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843800.9198Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs. BACKGROUND: We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. METHODS: We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. RESULTS: The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. CONCLUSIONS: These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.201626431369
600310.9133Contact Lens Wear Alters Transcriptional Responses to Pseudomonas aeruginosa in Both the Corneal Epithelium and the Bacteria. PURPOSE: Healthy corneas resist colonization by virtually all microbes yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. METHODS: Male and female C57BL/6J mice were fitted with a contact lens on one eye for 24 h. After lens removal, corneas were immediately challenged for 4 h with P. aeruginosa. A separate group of naïve mice were similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naive controls as was lens wear alone. Total RNA-sequencing determined corneal epithelium and bacterial gene expression. RESULTS: Prior lens wear profoundly altered the corneal response to P. aeruginosa, including: upregulated pattern-recognition receptors (tlr3, nod1), downregulated lectin pathway of complement activation (masp1), amplified upregulation of tcf7, gpr55, ifi205, wfdc2 (immune defense) and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, gpr55 (immune defense) and downregulated Ca2(+)-dependent genes necab1, snx31 and npr3. P. aeruginosa exposure to prior lens wearing vs. naïve corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226) and antimicrobial resistance (arnB, oprR). CONCLUSION: Prior lens wear impacts corneal epithelium gene expression altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.202439677621
600420.9133Contact Lens Wear Alters Transcriptional Responses to Pseudomonas aeruginosa in Both the Corneal Epithelium and the Bacteria. PURPOSE: Healthy corneas resist colonization by virtually all microbes, yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. METHODS: Male and female C57BL/6J mice were fitted with a contact lens on one eye for 24 hours. After lens removal, corneas were immediately challenged for 4 hours with P. aeruginosa. A separate group of naïve mice was similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naïve controls, as was lens wear alone. Total RNA sequencing determined corneal epithelium and bacterial gene expression. RESULTS: Prior lens wear profoundly altered the corneal response to P. aeruginosa, including upregulated pattern recognition receptors (tlr3, nod1); downregulated lectin pathway of complement activation (masp1); amplified upregulation of tcf7, gpr55, ifi205, and wfdc2 (immune defense); and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, and gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31, and npr3. P. aeruginosa exposure to prior lens wearing versus naïve corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226), and antimicrobial resistance (arnB, oprR). CONCLUSIONS: Prior lens wear impacts corneal epithelium gene expression, altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival, and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.202539932472
601530.9133Integrative genome analysis of bacteriocin-producing Lactiplantibacillus pentosus LNP1-39 and its synbiotic role in suppressing food-borne pathogens. Lactic acid bacteria were isolated from traditional Thai-fermented foods. Among these, the strain LNP1-39, closely related to Lactiplantibacillus pentosus, was selected for further study because of its non-pathogenic profile. The bacteriocins produced by L. pentosus LNP1-39 were proteinaceous substances that exhibited strong antimicrobial activity across a wide pH range (pH 2-11; 6400-2400 AU/mL) and thermal stability at 100 °C for 40 min (400 AU/mL). These bacteriocins showed a narrow antimicrobial spectrum, effectively targeting Gram-positive pathogens, such as Kocuria rhizophila MIII, Enterococcus faecalis JCM 5803( T), and Listeria monocytogenes ATCC 19115. Comprehensive safety assessments, including whole-genome analysis and in vitro tests, confirmed a low risk of antibiotic resistance and the absence of virulence factors. Strain LNP1-39 was confirmed to be closely related to L. pentosus DSM 20314( T) via digital DNA‒DNA hybridization (dDDH; 75.4%), with average nucleotide identity (ANI) at 96.56% ANIb and 97.22% ANIm values. Additionally, LNP1-39 produces pediocin with notable similarity (76.29% identity to pediocin) and presents low risks for antibiotic-resistance genes or transfer genes while providing antioxidant properties. Strain LNP1-39 survived harsh gastrointestinal tract conditions and exhibited a favorable prebiotic index and positive prebiotic activity score when paired with polydextrose or isomalto-oligosaccharide. These findings support L. pentosus LNP1-39 as potential bacteriocin-producing lactic acid bacteria for further application in food preservation and pathogen control or as a synbiotic.202540622670
519640.9131Phenomics and genomic features of Enterococcus avium IRMC1622a isolated from a clinical sample of hospitalized patient. BACKGROUND: Enterococcus avium (E. avium) is a Gram-positive nosocomial pathogen that is commonly isolated from the alimentary tract. The objective of this functional genomics study was to identify the resistant genes by analyzing the genome of E. avium IRMC1622a, a type of bacteria found in feces collected from a patient at a Saudi Arabian tertiary hospital. METHODS: The bacterial strain IRMC1622a was identified by 16 S rRNA sequencing as Enterococcus sp. The resistance phenomics were performed using VITEK® 2, and morphological analysis was achieved using a scanning electron microscope (SEM). Finally, the whole bacterial genome of the bacterial strain IRMC1622a was subjected to sequencing during October 2023 using Oxford Nanopore long-read sequencing technology, and mining for resistant genes. RESULTS: The results of antimicrobial resistant phenomics indicated that the IRMC1622a strain was sensitive to all tested antimicrobial agents except for erythromycin, and the same result was confirmed by genomic analysis in addition to other classes of antibiotics. SEM showed E. avium IRMC1622a is ovoid shape, in single cells (L 1.2797 ± 0.1490 µm), in pairs (L 1.7333 ± 0.1054 µm), and in chains (L 2.44033 ± 0.1978 µm). The E. avium IRMC1622a genome has 14 (in CARD) antimicrobial resistance genes that were identified with several mechanisms of antimicrobial resistance, such as the efflux pump and conferring antibiotic resistance. The present study revealed that the E. avium IRMC1622a genome contains a high number of genes associated with virulence factors, and 14 matched pathogenic protein families and predicted as human pathogen (probability score 0.855). We report two (ISEnfa4 and ISEfa5) mobile genetic elements for the first time in the E. avium genome. CONCLUSIONS: The study concludes that E. avium IRMC1622a is susceptible to all tested antibacterials except erythromycin. The IRMC1622a has 14 genes encoding antimicrobial resistance mechanisms, including the efflux pump and conferring antibiotic resistance. This could indicate a potential rise in E. avium resistance in healthcare facilities. These observations may raise concerns regarding E. avium resistance in healthcare. We need more research to understand the pathophysiology of E. avium, which leads to hospital-acquired infections.202438833914
519350.9127Antibiotic resistance genes prediction via whole genome sequence analysis of Stenotrophomonas maltophilia. BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is the first dominant ubiquitous bacterial species identified from the genus Stenotrophomonas in 1943 from a human source. S. maltophilia clinical strains are resistance to several therapies, this study is designed to investigate the whole genome sequence and antimicrobial resistance genes prediction in Stenotrophomonas maltophilia (S. maltophilia) SARC-5 and SARC-6 strains, isolated from the nasopharyngeal samples of an immunocompromised patient. METHODS: These bacterial strains were obtained from Pakistan Institute of Medical Sciences (PIMS) Hospital, Pakistan. The bacterial genome was sequenced using a whole-genome shotgun via a commercial service that used an NGS (Next Generation Sequencing) technology called as Illumina Hiseq 2000 system for genomic sequencing. Moreover, detailed in-silico analyses were done to predict the presence of antibiotic resistance genes in S. maltophilia. RESULTS: Results showed that S. maltophilia is a rare gram negative, rod-shaped, non sporulating bacteria. The genome assembly results in 24 contigs (>500 bp) having a size of 4668,850 bp with 65.8% GC contents. Phylogenetic analysis showed that SARC-5 and SARC-6 were closely related to S. maltophilia B111, S. maltophilia BAB-5317, S. maltophilia AHL, S. maltophilia BAB-5307, S. maltophilia RD-AZPVI_04, S. maltophilia JFZ2, S. maltophilia RD_MAAMIB_06 and lastly with S. maltophilia sp ROi7. Moreover, the whole genome sequence analysis of both SARC-5 and SARC-6 revealed the presence of four resistance genes adeF, qacG, adeF, and smeR. CONCLUSION: Our study confirmed that S. maltophilia SARC-5 and SARC-6 are one of the leading causes of nosocomial infection which carry multiple antibiotic resistance genes.202438128408
332760.9126Ribaxamase, an Orally Administered β-Lactamase, Diminishes Changes to Acquired Antimicrobial Resistance of the Gut Resistome in Patients Treated with Ceftriaxone. INTRODUCTION: Intravenous (IV) β-lactam antibiotics, excreted through bile into the gastrointestinal (GI) tract, may disrupt the gut microbiome by eliminating the colonization resistance from beneficial bacteria. This increases the risk for Clostridium difficile infection (CDI) and can promote antimicrobial resistance by selecting resistant organisms and eliminating competition by non-resistant organisms. Ribaxamase is an orally administered β-lactamase for use with IV β-lactam antibiotics (penicillins and cephalosporins) and is intended to degrade excess antibiotics in the upper GI before they can disrupt the gut microbiome and alter the resistome. METHODS: Longitudinal fecal samples (349) were collected from patients who participated in a previous Phase 2b clinical study with ribaxamase for prevention of CDI. In that previous study, patients were treated with ceftriaxone for a lower respiratory tract infection and received concurrent ribaxamase or placebo. Extracted fecal DNA from the samples was subjected to whole-genome shotgun sequencing and analyzed for the presence of antimicrobial resistance (AMR) genes by alignment of sequences against the Comprehensive Antibiotic Resistance Database. A qPCR assay was also used to confirm some of the results. RESULTS: Database alignment identified ~1300 acquired AMR genes and gene variants, including those encoding β-lactamases and vancomycin resistance which were significantly increased in placebo vs ribaxamase-treated patients following antibiotic exposure. qPCR corroborated the presence of these genes and supported both new acquisition and expansion of existing gene pools based on no detectable copy number or a low copy number in pre-antibiotic samples which increased post-antibiotics. Additional statistical analyses demonstrated significant correlations between changes in the gut resistome and clinical study parameters including study drug assignment and β-lactamase and vancomycin resistance gene frequency. DISCUSSION: These findings demonstrated that ribaxamase reduced changes to the gut resistome subsequent to ceftriaxone administration and may help limit the emergence of AMR.202032801790
604670.9124Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
516980.9122Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment. Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.202336475849
908290.9121GeneMates: an R package for detecting horizontal gene co-transfer between bacteria using gene-gene associations controlled for population structure. BACKGROUND: Horizontal gene transfer contributes to bacterial evolution through mobilising genes across various taxonomical boundaries. It is frequently mediated by mobile genetic elements (MGEs), which may capture, maintain, and rearrange mobile genes and co-mobilise them between bacteria, causing horizontal gene co-transfer (HGcoT). This physical linkage between mobile genes poses a great threat to public health as it facilitates dissemination and co-selection of clinically important genes amongst bacteria. Although rapid accumulation of bacterial whole-genome sequencing data since the 2000s enables study of HGcoT at the population level, results based on genetic co-occurrence counts and simple association tests are usually confounded by bacterial population structure when sampled bacteria belong to the same species, leading to spurious conclusions. RESULTS: We have developed a network approach to explore WGS data for evidence of intraspecies HGcoT and have implemented it in R package GeneMates ( github.com/wanyuac/GeneMates ). The package takes as input an allelic presence-absence matrix of interested genes and a matrix of core-genome single-nucleotide polymorphisms, performs association tests with linear mixed models controlled for population structure, produces a network of significantly associated alleles, and identifies clusters within the network as plausible co-transferred alleles. GeneMates users may choose to score consistency of allelic physical distances measured in genome assemblies using a novel approach we have developed and overlay scores to the network for further evidence of HGcoT. Validation studies of GeneMates on known acquired antimicrobial resistance genes in Escherichia coli and Salmonella Typhimurium show advantages of our network approach over simple association analysis: (1) distinguishing between allelic co-occurrence driven by HGcoT and that driven by clonal reproduction, (2) evaluating effects of population structure on allelic co-occurrence, and (3) direct links between allele clusters in the network and MGEs when physical distances are incorporated. CONCLUSION: GeneMates offers an effective approach to detection of intraspecies HGcoT using WGS data.202032972363
1797100.9120Genetic Characteristics of the Transmissible Locus of Stress Tolerance (tLST) and tLST Harboring Escherichia coli as Revealed by Large-Scale Genomic Analysis. The transmissible locus of stress tolerance (tLST) confers resistance to multiple stresses in E. coli. Utilizing 18,959 E. coli genomes available in the NCBI database, we investigated the prevalence, phylogenetic distribution, and configuration patterns of tLST, and correlations between tLST, and virulence and antimicrobial resistance (AMR) genes in E. coli. Four tLST variants were found in 2.7% of E. coli, with the most prevalent (77.1%) variant being tLST1 followed by tLST2 (8.3%), tLST3b (8.3%) and tLST3a (6.3%). The majority (93%) of those tLST were in E. coli belonging to phylogroup A in which the prevalence was 10.4%. tLST was also found in phylogroup B1 (0.5%) and C (0.5%) but not found in B2 or D-G. An additional 1% of the 18,959 E. coli genomes harbored tLST fragments to various extent. Phylogenetic analysis revealed both intra- and interspecies transmission of both chromosomal and plasmid-borne tLST, with E. coli showing a preference of chromosomal over plasmid-borne tLST. The presence of tLST and virulence genes in E. coli was overall negatively correlated, but tLST was found in all genomes of a subgroup of enterotoxigenic E. coli (ST2332). Of note, no Shiga toxin-producing E. coli (n = 3,492) harbored tLST. The prevalence of tLST and AMR genes showed different temporal trends over the period 1985 to 2019. However, a substantial fraction of tLST positive E. coli harbor AMR genes, posing a threat to public health. In conclusion, this study improves our understanding of the genetic characteristics of tLST and E. coli harboring tLST. IMPORTANCE This study, through a large-scale genomic analysis, demonstrated that the genomic island tLST related to multiple stress resistance (such as extreme heat resistance and oxidative stress tolerance) in E. coli is differentially present in subgroups of E. coli and is strongly associated with certain phylogenetic background of the host strain. The study also shows the transmission mechanisms of tLST in E. coli and other bacterial species. The overall negative association of tLST, and virulence genes and antimicrobial (AMR) genes suggest the selective pressures for the acquisition and transmission of these traits likely differ. Even so, the high prevalence of tLST in the enterotoxigenic E. coli clone ST2332 and co-occurrence of tLST and AMR genes in E. coli are concerning. Thus, the findings better our understanding of tLST evolution and provide information for risk assessment of tLST harboring bacteria.202235285715
5230110.9120Characterization of Fosfomycin and Nitrofurantoin Resistance Mechanisms in Escherichia coli Isolated in Clinical Urine Samples. Fosfomycin and nitrofurantoin are antibiotics of choice to orally treat non-complicated urinary tract infections (UTIs) of community origin because they remain active against bacteria resistant to other antibiotics. However, epidemiologic surveillance studies have detected a reduced susceptibility to these drugs. The objective of this study was to determine possible mechanisms of resistance to these antibiotics in clinical isolates of fosfomycin- and/or nitrofurantoin-resistant UTI-producing Escherichia coli. We amplified and sequenced murA, glpT, uhpT, uhpA, ptsI, cyaA, nfsA, nfsB, and ribE genes, and screened plasmid-borne fosfomycin-resistance genes fosA3, fosA4, fosA5, fosA6, and fosC2 and nitrofurantoin-resistance genes oqxA and oqxB by polymerase chain reaction. Among 29 isolates studied, 22 were resistant to fosfomycin due to deletion of uhpT and/or uhpA genes, and 2 also possessed the fosA3 gene. Some modifications detected in sequences of NfsA (His11Tyr, Ser33Arg, Gln67Leu, Cys80Arg, Gly126Arg, Gly154Glu, Arg203Cys), NfsB (Gln44His, Phe84Ser, Arg107Cys, Gly192Ser, Arg207His), and RibE (Pro55His), and the production of truncated NfsA (Gln67 and Gln147) and NfsB (Glu54), were associated with nitrofurantoin resistance in 15/29 isolates; however, the presence of oqxAB plasmid genes was not detected in any isolate. Resistance to fosfomycin was associated with the absence of transporter UhpT expression and/or the presence of antibiotic-modifying enzymes encoded by fosA3 plasmid-mediated gene. Resistance to nitrofurantoin was associated with modifications of NfsA, NfsB, and RibE proteins. The emergence and spread of these resistance mechanisms, including transferable resistance, could compromise the future usefulness of fosfomycin and nitrofurantoin against UTIs. Furthermore, knowledge of the genetic mechanisms underlying resistance may lead to rapid DNA-based testing for resistance.202032847131
8472120.9119Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
5033130.9118A novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Imipenem-resistant Escherichia coli strains represent a growing public health concern, posing a threat due to their resistance to last-resort antibiotics. Here, we present the discovery of the Imipenem-Linked Resistance Gene VIN (ILR-VIN) within E. coli isolates from Vietnam, revealing its absence in non-resistant E. coli and local bacterial species. ILR-VIN constitutes a previously unrecognized genetic element potentially linked to Imipenem resistance, with notable prevalence in Vietnamese E. coli strains.We conducted an in-depth examination of the genetic basis of Carbapenem resistance in E. coli strains causing urinary tract infections. In a set of 47 UTI strains, we identified five displaying Imipenem resistance, with four of them carrying known resistance genes. Interestingly, ECV219, despite exhibiting Imipenem resistance, lacked known resistance genes, suggesting an unreported resistance mechanism. Comparative genetic analysis revealed distinct genes in ECV219, indicating a novel Imipenem resistance gene. To assess its function, we conducted transformation experiments in E. coli Rosetta™(DE3)pLysS and performed bioinformatics analyses using BLASTp, InterProScan, and Pfam to characterize the gene's structure and potential functions.Our study identifies ILR-VIN as a novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Experimental evidence confirmed that ILR-VIN expression enhances bacterial survival under Imipenem stress, providing direct evidence of its role in resistance. This discovery highlights the importance of ongoing research into antibiotic resistance genes to develop effective treatment strategies against antibiotic-resistant bacterial infections.202540097570
4761140.9117Antimicrobial resistance and biofilm formation of penile prosthesis isolates: insights from in-vitro analysis. BACKGROUND: Inflatable penile prostheses (IPPs) have been shown to harbor biofilms in the presence and absence of infection despite exposure to various antimicrobials. Microbes persisting on IPPs following antibiotic exposure have not been adequately studied to assess biofilm formation capacity and antibiotic resistance. AIM: In this study, we aimed to assess these properties of microbes obtained from explanted infected and non-infected IPPS using an in vitro model. METHODS: 35 bacterial isolates were grown and tested against various single-agent or multiple agent antibiotic regimens including: bacitracin, cefaclor, cefazolin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tobramycin, vancomycin, piperacillin/tazobactam, gentamicin + piperacillin/tazobactam, gentamicin + cefazolin, and gentamicin + vancomycin. Zones of inhibition were averaged for each sample site and species. Statistics were analyzed with Holm's corrected, one-sample t-tests against a null hypothesis of 0. Isolates were also allowed to form biofilms in a 96-well polyvinyl plate and absorbance was tested at 570 nm using a microplate reader. OUTCOMES: Resistance was determined via clinical guidelines or previously established literature, and the mean and standard deviation of biofilm absorbance values were calculated and normalized to the optical density600 of the bacterial inoculum. RESULTS: Every species tested was able to form robust biofilms with the exception of Staphylococcus warneri. As expected, most bacteria were resistant to common perioperative antimicrobial prophylaxis. Gentamicin dual therapy demonstrated somewhat greater efficacy. STRENGTHS AND LIMITATIONS: This study examines a broad range of antimicrobials against clinically obtained bacterial isolates. However, not all species and antibiotics tested had standardized breakpoints, requiring the use of surrogate values from the literature. The microbes included in this study and their resistance genes are expectedly biased towards those that survived antibiotic exposure, and thus reflect the types of microbes which might "survive" in vivo exposure following revisional surgery. CLINICAL TRANSLATION: Despite exposure to antimicrobials, bacteria isolated during penile prosthesis revision for both infected and non-infected cases exhibit biofilm forming capacity and extensive antibiotic resistance patterns in vitro. These microbes merit further investigation to understand when simple colonization vs re-infection might occur. CONCLUSIONS: Although increasing evidence supports the concept that all IPPs harbor biofilms, even in the absence of infection, a deeper understanding of the characteristics of bacteria that survive revisional surgery is warranted. This study demonstrated extensive biofilm forming capabilities, and resistance patterns among bacteria isolated from both non-infected and infected IPP revision surgeries. Further investigation is warranted to determine why some devices become infected while others remain colonized but non-infected.202540062463
5803150.9117Face mask sampling reveals antimicrobial resistance genes in exhaled aerosols from patients with chronic obstructive pulmonary disease and healthy volunteers. INTRODUCTION: The degree to which bacteria in the human respiratory tract are aerosolised by individuals is not established. Building on our experience sampling bacteria exhaled by individuals with pulmonary tuberculosis using face masks, we hypothesised that patients with conditions frequently treated with antimicrobials, such as chronic obstructive pulmonary disease (COPD), might exhale significant numbers of bacteria carrying antimicrobial resistance (AMR) genes and that this may constitute a previously undefined risk for the transmission of AMR. METHODS: Fifteen-minute mask samples were taken from 13 patients with COPD (five paired with contemporaneous sputum samples) and 10 healthy controls. DNA was extracted from cell pellets derived from gelatine filters mounted within the mask. Quantitative PCR analyses directed to the AMR encoding genes: blaTEM (β-lactamase), ErmB (target methylation), mefA (macrolide efflux pump) and tetM (tetracycline ribosomal protection protein) and six additional targets were investigated. Positive signals above control samples were obtained for all the listed genes; however, background signals from the gelatine precluded analysis of the additional targets. RESULTS: 9 patients with COPD (69%), aerosolised cells containing, in order of prevalence, mefA, tetM, ErmB and blaTEM, while three healthy controls (30%) gave weak positive signals including all targets except blaTEM. Maximum estimated copy numbers of AMR genes aerosolised per minute were mefA: 3010, tetM: 486, ErmB: 92 and blaTEM: 24. The profile of positive signals found in sputum was not concordant with that in aerosol in multiple instances. DISCUSSION: We identified aerosolised AMR genes in patients repeatedly exposed to antimicrobials and in healthy volunteers at lower frequencies and levels. The discrepancies between paired samples add weight to the view that sputum content does not define aerosol content. Mask sampling is a simple approach yielding samples from all subjects and information distinct from sputum analysis. Our results raise the possibility that patient-generated aerosols may be a significant means of AMR dissemination that should be assessed further and that consideration be given to related control measures.201830271606
5098160.9116Feature selection and aggregation for antibiotic resistance GWAS in Mycobacterium tuberculosis: a comparative study. INTRODUCTION: Drug resistance (DR) of pathogens remains a global healthcare concern. In contrast to other bacteria, acquiring mutations in the core genome is the main mechanism of drug resistance for Mycobacterium tuberculosis (MTB). For some antibiotics, the resistance of a particular isolate can be reliably predicted by identifying specific mutations, while for other antibiotics the knowledge of resistance mechanisms is limited. Statistical machine learning (ML) methods are used to infer new genes implicated in drug resistance leveraging large collections of isolates with known whole-genome sequences and phenotypic states for different drugs. However, high correlations between the phenotypic states for commonly used drugs complicate the inference of true associations of mutations with drug phenotypes by ML approaches. METHODS: Recently, several new methods have been developed to select a small subset of reliable predictors of the dependent variable, which may help reduce the number of spurious associations identified. In this study, we evaluated several such methods, namely, logistic regression with different regularization penalty functions, a recently introduced algorithm for solving the best-subset selection problem (ABESS) and "Hungry, Hungry SNPos" (HHS) a heuristic algorithm specifically developed to identify resistance-associated genetic variants in the presence of resistance co-occurrence. We assessed their ability to select known causal mutations for resistance to a specific drug while avoiding the selection of mutations in genes associated with resistance to other drugs, thus we compared selected ML models for their applicability for MTB genome wide association studies. RESULTS AND DISCUSSION: In our analysis, ABESS significantly outperformed the other methods, selecting more relevant sets of mutations. Additionally, we demonstrated that aggregating rare mutations within protein-coding genes into markers indicative of changes in PFAM domains improved prediction quality, and these markers were predominantly selected by ABESS, suggesting their high informativeness. However, ABESS yielded lower prediction accuracy compared to logistic regression methods with regularization.202540606161
5235170.9115Draft 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
8739180.9113LCT-EF258 with S17I Mutation in DprA Exhibits Horizontal Gene Transfer Deficiency After Spaceflight. BACKGROUND: Space is a special environment in which microgravity and cosmic rays are the primary factors that induce gene mutations of microorganisms. In our previous studies, a single point mutation in the gene dprA was found in an Enterococcus faecium strain of LCT-EF258 after spaceflight. DNA processing protein A (DprA) plays a prominent role in the horizontal transfer of genes among bacteria (such as Streptococcus pneumoniae, Helicobacter pylori, Bacillus subtilis, and Rhodobacter capsulatus). However, the function of DprA in E. faecium remains unknown. Furthermore, E. faecium could acquire antibiotic resistance through the horizontal transfer of antibiotic resistance genes, but it is unclear whether dprA mutants could affect this process in E. faecium.METHODS: In this study, we constructed a plasmid containing the vancomycin resistance gene vanA and then transferred the gene vanA into the dprA-mutant strain LCT-EF258 and the control strain LCT-EF90 using the electroporation technique. We then used Discovery Studio(TM) software to construct the 3D protein structure.RESULTS: The results showed that the horizontal transfer efficiency of the vancomycin resistance gene vanA in the dprA-mutant E. faecium decreased. And the hydrophobic core of the mutant DprA became stable and the binding affinity between the mutant DprA and ssDNA reduced.DISCUSSION: This study is an exploration of bacterial gene mutation after spaceflight. The dprA mutant could affect the ability of E. faecium to acquire exogenous resistance gene vanA, which offered us an interesting path to block the dissemination of resistance genes between strains.Yu Y, Chang D, Guo Q, Wang J, Liu C. LCT-EF258 with S171 mutation in DprA exhibits horizontal gene transfer deficiency after spaceflight. Aerosp Med Hum Perform. 2019; 90(2):116-122.201930670121
5143190.9113Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles. Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.202133737921