSA Carriage and Their Association with Decolonization Outcome.

Methicillin-resistant Staphylococcus aureus (MRSA) colonization increases the risk of infection. Response to decolonization treatment is highly variable and determinants for successful decolonization or failure of eradication treatment are largely unknown. Insight into genetic predictors of eradication failure is potentially useful in clinical practice. The aim of this study was to explore genetic characteristics that are associated with MRSA decolonization failure. This cohort study was performed in a tertiary care hospital in the Netherlands. Patients with ≥ 1 positive MRSA culture from any site and with available whole -genome sequencing data of the MRSA isolate between 2017 and 2022 were included. Lineages, resistance, and virulence factors were stratified by MRSA decolonization outcome. In total, 56 patients were included: 12/56 (21%) with treatment failure and 44/56 (79%) with successful decolonization (with or without preceding treatment). A significant association was found between ciprofloxacin-resistant lineages and failure of eradication (OR 4.20, 95%CI 1.11-15.96, P = 0.04). Furthermore, livestock-associated MRSA and the major community-associated MRSA lineages ST6-t304 and ST8-t008 were associated with successful eradication treatment or spontaneous clearance. In conclusion, this explorative study showed a higher eradication failure rate in complicated MRSA carriers with ciprofloxacin-resistant MRSA lineages, which are predominantly healthcare-associated. Further studies are warranted to confirm the higher eradication failure risk of ciprofloxacin-resistant lineages, and identify the underlying mechanisms.

Enhancement and Comparison of (Ceftazidime-)Avibactam Plus Aztreonam Susceptibility Tests for Stenotrophomonas maltophilia in Clinical Diagnostics.

Stenotrophomonas maltophilia is naturally resistant to many antimicrobials. We evaluated the in vitro activity and reproducibility of two different super-position methods of aztreonam in combination with ceftazidime-avibactam for S. maltophilia and compared these results with the recently available aztreonam-avibactam gradient strip. We recommend an improved super-position method that avoids the possible risk of handling a contaminated aztreonam strip. In addition, we report that the cefazidime-avibactam and aztreonam super-position method showed increased in vitro activity in comparison with aztreonam-avibactam indicating activity of the ceftazidime component in vitro.

Review on infection control strategies to minimize outbreaks of the emerging pathogen Elizabethkingia anophelis.

BACKGROUND: Elizabethkingia anophelis is a multi-drug resistant emerging opportunistic pathogen with a high mortality rate, causing healthcare-associated outbreaks worldwide. METHODS: We report a case of E. anophelis pleuritis, resulting from transmission through lung transplantation, followed by a literature review of outbreak reports and strategies to minimize E. anophelis transmission in healthcare settings. RESULTS: From 1990 to August 2022, 14 confirmed E. anophelis outbreak cohorts and 21 cohorts with suspected E. anophelis outbreaks were reported in literature. A total of 80 scientific reports with recommendations on diagnostics and infection control measures were included and summarized in our study. CONCLUSION: Strategies to prevent and reduce spread of E. anophelis include water-free patient rooms, adequate hygiene and disinfection practices, and optimized diagnostic techniques for screening, identification and molecular typing.

Zoonotic Transmission of Vaccine-Derived Bordetella bronchiseptica.

We describe a unique case of a 43-year-old-female with a Bordetella bronchiseptica infection caused by zoonotic transmission following vaccination of her dog. With this report, we want to raise awareness of potential zoonotic transmission of live attenuated vaccines from animals to patients with impaired immunity.

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.

Cross-site collaboration on infection prevention and control research-room for improvement? A 7-year comparative study in five European countries.

BACKGROUND: The spread of SARS-CoV-2, multidrug-resistant organisms and other healthcare-associated pathogens represents supra-regional challenges for infection prevention and control (IPC) specialists in every European country. To tackle these problems, cross-site research collaboration of IPC specialists is very important. This study assesses the extent and quality of national research collaborations of IPC departments of university hospitals located in Austria, England, France, Germany, and the Netherlands, identifies network gaps, and provides potential solutions. METHODS: Joint publications of IPC heads of all university hospitals of the included countries between 1st of June 2013 until 31st of May 2020 were collected by Pubmed/Medline search. Further, two factors, the journal impact factor and the type/position of authorship, were used to calculate the Scientific Collaboration Impact (SCI) for all included sites; nationwide network analysis was performed. RESULTS: In five European countries, 95 sites and 125 responsible leaders for IPC who had been in charge during the study period were identified. Some countries such as Austria have only limited national research cooperations, while the Netherlands has established a gapless network. Most effective collaborating university site of each country were Lille with an SCI of 1146, Rotterdam (408), Berlin (268), Sussex (204), and Vienna/Innsbruck (18). DISCUSSION: The present study indicates major differences and room for improvement in IPC research collaborations within each country and underlines the potential and importance of collaborating in IPC.

Case Report: Necrotizing fasciitis caused by Staphylococcus aureus positive for a new sequence variant of exfoliative toxin E.

Objectives: Necrotizing fasciitis (NF) caused by S. aureus is a rare, aggressive and rapidly progressing superficial fascia infection with a high mortality rate. The aim of this study was to identify virulence-related genes from a complete genome sequence of a methicillin-susceptible S. aureus (MSSA) isolate recovered from a monomicrobial case of NF. Materials and methods: The MSSA isolate UMCG579 was cultured from a pus collection from the subcutis of a patient with NF. The genome of isolate UMCG579 was sequenced using MinION (Oxford Nanopore) and MiSeq (illumina) platforms. Results: The genome of the UMCG579 isolate was composed of a 2,741,379 bp chromosome and did not harbor any plasmids. Virulence factor profiling identified multiple pore-forming toxin genes in the UMCG579 chromosome, including the Panton-Valentine leukocidin (PVL) genes, and none of the superantigen genes. The UMCG579 isolate harbored a new sequence variant of the recently described ete gene encoding exfoliative toxin (type E). A search in the GenBank database revealed that the new sequence variant (ete2) was exclusively found among isolates (n = 115) belonging to MLST CC152. While the majority of S. aureus ete-positive isolates were recovered from animal sources, S. aureus ete2-positive isolates originated from human carriers and human infections. Comparative genome analysis revealed that the ete2 gene was located on a 8777 bp genomic island. Conclusion: The combination of two heterogeneously distributed potent toxins, ETE2 and PVL, is likely to enhance the pathogenic ability of S. aureus isolates. Since anti-virulence therapies for the treatment of S. aureus infections continue to be explored, the understanding of specific pathogenetic mechanisms may have an important prophylactic and therapeutic value. Nevertheless, the exact contribution of ETE sequence variants to S. aureus virulence in NF infections must be determined.

Brief report: community-acquired Friedlander's pneumonia and pulmonary metastatic Klebsiella pneumoniae infection caused by hypervirulent ST23 in the Netherlands.

Infections with hypervirulent Klebsiella pneumoniae (hvKp) commonly presents with primary liver infection, bacteremia, and metastatic abscesses. Here, we present 2 cases of severe community-acquired pulmonary infections by hvKp in patients in the Netherlands without recent travel history. Both bacterial isolates are closely related to an archetype ST23 hvKp reference isolate. Based on these findings, surveillance programs on hvKp may consider to include isolates from community-acquired pneumonia by K. pneumoniae.

Molecular Characterisation of Vancomycin-Resistant Enterococcus faecium Isolates Belonging to the Lineage ST117/CT24 Causing Hospital Outbreaks.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis. Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content. Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster's closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes. Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients.

Long-read sequencing-based in silico phage typing of vancomycin-resistant Enterococcus faecium.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome's structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates. RESULTS: Analysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity. CONCLUSIONS: For rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period.

Misidentification of meticillin-resistant Staphylococcus aureus by the Cepheid Xpert MRSA NxG assay, the Netherlands, February to March 2021.

We describe two false-negative results in the detection of meticillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 and spa type t011 using the Cepheid Xpert MRSA NxG assay. The isolates were recovered in late February and early March 2021 from two patients in different hospitals in the northern Netherlands. Variations between the two isolate genomes indicate that this MRSA strain might have been spreading for some time and could have disseminated to other regions of the Netherlands and other European countries.

Pseudomonas aeruginosa and Staphylococcus aureus virulence factors as biomarkers of infection.

The gold standard for the diagnosis of bacterial infections in clinical samples is based on culture tests that are time-consuming and labor-intense. For these reasons, an extraordinary effort has been made to identify biomarkers as the tools for sensitive, rapid and accurate identification of pathogenic microorganisms. Moreover, biomarkers have been tested to distinguish colonization from infection, monitor disease progression, determine the clinical status of patients or predict clinical outcomes. This mini-review describes Pseudomonas aeruginosa and Staphylococcus aureus biomarkers, which contribute to pathogenesis and have been used in culture-independent bacterial identification directly from patient samples.

The sputum transcriptome better predicts COPD exacerbations after the withdrawal of inhaled corticosteroids than sputum eosinophils.

INTRODUCTION: Continuing inhaled corticosteroid (ICS) use does not benefit all patients with COPD, yet it is difficult to determine which patients may safely sustain ICS withdrawal. Although eosinophil levels can facilitate this decision, better biomarkers could improve personalised treatment decisions. METHODS: We performed transcriptional profiling of sputum to explore the molecular biology and compared the predictive value of an unbiased gene signature versus sputum eosinophils for exacerbations after ICS withdrawal in COPD patients. RNA-sequencing data of induced sputum samples from 43 COPD patients were associated with the time to exacerbation after ICS withdrawal. Expression profiles of differentially expressed genes were summarised to create gene signatures. In addition, we built a Bayesian network model to determine coregulatory networks related to the onset of COPD exacerbations after ICS withdrawal. RESULTS: In multivariate analyses, we identified a gene signature (LGALS12, ALOX15, CLC, IL1RL1, CD24, EMR4P) associated with the time to first exacerbation after ICS withdrawal. The addition of this gene signature to a multiple Cox regression model explained more variance of time to exacerbations compared to a model using sputum eosinophils. The gene signature correlated with sputum eosinophil as well as macrophage cell counts. The Bayesian network model identified three coregulatory gene networks as well as sex to be related to an early versus late/nonexacerbation phenotype. CONCLUSION: We identified a sputum gene expression signature that exhibited a higher predictive value for predicting COPD exacerbations after ICS withdrawal than sputum eosinophilia. Future studies should investigate the utility of this signature, which might enhance personalised ICS treatment in COPD patients.

Staphylococcal cassette chromosome mec containing a novel mec gene complex, B4.

OBJECTIVES: To describe a new subclass of mec class B complex identified in Staphylococcus epidermidis. METHODS: Four S. epidermidis isolates obtained from bloodstream infections in patients at University Medical Center Groningen (UMCG) were analysed by phenotypic antibiotic susceptibility testing and WGS. RESULTS: Sequence analysis revealed a new staphylococcal cassette chromosome mec (SCCmec) structure in isolate UMCG335. In this structure, plasmid pUB110 was found to be integrated into SCCmec IVc, creating a new SCCmec subtype, IVUMCG335. SCCmec IVc and a copy of plasmid pUB110 were found in other isolates, UMCG364 and UMCG341, respectively, indicating a probability that SCCmec IVUMCG335 could have evolved at the UMCG. SCCmec of UMCG337 contained a new genetic organization of the mec complex (IS431-ΔmecR1-mecA-IS431-pUB110-IS431-ψIS1272) that we have named B4. This new subclass of mec class B complex originated by IS431-mediated inversion of the DNA segment encompassing the plasmid and most of the genes of the mec complex with the exception of IS1272. As the SCCmec organization in UMCG337 differed by the inversion of an ∼10 kb sequence compared with SCCmec IVUMCG335, we have named it SCCmec subtype IVUMCG337. Isolates UMCG335 and UMCG337 carrying SCCmec IVUMCG335 and IVUMCG337, respectively, were associated with a restriction-modification system and a CRISPR-Cas system, creating a composite island of almost 70 kb. CONCLUSIONS: Our findings highlight the importance of IS431 in the evolution of the SCCmec region. The increasing genetic diversity identified in the SCCmec elements imposes a great challenge for SCCmec typing methods and highlights possible difficulties with the SCCmec nomenclature.

Proteomic Charting of Imipenem Adaptive Responses in a Highly Carbapenem Resistant Clinical Enterobacter roggenkampii Isolate.

Gram-negative bacteria belonging to the Enterobacter cloacae complex are increasingly implicated in difficult-to-treat nosocomial infections, as exemplified by a recently characterized highly carbapenem-resistant clinical Enterobacter roggenkampii isolate with sequence type (ST) 232. While mechanisms of carbapenem resistance are well-understood, little is known about the responses of highly drug-resistant bacteria to these antibiotics. Our present study was therefore aimed at charting the responses of the E. roggenkampii ST232 isolate to the carbapenem imipenem, using a 'stable isotope labeling of amino acids in cell culture' approach for quantitative mass spectrometry. This unveiled diverse responses of E. roggenkampii ST232 to imipenem, especially altered levels of proteins for cell wall biogenesis, central carbon metabolism, respiration, iron-sulfur cluster synthesis, and metal homeostasis. These observations suggest a scenario where imipenem-challenged bacteria reduce metabolic activity to save resources otherwise used for cell wall biogenesis, and to limit formation of detrimental reactive oxygen species at the cytoplasmic membrane due to respiration and Fenton chemistry. We consider these observations important, because knowing the adaptive responses of a highly resistant bacterium of the E. cloacae complex to last-resort antibiotics, such as imipenem, provides a 'sneak preview' into the future development of antibiotic resistance in this emerging group of pathogens.