Plasmid-encoded gene duplications of extended-spectrum ß-lactamases in clinical bacterial isolates.

Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Characterization of blaOXA-48-carrying plasmids and small non-AMR-coding plasmids collected from Ukrainian patients.

PURPOSE: Carbapenemase-producing Enterobacterales (CPE) pose a serious threat for healthcare facilities worldwide, yet the mode of transmission is often unclear. Recently, we recorded an increase of blaOXA-48-harboring isolates at our hospital associated with patients with previous medical treatment in the Ukraine. We used long-read whole genome sequencing (lrWGS) to characterize these isolates including their plasmids. METHODS: Samples were collected as part of clinical routine diagnostic or screening of multi-drug resistance bacteria (MDRB). Antimicrobial susceptibility testing was performed and all MDRB (n = 10) were sequenced by lrWGS for genotyping, identification of antimicrobial resistance (AMR) genes, and characterization of plasmids. RESULTS: While routine analysis of core genome multilocus sequence typing (cgMLST) did not show any genetic similarities between isolates, we found an unexpected high similarity in the plasmid diversity of different Enterobacterales in patients with previous medical treatment in the Ukraine. This included an IncL/M plasmid carrying blaOXA-48 and additional small non-AMR-coding plasmids. CONCLUSION: Our results show that lrWGS can be used in the routine setting to uncover similarities in plasmids and may give further information about potential epidemiologic associations. In the future, analysis of both AMR and non-AMR plasmids may provide an additional layer of information for molecular surveillance of CPE.

Development of prevalence and incidence of non-tuberculous mycobacteria in German laboratories from 2016 to 2020.

Numbers of non-tuberculous mycobacteria (NTM) pulmonary diseases (PD) have been repeatedly reported as increasing over the last decades, particularly in Europe. Sound epidemiological data are however missing for most European regions. This study calculated prevalence and incidence of NTM recovered from patients' lungs in Germany, the largest Central European country, over a five-year period. It furthermore determined regional particularities of NTM species and results from susceptibility testing. 22 German NTM laboratories provided their mycobacteriological diagnostic data of 11,430 NTM isolates recovered from 5998 pulmonary patients representing 30% of all notified NTM-PD cases of Germany from 2016 to 2020. NTM incidence and prevalence were calculated for every study year. The presented epidemiological indicators are particularly reliant as TB surveillance data were used as a reference and TB notification reaches almost 100% in Germany. Laboratory incidence and prevalence of NTM recovered from respiratory samples ranged from 4.5-4.9 and from 5.3-5.8/100,000 for the population of Germany, respectively, and did not change over the five-year study period. Prevalence and incidence were stable also when stratifying for facultative pathogenic NTM, M. avium/intracellulare complex (MAIC), and M. abscessus/chelonae complex (MABSC). The proportion of NTM with drug susceptibility testing (DST) increased from 27.3% (2016) to 43.8% (2020). The unchanging laboratory NTM prevalence/incidence in Germany represents a "ceiling" of possible NTM-PD notification when diagnostic strategies do not change in the coming years. A notable increase in NTM-DST may indicate better notification of NTM-PD and/or awareness of new clinical guidelines but still remains below clinical needs.

Molecular Characterization of Clinical Linezolid-Resistant Staphylococcus epidermidis in a Tertiary Care Hospital.

Staphylococcus epidermidis (S. epidermidis) is part of the human skin flora but can also cause nosocomial infections, such as device-associated infections, especially in vulnerable patient groups. Here, we investigated clinical isolates of linezolid-resistant S. epidermidis (LRSE) collected from blood cultures at the University Hospital Münster (UHM) during the period 2020-2022. All detected isolates were subjected to whole genome sequencing (WGS) and the relatedness of the isolates was determined using core genome multilocus sequence typing (cgMLST). The 15 LRSE isolates detected were classified as multilocus sequence type (ST) 2 carrying the staphylococcal cassette chromosome mec (SCCmec) type III. All isolates showed high-level resistance for linezolid by gradient tests. However, no isolate carried the cfr gene that is often associated with linezolid resistance. Analysis of cgMLST data sets revealed a cluster of six closely related LRSE isolates, suggesting a transmission event on a hematological/oncological ward at our hospital. Among the included patients, the majority of patients affected by LRSE infections had underlying hematological malignancies. This confirms previous observations that this patient group is particularly vulnerable to LRSE infection. Our data emphasize that the surveillance of LRSE in the hospital setting is a necessary step to prevent the spread of multidrug-resistant S. epidermidis among vulnerable patient groups, such as patients with hematological malignancies, immunosuppression or patients in intensive care units.

Does pre-incubation in selective-enrichment media improve the detection of diarrheagenic Escherichia coli using the RIDA®GENE PCR?

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70-93) and 89% (stx1/2, 95%CI 76-96), and 71% (95%CI 58-81) and 72% (eae, 95%CI 60-82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

Ejaculate for Microbiological Culture: To Wash or Not To Wash?

Bacteria can be associated with male infertility. Antibacterial substances (e.g., zinc-containing proteins, antimicrobial peptides) in ejaculates might impair the growth of bacteria in culture. We therefore wanted to test if removing antibacterial substances by washing the ejaculate could improve the detection of bacteria in culture. All ejaculates from patients ≥18 years old, which were obtained for routine diagnostics to assess male infertility were included in this study (no exclusion criteria were applied). Test samples were diluted with 2 mL sterile 0.45% saline, vortexed, and centrifuged (5 min; 7.5 × g). After the removal of 2 mL of the supernatant and resuspension, 10 µL of the pellet was used for aerobic and anaerobic culture. Control samples were cultured identically but without washing. Species identification was done with matrix-assisted laser desorption ionization-time of flight mass spectrometry. A total of 186 samples were included. The data set was stratified into five groups (Gram-negative rods [GNR], anaerobes [AN], Enterococcus spp. [EC], coagulase-negative staphylococci [CNS], and viridans streptococci [VS]). Compared to the control arm, the test arm revealed significant lower proportions for CNS (59.1% versus 44.6%, P < 0.01) and VS (53.8% versus 41.9%, P = 0.03). Similarly, slightly lower proportions of GNR (16.1% versus 15.1%, P = 0.89), AN (19.9% versus 17.2%, P = 0.5), and EC (25.3% versus 23.1%, P = 0.63) were observed. The medians of CFU were lower in test samples compared to the control samples (6.5 × 103 versus 2.5 × 103, P < 0.01) for any bacterial growth. Lower colony counts were also observed for individual bacterial groups. In conclusion, preculture washing of ejaculates results in a decrease in total bacteria count and culture-positive samples. IMPORTANCE This study compares two methods for processing ejaculate samples from men undergoing investigations for infertility. The method of sample washing and centrifugation was compared to the standard method of direct inoculation and culture. The study hypothesis was that preprocessing of samples may increase bacterial yield by removing bactericidal substances from semen. However, we found that washing ejaculate samples before microbiological culture did not improve the detection of bacteria and led to a reduction in colony counts.

Lethal Waterhouse-Friderichsen syndrome caused by Capnocytophaga canimorsus in an asplenic patient.

BACKGROUND: Capnocytophaga canimorsus, a Gram-negative rod, belongs to the Flavobacteriaceae family and colonizes the oropharynx of dogs and cats. Infections with C. canimorsus are rare and can induce a systemic infection with a severe course of the disease. So far, only five case reports of C. canimorsus infections associated with Waterhouse-Friderichsen Syndrome (WFS) have been reported with only two of the patients having a history of splenectomy. CASE PRESENTATION: Here, we report a fatal case of WFS due to C. canimorsus bacteremia and mycetal superinfection in a 61-year-old female asplenic patient. Despite extensive therapy including mechanical ventilation, antibiotic coverage with meropenem, systemic corticosteroids medication, vasopressor therapy, continuous renal replacement therapy, therapeutic plasma exchange, multiple transfusions of blood products and implantation of a veno-arterial extracorporeal membrane oxygenation the patient died 10 days after a dog bite. The autopsy showed bilateral hemorrhagic necrosis of the adrenal cortex and septic embolism to heart, kidneys, and liver. Diagnosis of C. canimorsus was prolonged due to the fastidious growth of the bacteria. CONCLUSIONS: The occurrence of a severe sepsis after dog bite should always urge the attending physician to consider C. canimorsus as the disease-causing pathogen. A therapeutic regimen covering C. canimorsus such as aminopenicillins or carbapenems should be chosen. However, despite maximum therapy, the prognosis of C. canimorsus-induced septic shock remains very poor. Asplenic or otherwise immunocompromised patients are at higher risk for a severe course of disease and should avoid exposure to dogs and cats and consider antibiotic prophylaxis after animal bite.

Outbreak of MRSA in a Gynecology/Obstetrics Department during the COVID-19 Pandemic: A Cautionary Tale.

Since March 2020, the COVID-19 pandemic forced hospitals worldwide to intensify their infection control measures to prevent health care-associated transmission of SARS-CoV-2. The correct use of personal protective equipment, especially the application of masks, was quickly identified as priority to reduce transmission with this pathogen. Here, we report a nosocomial cluster of methicillin-resistant Staphylococcus aureus (MRSA) that occurred during the COVID-19 pandemic in a gynecology/obstetrics department, despite these intensified contact precautions. Five MRSA originating from clinical samples after surgical intervention led to an outbreak investigation. Firstly, this included environmental sampling of the operation theatre (OT) and, secondly, a point prevalence screening of patients and health care workers (HCW). All detected MRSA were subjected to whole genome sequencing (WGS) and isolate relatedness was determined using core genome multilocus sequence typing (cgMLST). WGS revealed one MRSA cluster with genetically closely related five patient and two HCW isolates differing in a single cgMLST allele at maximum. The outbreak was terminated after implementation of infection control bundle strategies. Although contact precaution measures, which are also part of MRSA prevention bundle strategies, were intensified during the COVID-19 pandemic, this MRSA outbreak could take place. This illustrates the importance of adherence to classical infection prevention strategies.

The Use of Long-Read Sequencing Technologies in Infection Control: Horizontal Transfer of a blaCTX-M-27 Containing lncFII Plasmid in a Patient Screening Sample.

Plasmid transfer is one important mechanism how antimicrobial resistance can spread between different species, contributing to the rise of multidrug resistant bacteria (MDRB) worldwide. Here were present whole genome sequencing (WGS) data of two MDRB isolates, an Escherichia coli and a Klebsiella quasipneumoniae, which were isolated from a single patient. Detailed analysis of long-read sequencing data identified an identical F2:A-:B- lncFII plasmid containing blaCTX-M-27 in both isolates, suggesting horizontal plasmid exchange between the two species. As the plasmid of the E. coli strain carried multiple copies of the resistance cassette, the genomic data correlated with the increased antimicrobial resistance (AMR) detected for this isolate. Our case report demonstrates how long-read sequencing data of MDRB can be used to investigate the role of plasmid mediate resistance in the healthcare setting and explain resistance phenotypes.

Development and Validation of a Tool for the Prediction of Vancomycin-Resistant Enterococci Colonization Persistence-the PREVENT Score.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with increasing prevalence worldwide. Extensive hygiene measures have been established to prevent infection transmission in hospitals. Here, we developed a predictive score system (the predictive vancomycin-resistant enterococci [PREVENT] score) to identify the clearance or persistence in patients with a history of VRE carrier status at readmission. Over a cumulative 3-year period, patients with a positive VRE carrier status were included. The study population was recruited in two successive time periods and separated into training data for predictive score development and validation data for evaluation of the predictive power. The risk factors for persistent VRE colonization were analyzed in a univariable analysis before development of a logistic regression model based on the potential risk factors. The score points were determined proportionally to the beta coefficients of the logistic regression model. The data from 448 (79%) patients were used as the training data, and those from 119 (21%) as the validation data. Multivariable analysis revealed the following variables as independent risk factors: age of ≥60 years, hemato-oncological disease, cumulative antibiotic treatment for >4 weeks, and a VRE infection. The resulting logistic regression model exhibited an acceptable area under the curve (AUC) of 0.81 (95% confidence interval [CI], 0.72 to 0.91). The predictive score system had a sensitivity of 82% (95% CI, 65 to 93%) and a specificity of 77% (95% CI, 66 to 85%). The developed predictive score system is a useful tool to assess the VRE carrier status of patients with a history of VRE colonization. On the basis of this risk assessment, more focused and cost-effective infection control measures can be implemented. IMPORTANCE Given the increasing relevance of VRE as nosocomial pathogens worldwide, infection prevention and control measures, including patient isolation and contact precautions, are indispensable to avoid their spread in the hospital setting. In this study, we developed and validated the PREVENT score, a tool for rapid risk assessment of VRE persistence in patients with a history of previous VRE colonization. The score is designed to be easily performed, employing clinical information available in a regular admission setting and immediately providing information to inform the decision of whether to adopt patient isolation and contact precautions during the hospital stay. After validation, the score was shown to accurately identify patients with persistent VRE colonization upon admission, representing a suitable option as (i) a complementary method yielding preliminary results significantly more quickly than culture-based VRE detection techniques and (ii) an alternative strategy for VRE detection in settings in which microbiological VRE screening is not routinely performed due to limited resources.

Phenotypic Variants of Bacterial Colonies in Microbiological Diagnostics: How Often Are They Indicative of Differing Antimicrobial Susceptibility Patterns?

Phenotypic variants (PV) are colonies of the same species in the same specimen with different morphological features. It is controversial whether antimicrobial susceptibility testing (AST) should be done for all PV. The objectives of this study were to quantify the proportion of differing antimicrobial susceptibility patterns (dASP) among PV and to identify species and antimicrobial compounds that are mostly affected. All PV from routine diagnostics (University Hospital Münster, Germany; 1 September 2019 to 31 August 2020) were subjected to species identification (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) and AST (Vitek 2). To assess the dASP, only antimicrobial agents were considered for which Vitek-derived MIC were available (interpreted according to the EUCAST clinical breakpoints). The categorical agreement (CA; agreement with the AST categories S [susceptible, standard dosing regimen], I [susceptible, increased exposure], R [resistant]) of the PV was calculated. The PV of Escherichia coli (n = 260), Pseudomonas aeruginosa (n = 86), Klebsiella pneumoniae (n = 47), Enterobacter cloacae complex (n = 45), and Staphylococcus aureus (n = 38) were included. The median CA was 95% (range, 80 to 100%, depending on the species). The colony characteristics (e.g., form/size, color, margin, hemolysis) were not indicative for dASP. PV showed a high categorical agreement in the AST categories. This observation supports a test strategy to perform AST for only one colony of PV. IMPORTANCE Phenotypic variants of bacteria are frequent in routine diagnostics and can display differing antimicrobial susceptibility patterns. We found that the likelihood of different antimicrobial susceptibility is low among PV. To save laboratory resources, only one isolate per PV could be tested to guide the antimicrobial treatment of patients.

A Narrative Review on the Role of Staphylococcus aureus Bacteriuria in S. aureus Bacteremia.

Staphylococcus aureus bacteriuria (SABU) can occur in patients with S. aureus bacteremia (SAB). However, little is known on the (molecular) pathomechanisms of the renal passage of S. aureus. This review discusses the epidemiology and pathogenesis of SABU in patients with SAB and identifies knowledge gaps. The literature search was restricted to the English language. The prevalence of SABU in patients with SAB is 7.8%-39% depending on the study design. The main risk factor for SABU is urinary tract catheterization. SABU in SAB patients is associated with increased mortality. Given present evidence, hematogenous seeding-as seen in animal models-and the development of micro-abscesses best describe the translocation of S. aureus from blood to urine. Virulence factors that might be involved are adhesion factors, sortase A, and coagulase, among others. Other potential routes of bacterial translocation (eg, transcytosis, paracytosis, translocation via "Trojan horses") were identified as knowledge gaps.

Sex differences in vancomycin-resistant enterococci bloodstream infections-a systematic review and meta-analysis.

BACKGROUND: Vancomycin-resistant enterococci (VRE) have emerged in the healthcare setting worldwide. Infections with these pathogens, i.e., bloodstream infections (BSI), are accompanied with an impaired patient outcome. Diverse factors comprising patient characteristics, therapeutic strategies, and infection control measures are positively or negatively associated with VRE BSI occurrence. However, whether sex-specific differences influence the frequency of VRE BSI is yet unknown. The aim of this systematic review was to comprehensively summarize and analyze sex prevalence in VRE BSI patients. MAIN TEXT: A systematic search for relevant articles was conducted in PubMed and Web of Science. After screening for eligibility, data extraction from included articles and risk of bias assessment were processed. The prevalence of male/female sex in VRE BSI patients and 95% CI were calculated for each study and summarized as pooled estimated effect. In total, nine articles met the inclusion criteria. Risk of bias assessment resulted in low (six studies) to moderate bias (three studies). The pooled prevalence of male patients suffering from VRE BSI was 59% resulting in a 1.4 male/female prevalence ratio. CONCLUSIONS: Current literature suggests sex differences with male preference (59%) in the distribution of VRE BSI cases. Further primary studies should address the question of male-specific factors favoring the enhanced frequency of VRE BSI.

Variability of antibiograms: how often do changes in the antimicrobial susceptibility pattern occur in isolates from one patient?

OBJECTIVES: Many laboratories do not perform antimicrobial susceptibility testing (AST) for all consecutive isolates of the same species from one patient. The objective of this study was to assess how often changes in antimicrobial susceptibility patterns (cASP) occur in such isolates. METHODS: AST was performed for all isolates of defined species obtained from clinical routine (2015-2018) without restrictions for consecutive or sequential isolates of one patient. Occurrence of cASP and time between the first sampling date and first cASP were determined by combining antibiograms from all specimens and after stratification into species and specimens. RESULTS: A total of 35 473 AST results were included (range 2-71 per case). Combining pathogens and specimens, 1991 cASP occurred in 1269/8502 (14.9%) of all cases after a median time until cASP of 5 days (range 0-364). Of these, 628/1991 (31.5%) occurred on the day of first sampling (predominantly due to phenotypic variants in the same specimen). Excluding isolates with differing AST pattern on the first day of sampling, the median time until cASP was 12 days (range 1-364). Stratification into species and specimen revealed a large variance of the median time until cASP (e.g. in Escherichia coli: 5 days; range 1-48 for blood cultures or 16 days; 1-364 for urine). DISCUSSION: Using routine microbiological data in a large tertiary hospital, cASP occurs occasionally. The time to perform subsequent AST to detect cASP depends on the species and type of specimen. Other studies are needed to evaluate whether ideal time intervals applicable beyond local settings can be defined.

Gram Staining: a Comparison of Two Automated Systems and Manual Staining.

Various Gram staining automated systems are available to accelerate and standardize the staining process, but a systematic comparison of different systems is largely lacking. The objective of this study was to evaluate two devices in comparison to manual Gram staining. Clinical samples (n = 500; University Hospital Münster, Germany; May to June 2020) were simultaneously Gram stained manually and with two automated Gram stainers (Previ Color Gram, bioMérieux, and ColorAX2, Axonlab). The quality was assessed based on four criteria: (i) homogeneous staining of bacteria/fungi, (ii) uniform staining of the background, (iii) absence of staining artifacts, and (iv) congruency between culture and microscopy. Each criterion was rated with 0 (absence) or 1 (presence) point to calculate a quality score (0 to 4 points). The costs for each staining procedure were calculated based on consumables and hands-on time (applying the average wage of a laboratory technician in the public service for Germany and the United States). The mean (± standard deviation [SD]) quality scores were comparable for manual staining (3.06 ± 0.91) and Previ Color Gram (3.04 ± 0.90; P = 0.6), while significantly lower scores were achieved by ColorAX2 (2.57 ± 1.09; P < 0.0001). The total cost per Gram stain was €1.13/$1.34 for Previ Color Gram, €0.80/$0.83 for manual, and €0.60/$0.71 for ColorAX2, respectively. The quality and costs per slide vary significantly between instruments of different manufacturers.

Prevalence and risk factors for bacteremia in patients with Staphylococcus aureus bacteriuria: A retrospective cohort study.

OBJECTIVES: Staphylococcus aureus bacteriuria (SABU) is rare but can be an indicator of S. aureus bacteremia (SAB). The objective of this study was to assess the proportion of SAB in patients with SABU and identify risk factors in a hospital-based population. METHODS: We used electronic databases to identify eligible patients to be enrolled in a retrospective cohort study. Inclusion criteria were (i) SABU, (ii) ≥18 years of age, and (iii) ≥1 blood culture sampled ±3 months of SABU. Patients with missing values for demographic (e.g., age, sex) or clinical characteristics (e.g., comorbidities) and laboratory analyses were excluded. RESULTS: In total, 245 patients attending the University Hospital Münster, Germany, between 1 January 2012 and 31 December 2019 met the inclusion/exclusion criteria. Of the 245 patients with SABU, 66 had a concomitant SAB (26.9%). Elevated C-reactive protein (CRP) levels were associated with SAB. Other parameters (e.g., leukocytes, comorbidities) were not associated with SAB in a multivariate analysis. CONCLUSION: The frequency of SAB in patients with SABU was high and warrants active screening for bloodstream infections in hospitalized patients, particularly if CRP levels are elevated.

Moraxella nonliquefaciens bloodstream infection and sepsis in a pediatric cancer patient: case report and literature review.

BACKGROUND: Moraxella nonliquefaciens is a usually non-pathogenic biofilm-producing Gram-negative coccobacillus which may colonize the upper respiratory tract, rarely causing invasive disease. Although very rare, bloodstream infections caused by this organism have been described, showing often a fatal outcome. Here, we report the case of a pediatric cancer patient with bloodstream infection and sepsis due to M. nonliquefaciens showing full recovery after appropriate antibiotic treatment. CASE PRESENTATION: A three-year-old boy with stage IV neuroblastoma was admitted for high-dose chemotherapy with autologous stem cell rescue after standard neuroblastoma treatment. Despite receiving antimicrobial prophylaxis with trimethoprim/sulfamethoxazole, acyclovir and amphothericin B, the patient presented with fever of up to 39.5 °C and neutropenia. Besides a chemotherapy-related mucositis and an indwelling Broviac catheter (removed), no infection focus was identified on physical examination. Moraxella nonliquafaciens was identified in blood cultures. After antibiotic treatment and neutrophil recovery, the patient was fit for discharge. CONCLUSIONS: The case described highlights the importance of an otherwise non-pathogenic microorganism, especially in immunosupressed cancer patients. It should be kept in mind that, although very infrequently, Moraxella nonliquefaciens may cause bloodstream infections that can be successfully treated with prompt focus identification and antibiotic therapy.