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255000.9520Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects. BACKGROUND: The gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed "selective decontamination of the digestive tract" (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes ("the resistome") of ICU patients during SDD and of healthy subjects. RESULTS: From ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6')-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. CONCLUSIONS: The gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.201728803549
157610.9512Emergence of multidrug-resistant Gram-negative bacteria during selective decontamination of the digestive tract on an intensive care unit. OBJECTIVES: During treatment with selective decontamination of the digestive tract (SDD), four multidrug-resistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum beta-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes. METHODS: The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis. RESULTS: The strains were genetically unrelated and contained identical plasmids with ESBL genes. CONCLUSIONS: We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs.200616891326
254920.9511Effects of selective digestive decontamination (SDD) on the gut resistome. OBJECTIVES: Selective digestive decontamination (SDD) is an infection prevention measure for critically ill patients in intensive care units (ICUs) that aims to eradicate opportunistic pathogens from the oropharynx and intestines, while sparing the anaerobic flora, by the application of non-absorbable antibiotics. Selection for antibiotic-resistant bacteria is still a major concern for SDD. We therefore studied the impact of SDD on the reservoir of antibiotic resistance genes (i.e. the resistome) by culture-independent approaches. METHODS: We evaluated the impact of SDD on the gut microbiota and resistome in a single ICU patient during and after an ICU stay by several metagenomic approaches. We also determined by quantitative PCR the relative abundance of two common aminoglycoside resistance genes in longitudinally collected samples from 12 additional ICU patients who received SDD. RESULTS: The patient microbiota was highly dynamic during the hospital stay. The abundance of antibiotic resistance genes more than doubled during SDD use, mainly due to a 6.7-fold increase in aminoglycoside resistance genes, in particular aph(2″)-Ib and an aadE-like gene. We show that aph(2″)-Ib is harboured by anaerobic gut commensals and is associated with mobile genetic elements. In longitudinal samples of 12 ICU patients, the dynamics of these two genes ranged from a ∼10(4) fold increase to a ∼10(-10) fold decrease in relative abundance during SDD. CONCLUSIONS: ICU hospitalization and the simultaneous application of SDD has large, but highly individualized, effects on the gut resistome of ICU patients. Selection for transferable antibiotic resistance genes in anaerobic commensal bacteria could impact the risk of transfer of antibiotic resistance genes to opportunistic pathogens.201424710024
149230.9509Characterization of the tet(M)-bearing transposon Tn7125 of Escherichia coli strain A13 isolated from an intensive pig farm located in Henan province, China. BACKGROUND: Transposons carrying tet(M) in Gram-positive bacteria have been reported extensively, while there is a paucity of data on the transmission characteristics of tet(M) in Gram-negative bacteria. Therefore, the aim of this study was to investigate the genetic characteristics of the tet(M)-bearing transposon Tn7125, and to clarify the transmission mechanism of the plasmids pTA13-1 and pTA13-3 in Escherichia coli strain A13. METHODS: Plasmids from strain A13 and a corresponding transconjugant were determined by whole genome sequencing and analyzed using bioinformatics tools. The plasmids pTA13-1 and pTA13-3 of the transconjugant TA13 were characterized by S1-pulse-field gel electrophoresis, Southern hybridization, stability experiments, and direct competition assays. RESULTS: The conjugated IncF2:A6:B20 plasmid pTA13-1 co-transferred with the 41-kb plasmid pTA13-3, which carried no resistance genes; plasmid pTA13-2, which harbored the replication initiator PO111; and the IncX4 plasmid pTA13-4, which harbored the antibiotic resistance gene mcr-1. The novel IS26-bracked composite transposon Tn7125 was located on plasmid pTA13-1, which mainly consists of three resistance modules: IS26-ctp-lp-tet(M)-hp-IS406tnp, qac-aadA1-cmlA1-aadA2-DUF1010-dfrA12, and ∆ISVSa3-VirD-floR-LysR-ISVSa3. The plasmid pTA13-1 was highly stable in E. coli strain J53 with no fitness cost to the host or disadvantage in growth competition. CONCLUSION: Evolution of co-integrated transposons, such as Tn7125, may convey antibiotic resistance to a wide spectrum of hosts via the plasmids pTA13-1 and pTA13-3, which acts as an adaptable and mobile multidrug resistance reservoir to accelerate dissemination of other genes by co-selection, thereby posing a potentially serious barrier to clinical treatment regimens.202540639501
306440.9509High Diversity but Monodominance of Multidrug-Resistant Bacteria in Immunocompromised Pediatric Patients with Acute Lymphoblastic Leukemia Developing GVHD Are Not Associated with Changes in Gut Mycobiome. Graft-versus-host disease (GvHD) is a severe complication after hematopoietic stem cell transplantation (HSCT). Our study focused on identifying multidrug-resistant (MDR) gut bacteria associated with GvHD-prone guts and association with gut microbiota (GM) diversity, bacteriome, and mycobiome composition in post-HSCT patients. We examined 11 pediatric patients with acute lymphoblastic leukemia (ALL), including six with GvHD, within three time points: seven days pre-HSCT, seven days post-, and 28 days post-HSCT. The gut microbiome and its resistome were investigated using metagenomic sequencing, taxonomically classified with Kraken2, and statistically evaluated for significance using appropriate tests. We observed an increase in the abundance of MDR bacteria, mainly Enterococcus faecium strains carrying msr(C), erm(T), aac(6')-li, dfrG, and ant(6)-la genes, in GvHD patients one week post-HSCT. Conversely, non-GvHD patients had more MDR beneficial bacteria pre-HSCT, promoting immunosurveillance, with resistance genes increasing one-month post-HSCT. MDR beneficial bacteria included the anti-inflammatory Bacteroides fragilis, Ruminococcus gnavus, and Turicibacter, while most MDR bacteria represented the dominant species of GM. Changes in the gut mycobiome were not associated with MDR bacterial monodominance or GvHD. Significant α-diversity decline (Shannon index) one week and one month post-HSCT in GvHD patients (p < 0.05) was accompanied by increased Pseudomonadota and decreased Bacteroidota post-HSCT. Our findings suggest that MDR commensal gut bacteria may preserve diversity and enhance immunosurveillance, potentially preventing GvHD in pediatric ALL patients undergoing HSCT. This observation has therapeutic implications.202338136701
248050.9507GLO1 Contributes to the Drug Resistance of Escherichia coli Through Inducing PER Type of Extended-Spectrum β-Lactamases. BACKGROUND: Escherichia coli-associated antimicrobial resistance (AMR) issue so far needs urgent considerations. This study aims to screen the potent genes associated with extended-spectrum β-lactamases (ESBLs) in drug-resistant Escherichia coli and elucidate the specific drug-resistant mechanism. METHODS: Clinical ESBLs-EC samples were obtained based on the microbial identification, and the whole genome was sequenced. In combination with the significantly enriched pathways, several differently expressed genes were screened and verified by RT-PCR. Furthermore, through knocking out glyoxalase 1 (GLO1) gene and transfecting overexpressed plasmids, the potential relationship between GLO1 and ESBLs was then investigated. Lastly, the concentrations of β-lactamases in bacteria and supernatant from different groups were examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: After successful isolation and identification of ESBLs-EC, the whole genome and eighteen differential metabolic pathways were analyzed to select differently expressed genes, including add, deoD, guaD, speG, GLO1, VNN1, etc. RT-PCR results showed that there were no differences in these genes between the standard bacteria and susceptible Escherichia coli. Remarkably, the relative levels of four genes including speG, Hdac10, GLO1 and Ppcdc were significantly increased in ESBLs-EC in comparison with susceptible strains, whereas other gene expression was decreased. Further experiments utilizing gene knockout and overexpression strains confirmed the role of GLO1. At last, a total of 10 subtypes of β-lactamases were studied using ELISA, including BES-, CTX-M1-, CTX-M2-, OXA1-, OXA2-, OXA10-, PER-, SHV-, TEM-, and VEB-ESBLs, and results demonstrated that GLO1 gene expression only affected PER-β-lactamases but had no effects on other β-lactamases. CONCLUSION: SpeG, Hdac10, GLO1 and Ppcdc might be associated with the drug-resistant mechanism of Escherichia coli. Of note, this study firstly addressed the role of GLO1 in the drug resistance of ESBLs-EC, and this effect may be mediated by increasing PER-β-lactamases.202235414749
246360.9505Characterization of Antibiotic-Resistant Stenotrophomonas Isolates from Painted Turtles Living in the Wild. Stenotrophomonas maltophilia is a ubiquitous multidrug-resistant opportunistic pathogen commonly associated with nosocomial infections. The purpose of this study was to isolate and characterize extended-spectrum beta-lactamase (ESBL) producing bacteria from painted turtles (Chrysemys picta) living in the wild and captured in southeastern Wisconsin. Fecal samples from ten turtles were examined for ESBL producing bacteria after incubation on HardyCHROM™ ESBL agar. Two isolates were cultivated and identified by 16S rRNA gene sequencing and whole genome sequencing (WGS) as Stenotrophomonas sp. 9A and S. maltophilia 15A. They were multidrug-resistant, as determined by antibiotic susceptibility testing. Stenotrophomonas sp. 9A was found to produce an extended spectrum beta-lactamase (ESBL) and both isolates were found to be carbapenem-resistant. EDTA-modified carbapenem inactivation method (eCIM) and the modified carbapenem inactivation method (mCIM) tests were used to examine the carbapenemase production and the test results were negative. Through WGS several antimicrobial resistance genes were identified in S. maltophilia 15A. For example a chromosomal L1 β-lactamase gene, which is known to hydrolyze carbapenems, a L2 β-lactamase gene, genes for the efflux systems smeABC and smeDEF and the aminoglycosides resistance genes aac(6')-lz and aph(3')-llc were found. An L2 β-lactamase gene in Stenotrophomonas sp. 9A was identified through WGS.202336729340
249570.9504Transmission of Mobile Colistin Resistance (mcr-1) by Duodenoscope. BACKGROUND: Clinicians increasingly utilize polymyxins for treatment of serious infections caused by multidrug-resistant gram-negative bacteria. Emergence of plasmid-mediated, mobile colistin resistance genes creates potential for rapid spread of polymyxin resistance. We investigated the possible transmission of Klebsiella pneumoniae carrying mcr-1 via duodenoscope and report the first documented healthcare transmission of mcr-1-harboring bacteria in the United States. METHODS: A field investigation, including screening targeted high-risk groups, evaluation of the duodenoscope, and genome sequencing of isolated organisms, was conducted. The study site included a tertiary care academic health center in Boston, Massachusetts, and extended to community locations in New England. RESULTS: Two patients had highly related mcr-1-positive K. pneumoniae isolated from clinical cultures; a duodenoscope was the only identified epidemiological link. Screening tests for mcr-1 in 20 healthcare contacts and 2 household contacts were negative. Klebsiella pneumoniae and Escherichia coli were recovered from the duodenoscope; neither carried mcr-1. Evaluation of the duodenoscope identified intrusion of biomaterial under the sealed distal cap; devices were recalled to repair this defect. CONCLUSIONS: We identified transmission of mcr-1 in a United States acute care hospital that likely occurred via duodenoscope despite no identifiable breaches in reprocessing or infection control practices. Duodenoscope design flaws leading to transmission of multidrug-resistant organsisms persist despite recent initiatives to improve device safety. Reliable detection of colistin resistance is currently challenging for clinical laboratories, particularly given the absence of a US Food and Drug Administration-cleared test; improved clinical laboratory capacity for colistin susceptibility testing is needed to prevent the spread of mcr-carrying bacteria in healthcare settings.201930204838
516180.9498Genomic analysis of contaminant Stenotrophomonas maltophilia, from placental swab culture, carrying antibiotic resistance: a potential hospital laboratory contaminant. Acute chorioamnionitis has been considered as reflective of amniotic fluid infection. Standard microbiological work ups for causative microorganism of intra-amniotic infection is based on microbial identification. However, frequency of positive placental culture is varied depending on placental sampling techniques, contaminations, methods of microbiologic work ups or comprehensive microbiologic work ups. In this report, we performed a hybrid whole genome sequencing of a proven bacterial contaminant obtained from placental culture in a patient with preterm labor and acute chorioamnionitis. This is to unveil genetic characterization of contaminant Stenotrophomonas maltophilia habouring antibiotic resistance genes. Stenotrophomonas maltiphilia was proven to be bacterial contaminant since Ureaplasma urealyticum was subsequently demonstrated in amniotic fluid by 16 S rRNA gene Sanger sequencing. Cultivation results from other sources were no growth. We identified Stenotrophomonas maltiphilia strain RAOG732 which carried several antibiotic resistance genes, including aminoglycoside, fluoroquiolone and beta-lactam. Biofilm production genes were also identified in this genome. We firstly utilized a hybrid sequencing approach to investigate the genome of S. maltiphilia in the patient with preterm and acute chorioamnionitis, a proven bacterial laboratory contaminant. The analysis provided several antibiotic resistance-associated and genes biofilm-associated genes. The detection of S. maltiphilia raised the awareness of the colonization of biofilm-producing bacteria in hospitals, where surveillance for decontamination is necessary.202540594762
209390.9496Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis. Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, bla(ACT-)(7), bla(ACT-)(14,)qacJ, oqxAB(,)aac(6')-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics-together with resistance and virulence genes-pose a health risk for infants consuming these food products.202236429148
5185100.9496Genomic characterisation of nasal isolates of coagulase-negative Staphylococci from healthy medical students reveals novel Staphylococcal cassette chromosome mec elements. Coagulase-negative staphylococci (CoNS) are a diverse group of Gram-positive bacteria that are part of the normal human microbiota. Once thought to be non-pathogenic, CoNS has emerged in recent years as opportunistic pathogens of concern particularly in healthcare settings. In this study, the genomes of four methicillin-resistant CoNS isolates obtained from the nasal swabs of healthy university medical students in Malaysia were sequenced using the Illumina short-read platform. Genome sequencing enabled the identification of the four isolates as Staphylococcus warneri UTAR-CoNS1, Staphylococcus cohnii subsp. cohnii UTAR-CoNS6, Staphylococcus capitis subsp. urealyticus UTAR-CoNS20, and Staphylococcus haemolyticus UTAR-CoNS26. The genome of S. cohnnii UTAR-CoNS6 harboured the mecA methicillin-resistance gene on a Staphylococcal cassette chromosome mec (SCCmec) element similar to SCCmec type XIV (5 A) but the SCCmec cassettes identified in the other three CoNS genomes were novel and untypeable. Some of these SCCmec elements also encoded heavy metal resistance genes while the SCCmec type XIV (5 A) variant in S. cohnii UTAR-CoNS6 harboured the complete ica operon, a known virulence factor that functions in biofilm formation. In S. cohnii UTAR-CoNS6, the macrolide resistance genes msrA and mphC along with copper and cadmium resistance genes were located on a 26,630 bp plasmid, pUCNS6. This study showcased the diversity of CoNS in the nasal microbiota of medical students but the discovery of novel SCCmec elements, various antimicrobial and heavy metal resistance along with virulence genes in these isolates is of concern and warrants vigilance due to the likelihood of spread, especially to hospitalised patients.202540595841
5195110.9496Genomic characteristics of antimicrobial resistance and virulence factors of carbapenem-resistant Stutzerimonas nitrititolerans isolated from the clinical specimen. BACKGROUND: Stutzerimonas nitrititolerans (S. nitrititolerans) is a rare human pathogenic bacterium and has been inadequately explored at the genomic level. Here, we report the first case of carbapenem-resistant S. nitrititolerans isolated from the peritoneal dialysis fluid of a patient with chronic renal failure. This study analyzed the genomic features, antimicrobial resistance, and virulence factors of the isolated strain through whole genome sequencing (WGS). METHODS: The bacterial isolate from the peritoneal dialysis fluid was named PDI170223, and preliminary identification was conducted through Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). WGS of the strain PDI170223 was performed using the Illumina platform, and a phylogenetic tree was constructed based on the 16S rRNA gene sequences. Antimicrobial susceptibility test (AST) was conducted using the TDR-200B2 automatic bacteria identification/drug sensitivity tester. RESULTS: S. nitrititolerans may emerge as a human pathogen due to its numerous virulence genes, including those encoding toxins, and those involved in flagellum and biofilm formation. The AST results revealed that the strain is multidrug- and carbapenem-resistant. The antimicrobial resistance genes of S. nitrititolerans are complex and diverse, including efflux pump genes and β⁃lactam resistance genes. CONCLUSION: The analysis of virulence factors and antimicrobial resistance of S. nitrititolerans provides clinical insight into the pathogenicity and potential risks of this bacterium. It is crucial to explore the mechanisms through which S. nitrititolerans causes diseases and maintains its antimicrobial resistance, thereby contributing to development of effective treatment and prevention strategies.202439358682
2445120.9496Isolation and characterisation of carbapenem-resistant Xanthomonas citri pv. mangiferaeindicae-like strain gir from the faecal material of giraffes. The purpose of this study was to determine if giraffes (Giraffa camelopardalis) living in captivity at the Jacksonville Zoo and Gardens, Jacksonville, FL were colonised with carbapenem-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to a carbapenem-resistant phenotype. Faecal samples from seven giraffes were examined for carbapenem-resistant bacteria. Only one isolate (a Xanthomondaceae) was found to be carbapenem-resistant by antibiotic susceptibility testing. This isolate was selected for additional characterization, including whole genome sequencing (WGS). Based on average nucleotide identity, the bacterium was identified as Xanthomonas citri pv. mangiferaeindicae-like strain gir. Phenotypic carbapenemase tests and PCR for the most common carbapenemase genes produced negative results, suggesting that carbapenem resistance was mediated by another mechanism. Resistance gene profile analysis of WGS results confirmed these results. Among identified resistance genes, a chromosomal class A beta-lactamase with 71% identity to the penP beta-lactamase gene from Xanthomonas citri ssp. citri was identified, which could contribute to a carbapenem-resistant phenotype.202031485840
5235130.9496Draft genome sequences of rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South Africa. OBJECTIVES: Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogens is a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 in Africa. METHODS: The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees. RESULTS: Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (bla(ACT-9)), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin. CONCLUSION: The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobei in Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance.202336948496
5122140.9495Clinical long-read metagenomic sequencing of culture-negative infective endocarditis reveals genomic features and antimicrobial resistance. BACKGROUND: Infective endocarditis (IE) poses significant diagnostic challenges, particularly in blood culture-negative cases where fastidious bacteria evade detection. Metagenomic-based nanopore sequencing enables rapid pathogen detection and provides a new approach for the diagnosis of IE. METHOD: Two cases of blood culture-negative infective endocarditis (IE) were analyzed using nanopore sequencing with an in silico host-depletion approach. Complete genome reconstruction and antimicrobial resistance gene annotation were successfully performed. RESULTS: Within an hour of sequencing, EPI2ME classified nanopore reads, identifying Corynebacterium striatum in IE patient 1 and Granulicatella adiacens in IE patient 2. After 18 h, long-read sequencing successfully reconstructed a single circular genome of C. striatum in IE patient 1, whereas short-read sequencing was used to compare but produced fragmented assemblies. Based on these results, long-read sequencing was exclusively used for IE patient 2, allowing for the complete and accurate assembly of G. adiacens, confirming the presence of these bacteria in the clinical samples. In addition to pathogen identification, antimicrobial resistance (AMR) genes were detected in both genomes. Notably, in C. striatum, regions containing a class 1 integron and multiple novel mobile genetic elements (ISCost1, ISCost2, Tn7838 and Tn7839) were identified, collectively harbouring six AMR genes. This is the first report of such elements in C. striatum, highlighting the potential of nanopore long-read sequencing for comprehensive pathogen characterization in IE cases. CONCLUSIONS: This study highlights the effectiveness of host-depleted, long-read nanopore metagenomics for direct pathogen identification and accurate genome reconstruction, including antimicrobial resistance gene detection. The approach enables same-day diagnostic reporting within a matter of hours. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-11741-5.202541087996
5831150.9494Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC. The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all β-lactam antibiotics. To avoid the use of β-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.201727569992
5494160.9494Molecular characterization of antimicrobial resistance in Brachyspira species isolated from UK chickens: Identification of novel variants of pleuromutilin and beta-lactam resistance genes. Brachyspira species are Gram negative, anaerobic bacteria that colonise the gut of many animals, including poultry. In poultry, Brachyspira species can be commensal (B. innocens, B. murdochii, 'B. pulli') or pathogenic (B. pilosicoli, B. intermedia, B. alvinipulli or rarely B. hyodysenteriae), the latter causing avian intestinal spirochaetosis (AIS). Antimicrobial therapy options for treatment is limited, frequently involving administration of the pleuromutilin, tiamulin, in water. In this study 38 Brachyspira isolates from chickens in the UK, representing both commensal and pathogenic species, were whole genome sequenced to identify antimicrobial resistance (AMR) mechanisms and the minimum inhibitory concentration (MIC) to a number of antimicrobials was also determined. We identified several new variants of bla(OXA) in B. pilosicoli and B. pulli isolates, and variations in tva which led to two new tva variants in B.murdochii and B.pulli. A number of isolates also harboured mutations known to encode AMR in the 16S and 23S rRNA genes. The percentage of isolates that were genotypically multi-drug resistance (MDR) was 16%, with the most common resistance profile being: tetracycline, pleuromutilin and beta-lactam, which were found in three 'B. pulli' and one B. pilosicoli. There was good correlation with the genotype and the corresponding antibiotic MIC phenotypes: pleuromutilins (tiamulin and valnemulin), macrolides (tylosin and tylvalosin), lincomycin and doxycycline. The occurrence of resistance determinants identified in this study in pathogenic Brachyspira, especially those which were MDR, is likely to impact treatment of AIS and clearance of infections on farm.202438306769
2486170.9494Virulence-associated genes and antimicrobial resistance patterns in bacteria isolated from pregnant and nonpregnant women with urinary tract infections: the risk of neonatal sepsis. Uropathogenic Escherichia coli (UPEC) is classified as the major causative agent of urinary tract infections (UTIs). UPEC virulence and antibiotic resistance can lead to complications in pregnant women and (or) newborns. Therefore, the aim of this study was to determine the etiological agents of UTIs, as well as to identify genes related to virulence factors in bacteria isolated from pregnant and nonpregnant women. A total of 4506 urine samples were collected from pregnant and nonpregnant women. Urine cultures were performed, and PCR was used to identify phylogroups and virulence-related genes. Antibiotic resistance profiles were determined. The incidence of UTIs was 6.9% (pregnant women, n = 206 and nonpregnant women, n = 57), and UPEC belonging to phylogroup A was the most prevalent. The presence of genes related to capsular protection, adhesins, iron acquisition, and serum protection in UPEC was associated with not being pregnant, while the presence of genes related to adhesins was associated with pregnancy. Bacteria isolated from nonpregnant women were more resistant to antibiotics; 36.5% were multidrug resistant, and 34.9% were extensively drug resistant. Finally, UTIs were associated with neonatal sepsis risk, particularly in pregnant women who underwent cesarean section while having a UTI caused by E. coli. In conclusion, UPEC isolated from nonpregnant women carried more virulence factors than those isolated from pregnant women, and maternal UTIs were associated with neonatal sepsis risk.202337815047
2365180.9494Vancomycin-resistant enterococci isolates colonizing and infecting haematology patients: clonality, and virulence and resistance profile. BACKGROUND: Vancomycin-resistant enterococci (VRE) are an important agent of colonization and infection in haematology patients. However, the role of virulence on VRE colonization and infection is controversial. AIM: To characterize the lineage, virulence and resistance profile of VRE infection and colonization isolates; as well as their impact on outcome of haematology patients using a regression logistic model. METHODS: Eighty-six isolates (80 Enterococcus faecium and six E. faecalis) from 76 patients were evaluated. Polymerase chain reaction for resistance and virulence genes, and pulsed-field gel electrophoresis and whole genome sequencing of the major clusters, were performed. Bivariate and multivariate analyses were carried out to evaluate the role of virulence genes on outcome. FINDINGS: All isolates harboured the vanA gene. Regarding the virulence genes, 96.5% of isolates were positive for esp, 69.8% for gelE and asa1 genes. VRE infection isolates were more virulent than colonization isolates and harboured more often the gelE gene (P = 0.008). Infections caused by VRE carrying asa1 gene resulted more frequently in death (P = 0.004), but only the predominant clone remained as protector in the multivariate model. The E. faecium strains were assigned to seven STs (ST78, ST412, ST478, ST792, ST896, ST987, ST963) that belonged to CC17. The E. faecalis sequenced belonged to ST9 (CC9). CONCLUSION: E. faecium was predominant, and infection isolates were more virulent than colonization isolates and harboured more often the gene gelE. Infections caused by VRE carrying the asa1 gene appeared to be associated with a fatal outcome.201829066140
2216190.9494Ultrafast detection of β-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing. Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1 h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.202337850345