A proof-of-principle study for the point-of-care detection of ESBL (CTX-M) by NG-Test® CTX-M MULTI lateral flow assay in urine samples using a simplified method for use in a resource-limited setting.

Background: The rise of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) in low- and middle-income countries limits treatment options, leading to the frequent use of broad-spectrum antibiotics. Reducing time-to-result for a urinary infection can facilitate correct antibiotic treatment and support antimicrobial and diagnostic stewardship measures. This study compared two simplified enrichment methods for detecting CTX-M directly from urine specimens. Methods: Two enrichment methods, namely centrifugation of 2 mL urine and filtration of 1 mL urine using the DirecTool adaptor, were compared using 20 culture-positive urine samples (20 suspected ESBL-E and 20 non-ESBL-E). CTX-M production was detected using a lateral flow assay (LFA), NG-Test® CTX-MMULTI. The presence of bla CTX-M genes was confirmed by whole-genome sequencing (WGS). Results: The results of both enrichment methods were identical, with a sensitivity of 87.5% and a specificity of 100%. In 19/20 (95%) of the urine samples, the results of the CTX-M LFA were identical with the phenotypic confirmation and WGS. Both methods could detect ESBL-E bacteriuria with ≥104 cfu/mL. All ESBL-E-negative samples were identified accurately. Both enrichment methods yielded negative results in one ESBL-E-positive (CTX-M-15) sample despite phenotypic and genotypic confirmation of ESBL production. High leukocyte count (>500 cells/µL), the presence of boric acid or polymicrobial samples did not appear to impact the performance of both enrichment methods. Conclusions: Our study underscores the feasibility of directly detecting CTX-M in urine. Simplified enrichment methods, particularly with a filtration kit, enhance the assay's practicality, rendering it suitable for use in primary care, emergency departments or remote laboratories without sophisticated equipment.

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.

Evaluation of screening algorithms to detect rectal colonization with carbapenemase-producing Enterobacterales in a resource-limited setting.

Objectives: To improve and rationalize the detection of carbapenemase-producing Enterobacterales (CPE) in rectal swabs in a high-prevalence and resource-constrained setting, addressing surveillance challenges typically encountered in laboratories with limited resources. Methods: A point prevalence survey (PPS) was conducted on 15 August 2022, in a provincial children's hospital in northern Vietnam. Rectal swab samples of all admitted children were collected and plated on a selective medium for carbapenem-resistant Enterobacterales (CRE). Species identification and antimicrobial susceptibility testing (AST) were performed by MALDI-TOF, and VITEK2 XL and interpreted according to CLSI breakpoints (2022). Carbapenemases were detected by the carbapenem inactivation method (CIM) and quantitative real-time PCR (qRT-PCR). Results: Rectal swab samples were obtained from 376 patients. Of 178 isolates growing on the CRE screening agar, 140 isolates were confirmed as Enterobacterales of which 118 (84.3%) isolates were resistant to meropenem and/or ertapenem. CIM and PCR showed that 90/118 (76.3%) were carbapenemase producers. Overall, 83/367 (22.6%) were colonized by CPE. Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae complex were the most common CPE detected, with NDM as the predominant carbapenemase (78/90; 86.7%). Phenotypic resistance to meropenem was the best predictor of CPE production (sensitivity 85.6%, specificity 100%) compared with ertapenem resistance (95.6% sensitivity, 36% specificity). CIM was 100% concordant with PCR in detecting carbapenemases. Conclusions: These findings underscore the effectiveness of meropenem resistance as a robust indicator of the production of carbapenemases and the reliability of the CIM method to detect such carbapenemases in resource-limited settings where the performance of molecular methods is not possible.

Genomic epidemiology of Streptococcus pyogenes from pharyngeal and skin swabs in Gabon.

The disease burden of Streptococcus pyogenes is particularly high in low- and middle-income countries. However, data on the molecular epidemiology of S. pyogenes in such regions, especially sub-Saharan Africa, are scarce. To address this, whole-genome sequencing (WGS) of S. pyogenes from Gabon was performed to identify transmission clusters and provide valuable genomic data for public repositories. A total of 76 S. pyogenes isolates from 73 patients, collected between September 2012 and January 2013, were characterized by short-read whole-genome sequencing. The predominant emm types were emm58.0, emm81.2 and emm223.0 with 9.2% (7 of 76), 7.9% (6 of 76), and 6.6% (5 of 76), respectively. Single-nucleotide polymorphism analysis revealed 16 putative transmission clusters. Four of these were household transmissions. Four antimicrobial genes (lmrP, tetM, tetL, and thfT) were found in the S. pyogenes isolates from this study. All strains carried lmrP. Of the 76 isolates, 64 (84.2%) carried at least one tetracycline resistance gene (tetM or tetL). Comparisons with other publicly available African genomic data revealed a significant correlation between geographical location and genetic diversity of S. pyogenes, with Gabonese strains showing similarities to those from Kenya and certain Oceanian regions. Our study showed that transmission of S. pyogenes can occur at the community/household level and that high-resolution molecular typing is needed to monitor changes in circulating clones and to detect community outbreaks. Advocacy for the adoption of WGS for comprehensive molecular characterization of S. pyogenes and data sharing through public repositories should be encouraged to understand the molecular epidemiology and evolutionary trajectory of S. pyogenes in sub-Saharan Africa. IMPORTANCE: The study conducted in Gabon underscores the critical importance of addressing the limited knowledge of the molecular epidemiology of Streptococcus pyogenes in low- and middle-income countries, particularly sub-Saharan Africa. Our molecular analysis identified predominant emm types and unveiled 16 putative transmission clusters, four involving household transmissions. Furthermore, the study revealed a correlation between geographical location and genetic diversity, emphasizing the necessity for a comprehensive understanding of the molecular epidemiology and evolutionary trajectory of S. pyogenes in various regions. The call for advocacy in adopting whole-genome sequencing for molecular characterization and data sharing through public repositories is crucial for advancing our knowledge and implementing effective strategies to combat the spread of S. pyogenes in sub-Saharan Africa.

Pulmonary Events in ICU patients with hyperoxia: is it possible to relate arterial partial pressure of oxygen to coded diseases? A retrospective analysis.

OBJECTIVE: Oxygen has been used liberally in ICUs for a long time to prevent hypoxia in ICU- patients. Current evidence suggests that paO2 >300 mmHg should be avoided, it remains uncertain whether an "optimal level" exists. We investigated how "mild" hyperoxia influences diseases and in-hospital mortality. DESIGN: This is a retrospective study. SETTING: 112 mechanically ventilated ICU-patients were enrolled. PATIENTS OR PARTICIPANTS: 112 ventilated patients were included and categorized into two groups based on the median paO2 values measured in initial 24 h of mechanical ventilation: normoxia group (paO2 ≤ 100 mmHg, n = 43) and hyperoxia group patients (paO2 > 100 mmHg, n = 69). INTERVENTIONS: No interventions were performed. MAIN VARIABLES OF INTEREST: The primary outcome was the incidence of pulmonary events, the secondary outcomes included the incidence of other new organ dysfunctions and in-hospital mortality. RESULTS: The baseline characteristics, such as age, body mass index, lactate levels, and severity of disease scores, were similar in both groups. There were no statistically significant differences in the incidence of pulmonary events, infections, and new organ dysfunctions between the groups. 27 out of 69 patients (39.1%) in the "mild" hyperoxia group and 12 out of 43 patients (27.9%) in the normoxia group died during their ICU or hospital stay (p = 0.54). The mean APACHE Score was 29.4 (SD 7.9) in the normoxia group and 30.0 (SD 6.7) in the hyperoxia group (p = 0.62). CONCLUSIONS: We found no differences in pulmonary events, other coded diseases, and in-hospital mortality between both groups. It remains still unclear what the "best oxygen regime" is for intensive care patients.

Tracing clinically-relevant antimicrobial resistances in Acinetobacter baumannii-calcoaceticus complex across diverse environments: A study spanning clinical, livestock, and wastewater treatment settings.

Acinetobacter baumannii has become a prominent nosocomial pathogen, primarily owing to its remarkable ability to rapidly acquire resistance to a wide range of antimicrobial agents and its ability to persist in diverse environments. However, there is a lack of data on the molecular epidemiology and its potential implications for public health of A. baumannii strains exhibiting clinically significant resistances that originate from non-clinical environments. Therefore, the genetic characteristics and resistance mechanisms of 80 A. baumannii-calcoaceticus (ABC) complex isolates, sourced from environments associated with poultry and pig production, municipal wastewater treatment plants (WWTPs), and clinical settings, were investigated. In total, our study classified 54 isolates into 29 previously described sequence types (STs), while 26 isolates exhibited as-yet-unassigned STs. We identified a broad range of A. baumannii STs originating from poultry and pig production environments (e.g., ST10, ST238, ST240, ST267, ST345, ST370, ST372, ST1112 according to Pasteur scheme). These STs have also been documented in clinical settings worldwide, highlighting their clinical significance. These findings also raise concerns about the potential zoonotic transmission of certain STs associated with livestock environments. Furthermore, we observed that clinical isolates exhibited the highest diversity of antimicrobial resistance genes (ARGs). In contrast to non-clinical isolates, clinical isolates typically carried a significantly higher number of ARGs, ranging from 10 to 15. They were also the exclusive carriers of biocide resistance genes and acquired carbapenemases (blaOXA-23, blaOXA-58, blaOXA-72, blaGIM-1, blaNDM-1). Additionally, we observed that clinical strains displayed an increased capacity for carrying plasmids and undergoing genetic transformation. This heightened capability could be linked to the intense selective pressures commonly found within clinical settings. Our study provides comprehensive insights into essential aspects of ABC isolates originating from livestock-associated environments and clinical settings. We explored their resistance mechanisms and potential implications for public health, providing valuable knowledge for addressing these critical issues.

Corrigendum: Inflammatory response against Staphylococcus aureus via intracellular sensing of nucleic acids in keratinocytes.

[This corrects the article DOI: 10.3389/fimmu.2022.828626.].

Monocentric observational cohort study to investigate the transmission of third-generation cephalosporin-resistant Enterobacterales in a neonatal intensive care unit in Heidelberg, Germany.

Third-generation cephalosporin-resistant Enterobacterales is a major threat for newborns in neonatal intensive care units (NICUs). The route of acquisition in a non-outbreak setting should be investigated to implement adequate infection prevention measures. To identify risk factors for colonization with and to investigate the transmission pattern of third-generation cephalosporin-resistant Enterobacterales in a NICU setting. This monocentric observational cohort study in a tertiary NICU in Heidelberg, Germany, enrolled all hospitalized neonates screened for cephalosporin-resistant Enterobacterales. Data were collected from 1 January 2018 to 31 December 2021. Weekly screening by rectal swabs for colonization with third-generation cephalosporin-resistant Enterobacterales was performed for all newborns until discharge. Whole-genome sequencing was performed for molecular characterization and transmission analysis. In total, 1,287 newborns were enrolled. The median length of stay was 20 (range 1-250) days. Eighy-eight infants (6.8%) were colonized with third-generation cephalosporin-resistant Enterobacterales. Low birth weight [<1500 g (adjusted odds ratio, 5.1; 95% CI 2.2-11.5; P < 0.001)] and longer hospitalization [per 30 days (adjusted odds ratio, 1.7; 95% CI 1.5-2.0; P < 0.001)] were associated with colonization or infection with drug-resistant Enterobacterales in a multivariate analysis. Enterobacter cloacae complex was the most prevalent third-generation cephalosporin-resistant Enterobacterales detected, 64.8% (59 of 91). Whole-genome sequencing, performed for the available 85 of 91 isolates, indicated 12 transmission clusters involving 37 patients. This cohort study suggests that transmissions of third-generation cephalosporin-resistant Enterobacterales in newborns occur frequently in a non-outbreak NICU setting, highlighting the importance of surveillance and preventive measures in this vulnerable patient group. IMPORTANCE Preterm newborns are prone to infections. Therefore, infection prevention should be prioritized in this vulnerable patient group. However, outbreaks involving drug-resistant bacteria, such as third-generation resistant Enterobacterales, are often reported. Our study aims to investigate transmission and risk factors for acquiring third-generation cephalosporin-resistant Enterobacterales in a non-outbreak NICU setting. Our data indicated that premature birth and low birth weight are significant risk factors for colonization/infection with third-generation cephalosporin-resistant Enterobacterales. Furthermore, we could identify putative transmission clusters by whole-genome sequencing, highlighting the importance of preemptive measures to prevent infections in this patient collective.

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.

Efficacy of an Antibiotic Loaded Ceramic-Based Bone Graft Substitute for the Treatment of Infected Non-Unions.

The treatment of non-unions is often complicated by segmental bone defects and bacterial colonization. Because of the limited availability of autologous bone grafts, tissue engineering focuses on antibiotic-loaded bone graft substitutes. HACaS+G is a resorbable calcium sulphate-hydroxyapatite loaded with gentamicin. The osteoinductive, osteoconductive, and anti-infective effect of HACaS+G has already been demonstrated in clinical studies on patients with chronic osteomyelitis. However, especially for the treatment of infected non-unions with segmental bone defects by HACaS+G, reliable clinical testing is difficult and sufficient experimental data are lacking. We used an already established sequential animal model in infected and non-infected rat femora to investigate the osteoinductive, osteoconductive, and anti-infective efficacy of HACaS+G for the treatment of infected non-unions. In biomechanical testing, bone consolidation could not be observed under infected and non-infected conditions. Only a prophylactic effect against infections, but no eradication, could be verified in the microbiological analysis. Using µ-CT scans and histology, osteoinduction was detected in both the infected and non-infected bone, whereas osteoconduction occurred only in the non-infected setting. Our data showed that HACaS+G is osteoinductive, but does not have added benefits in infected non-unions in terms of osteoconduction and mechanical bone stability, especially in those with segmental bone defects.

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.

Skin Infections Due to Panton-Valentine Leukocidin-Producing S. Aureus.

BACKGROUND: Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus (PVL-SA) strains are frequently associated with large, recurring abscesses in otherwise healthy young individuals. The typical clinical presentation and the recommended diagnostic evaluation and treatment are not widely known. METHODS: This review is based on pertinent publications retrieved by a selective search in PubMed, with special attention to international recommendations. RESULTS: PVL-SA can cause leukocytolysis and dermatonecrosis through specific cell-wall pore formation. Unlike other types of pyoderma, such conditions caused by PVL-SA have no particular site of predilection. In Germany, the PVL gene can be detected in 61.3% (252/411) of skin and soft tissue infections with S. aureus. Skin and soft tissue infections with PVL-SA recur three times as frequently as those due to PVL-negative S. aureus. They are diagnosed by S. aureus culture from wound swabs and combined nasal/pharyngeal swabs, along with PCR for gene detection. The acute treatment of the skin abscesses consists of drainage, followed by antimicrobial therapy if needed. Important secondary preventive measures include topical cleansing with mupirocin nasal ointment and whole-body washing with chlorhexidine or octenidine. The limited evidence (level IIb) concerning PVL-SA is mainly derived from nonrandomized cohort studies and experimental analyses. CONCLUSION: PVL-SA skin infections are easily distinguished from other skin diseases with targeted history-taking and diagnostic evaluation.

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.

Host genetic loci LZTFL1 and CCL2 associated with SARS-CoV-2 infection and severity of COVID-19.

OBJECTIVES: Host genetic factors contribute to the variable severity of COVID-19. We examined genetic variants from genome-wide association studies and candidate gene association studies in a cohort of patients with COVID-19 and investigated the role of early SARS-CoV-2 strains in COVID-19 severity. METHODS: This case-control study included 123 COVID-19 cases (hospitalized or ambulatory) and healthy controls from the state of Baden-Wuerttemberg, Germany. We genotyped 30 single nucleotide polymorphisms, using a custom-designed panel. Cases were also compared with the 1000 genomes project. Polygenic risk scores were constructed. SARS-CoV-2 genomes from 26 patients with COVID-19 were sequenced and compared between ambulatory and hospitalized cases, and phylogeny was reconstructed. RESULTS: Eight variants reached nominal significance and two were significantly associated with at least one of the phenotypes "susceptibility to infection", "hospitalization", or "severity": rs73064425 in LZTFL1 (hospitalization and severity, P <0.001) and rs1024611 near CCL2 (susceptibility, including 1000 genomes project, P = 0.001). The polygenic risk score could predict hospitalization. Most (23/26, 89%) of the SARS-CoV-2 genomes were classified as B.1 lineage. No associations of SARS-CoV-2 mutations or lineages with severity were observed. CONCLUSION: These host genetic markers provide insights into pathogenesis and enable risk classification. Variants which reached nominal significance should be included in larger studies.

Cefiderocol Protects against Cytokine- and Endotoxin-Induced Disruption of Vascular Endothelial Cell Integrity in an In Vitro Experimental Model.

The severe course of bloodstream infections with Gram-negative bacilli can lead to organ dysfunctions and compromise the integrity of the vascular barrier, which are the hallmarks of sepsis. This study aimed to investigate the potential effect of cefiderocol on the barrier function of vascular endothelial cells (vECs) in an in vitro experimental set-up. Human umbilical vein cells (HUVECs), co-cultured with erythrocyte-depleted whole blood for up to 48 h, were activated with tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) to induce endothelial damage in the absence or presence of cefiderocol (concentrations of 10, 40 and 70 mg/L). The endothelial integrity was quantified using transendothelial electrical resistance (TEER) measurement, performed at 0, 3, 24 and 48 h after stimulation. Stimulation with TNF-α and LPS increased the endothelial permeability assessed by TEER at 24 and 48 h of co-culture. Furthermore, cefiderocol reduces interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and TNF-α release in peripheral blood mononuclear cells (PBMCs) following LPS stimulation in a dose-dependent manner. Collectively, the data suggest that cefiderocol may have an influence on the cellular immune response and might support the maintenance of vEC integrity during inflammation associated with infection with Gram-negative bacteria, which warrants further investigations.