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54400.9365Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner. The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.202235044847
140010.9347Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
140220.9345Detection of β-lactam resistance genes in Gram-negative bacteria from positive blood cultures using a microchip-based molecular assay. BACKGROUND: Accurate detection of β-lactam resistance genes in bloodstream infections is critical for guiding antimicrobial therapy. This study evaluates the Alifax Gram-negative resistance (GNR) microchip assay for detecting β-lactam resistance genes directly from positive blood cultures (PBCs) for Gram-negative (GN) bacteria, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. METHODS: Simulated (n=146) and clinical (n=106) GN-PBC samples were tested for bla (KPC), bla (VIM), bla (NDM), bla (IMP), bla (OXA-23)-like, bla (OXA-48)-like, bla (SHV)-ESBL, bla (CTX-M-1/9) group, and bla (CMY-2)-like genes using the GNR microchip assay. Whole-genome sequencing (WGS) served as the reference assay for simulated samples and, selectively, for clinical samples. The bioMérieux BioFire Blood Culture Identification 2 (BCID2) panel assay was used as a comparator for clinical samples. RESULTS: The GNR microchip assay correctly identified 203 (99.5%) of 204 β-lactam resistance genes in simulated samples. One sample tested false negative for a bla (SHV)-ESBL gene but true positive for a bla (KPC) gene. In clinical samples, GNR results were concordant with BCID2 for 113 (100%) of 113 genes included in both assays. Additionally, the GNR assay detected bla (CMY-2) -like (n=6), bla (OXA-23)-like (n=5), and bla (SHV)-ESBL (n=2), which are not targeted by BCID2, all confirmed by WGS. In two β-lactam-resistant P. aeruginosa samples but negative by the GNR assay, WGS confirmed the absence of acquired β-lactam resistance genes, suggesting alternative resistance mechanisms. CONCLUSION: The GNR microchip assay demonstrated high concordance and broader β-lactam resistance gene coverage compared to BCID2, supporting its potential role in routine diagnostics. Further validation in larger, prospective studies is warranted.202540529307
82730.9338Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient. Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, bla(OXA-61), tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.202439208964
140940.9337Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya. Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.202133617559
537550.9336Mechanism of Eravacycline Resistance in Clinical Enterococcus faecalis Isolates From China. Opportunistic infections caused by multidrug-resistant Enterococcus faecalis strains are a significant clinical challenge. Eravacycline (Erava) is a synthetic fluorocycline structurally similar to tigecycline (Tige) that exhibits robust antimicrobial activity against Gram-positive bacteria. This study investigated the in vitro antimicrobial activity and heteroresistance risk of Eravacycline (Erava) in clinical E. faecalis isolates from China along with the mechanism of Erava resistance. A total of 276 non-duplicate E. faecalis isolates were retrospectively collected from a tertiary care hospital in China. Heteroresistance to Erava and the influence of tetracycline (Tet) resistance genes on Erava susceptibility were examined. To clarify the molecular basis for Erava resistance, E. faecalis variants exhibiting Erava-induced resistance were selected under Erava pressure. The relative transcript levels of six candidate genes linked to Erava susceptibility were determined by quantitative reverse-transcription PCR, and their role in Erava resistance and heteroresistance was evaluated by in vitro overexpression experiments. We found that Erava minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates ranged from ≤0.015 to 0.25 mg/l even in strains harboring Tet resistance genes. The detection frequency of Erava heteroresistance in isolates with MICs ≤ 0.06, 0.125, and 0.25 mg/l were 0.43% (1/231), 7.5% (3/40), and 0 (0/5), respectively. No mutations were detected in the 30S ribosomal subunit gene in Erava heteroresistance-derived clones, although mutations in this subunit conferred cross resistance to Tige in Erava-induced resistant E. faecalis. Overexpressing RS00630 (encoding a bone morphogenetic protein family ATP-binding cassette transporter substrate-binding protein) in E. faecalis increased the frequency of Erava and Tige heteroresistance, whereas RS12140, RS06145, and RS06880 overexpression conferred heteroresistance to Tige only. These results indicate that Erava has potent in vitro antimicrobial activity against clinical E. faecalis isolates from China and that Erava heteroresistance can be induced by RS00630 overexpression.202032523563
83060.9334Detection and characterisation of 16S rRNA methyltransferase-producing Pseudomonas aeruginosa from the UK and Republic of Ireland from 2003-2015. 16S rRNA methyltransferase (16S RMTase) genes confer high-level aminoglycoside resistance, reducing treatment options for multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa isolates (n = 221) exhibiting high-level pan-aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) were screened for 16S RMTase genes to determine their occurrence among isolates submitted to a national reference laboratory from December 2003 to December 2015. 16S RMTase genes were identified using two multiplex PCRs, and whole-genome sequencing (WGS) was used to identify other antibiotic resistance genes, sequence types (STs) and the genetic environment of 16S RMTase genes. 16S RMTase genes were found in 8.6% (19/221) of isolates, with rmtB4 (47.4%; 9/19) being most common, followed by rmtD3 (21.1%; 4/19), rmtF2 (15.8%; 3/19) and single isolates harbouring rmtB1, rmtC and rmtD1. Carbapenemase genes were found in 89.5% (17/19) of 16S RMTase-positive isolates, with bla(VIM) (52.9%; 9/17) being most common. 16S RMTase genes were found in 'high-risk' clones known to harbour carbapenemase genes (ST233, ST277, ST357, ST654 and ST773). Analysis of the genetic environment of 16S RMTase genes identified that IS6100 was genetically linked to rmtB1; IS91 to rmtB4, rmtC or rmtD3; ISCR14 to rmtD1; and rmtF2 was linked to Tn3, IS91 or Tn1721. Although 16S RMTase genes explained only 8.6% of pan-aminoglycoside resistance in the P. aeruginosa isolates studied, the association of 16S RMTase genes with carbapenemase-producers and 'high-risk' clones highlights that continued surveillance is required to monitor spread as well as the importance of suppressing the emergence of dually-resistant clones in hospital settings.202235176475
85770.9333Characterization of KPC-Producing Serratia marcescens in an Intensive Care Unit of a Brazilian Tertiary Hospital. Serratia marcescens has emerged as an important opportunistic pathogen responsible for nosocomial and severe infections. Here, we determined phenotypic and molecular characteristics of 54 S. marcescens isolates obtained from patient samples from intensive-care-unit (ICU) and neonatal intensive-care-unit (NIUC) of a Brazilian tertiary hospital. All isolates were resistant to beta-lactam group antibiotics, and 92.6% (50/54) were not susceptible to tigecycline. Furthermore, 96.3% showed intrinsic resistance to polymyxin E (colistin), a last-resort antibiotic for the treatment of infections caused by MDR (multidrug-resistant) Gram-negative bacteria. In contrast, high susceptibility to other antibiotics such as fluoroquinolones (81.5%), and to aminoglycosides (as gentamicin 81.5%, and amikacin 85.2%) was found. Of all isolates, 24.1% were classified as MDR. The presence of resistance and virulence genes were examined by PCR and sequencing. All isolates carried KPC-carbapenemase (bla (KPC) ) and extended spectrum beta-lactamase bla (TEM) genes, 14.8% carried bla (OXA-) (1), and 16.7% carried bla (CTX-M-) (1) (group) genes, suggesting that bacterial resistance to β-lactam antibiotics found may be associated with these genes. The genes SdeB/HasF and SdeY/HasF that are associated with efflux pump mediated drug extrusion to fluoroquinolones and tigecycline, respectively, were found in 88.9%. The aac(6')-Ib-cr variant gene that can simultaneously induce resistance to aminoglycoside and fluoroquinolone was present in 24.1% of the isolates. Notably, the virulence genes to (i) pore-forming toxin (ShlA); (ii) phospholipase with hemolytic and cytolytic activities (PhlA); (iii) flagellar transcriptional regulator (FlhD); and (iv) positive regulator of prodigiosin and serratamolide production (PigP) were present in 98.2%. The genetic relationship among the isolates determined by ERIC-PCR demonstrated that the vast majority of isolates were grouped in a single cluster with 86.4% genetic similarity. In addition, many isolates showed 100% genetic similarity to each other, suggesting that the S. marcescens that circulate in this ICU are closely related. Our results suggest that the antimicrobial resistance to many drugs currently used to treat ICU and NIUC patients, associated with the high frequency of resistance and virulence genes is a worrisome phenomenon. Our findings emphasize the importance of active surveillance plans for infection control and to prevent dissemination of these strains.202032670210
153880.9330KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: bla(KPC-80), bla(KPC-81), bla(KPC-96) and bla(KPC-97). Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of bla(KPC-2) resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant bla(KPC) genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for bla(KPC-80), bla(KPC-96), and bla(KPC-97) by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including bla(TEM-1), bla(OXA-18) and bla(OXA-1), were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of bla(KPC-2) and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, bla(KPC-2). The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.202438319084
82890.9330Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana. Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and bla(CTX-M), bla(IMP), bla(GES), bla(VIM), bla(OXA-58)-like, bla(NDM), bla(OXA-23)-like, bla(OXA-48)-like and bla(KPC) occurring in descending order of frequency. The beta-lactamase genes bla(OXA-40/24)-like, bla(NMC_A/IMI), bla(BIC), bla(SME), bla(GIM) and bla(DIM) were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.202337623959
8727100.9330Transcriptome Analysis of Rice Near-Isogenic Lines Inoculated with Two Strains of Xanthomonas oryzae pv. oryzae, AH28 and PXO99(A). Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a major threat to rice production and food security. Exploring new resistance genes and developing varieties with broad-spectrum and high resistance has been a key focus in rice disease resistance research. In a preliminary study, rice cultivar Fan3, exhibiting high resistance to PXO99(A) and susceptibility to AH28, was developed by enhancing the resistance of Yuehesimiao (YHSM) to BB. This study performed a transcriptome analysis on the leaves of Fan3 and YHSM following inoculation with Xoo strains AH28 and PXO99(A). The analysis revealed significant differential expression of 14,084 genes. Among the transcription factor (TF) families identified, bHLH, WRKY, and ERF were prominent, with notable differences in the expression of OsWRKY62, OsWRKY76, and OsbHLH6 across samples. Over 100 genes were directly linked to disease resistance, including nearly 30 NBS-LRR family genes. Additionally, 11 SWEET family protein genes, over 750 protein kinase genes, 63 peroxidase genes, and eight phenylalanine aminolysase genes were detected. Gene ontology (GO) analysis showed significant enrichment in pathways related to defense response to bacteria and oxidative stress response. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis and diterpenoid biosynthesis pathways. Gene expression results from qRT-PCR were consistent with those from RNA-Seq, underscoring the reliability of the findings. Candidate genes identified in this study that may be resistant to BB, such as NBS-LRR family genes LOC_Os11g11960 and LOC_Os11g12350, SWEET family genes LOC_Os01g50460 and LOC_Os01g12130, and protein kinase-expressing genes LOC_Os01g66860 and LOC_Os02g57700, will provide a theoretical basis for further experiments. These results suggest that the immune response of rice to the two strains may be more concentrated in the early stage, and there are more up-regulated genes in the immune response of the high-resistant to PXO99A and medium-resistant to AH28, respectively, compared with the highly susceptible rice. This study offers a foundation for further research on resistance genes and the molecular mechanisms in Fan3 and YHSM.202439599338
6003110.9329Contact 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
1215120.9328The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales. INTRODUCTION: Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. METHODS: A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, β-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay(®)) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. RESULTS: Of 170 β-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla (SHV ESBL) (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla (CTX-M-1-group ESBL) (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. CONCLUSIONS: Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.202337900312
5240130.9328Dynamics of Antimicrobial Resistance Carriage in Koalas (Phascolarctos Cinereus) and Pteropid Bats (Pteropus Poliocephalus) Before, During and After Wildfires. In the 2019-2020 summer, wildfires decimated the Australian bush environment and impacted wildlife species, including koalas (Phascolarctos cinereus) and grey headed flying fox pups (Pteropid bats, Pteropus poliocephalus). Consequently, hundreds of koalas and thousands of bat pups entered wildlife hospitals with fire-related injuries/illness, where some individuals received antimicrobial therapy. This study investigated the dynamics of antimicrobial resistance (AMR) in pre-fire, fire-affected and post-fire koalas and Pteropid bat pups. PCR and DNA sequencing were used to screen DNA samples extracted from faeces (koalas and bats) and cloacal swabs (koalas) for class 1 integrons, a genetic determinant of AMR, and to identify integron-associated antibiotic resistance genes. Class 1 integrons were detected in 25.5% of koalas (68 of 267) and 59.4% of bats (92 of 155). Integrons contained genes conferring resistance to aminoglycosides, trimethoprim and beta-lactams. Samples were also screened for blaTEM (beta-lactam) resistance genes, which were detected in 2.6% of koalas (7 of 267) and 25.2% of bats (39 of 155). Integron occurrence was significantly higher in fire-affected koalas in-care compared to wild pre-fire koalas (P < 0.0001). Integron and blaTEM occurrence were not significantly different in fire-affected bats compared to pre-fire bats (P > 0.05), however, their occurrence was significantly higher in fire-affected bats in-care compared to wild fire-affected bats (P < 0.0001 and P = 0.0488 respectively). The observed shifts of AMR dynamics in wildfire-impacted species flags the need for judicious antibiotic use when treating fire-affected wildlife to minimise unwanted selective pressure and negative treatment outcomes associated with carriage of resistance genes and antibiotic resistant bacteria.202438332161
6004140.9327Contact 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
6006150.9327Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae. NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.202333685902
1451160.9326Molecular Epidemiology of Extensively Drug-Resistant mcr Encoded Colistin-Resistant Bacterial Strains Co-Expressing Multifarious β-Lactamases. Plasmid-mediated colistin resistance (Col-R) conferred by mcr genes endangers the last therapeutic option for multifarious β-lactamase-producing bacteria. The current study aimed to explore the mcr gene molecular epidemiology in extensively drug-resistant (XDR) bacteria. Col-R gram-negative bacterial strains were screened using a minimum inhibitory concentration (MIC) breakpoint ≥4 µg/mL. Resistant isolates were examined for mcr variants, extended-spectrum β-lactamase, AmpC, and carbapenemase genes using polymerase chain reaction (PCR). The MIC breakpoints for mcr-positive strains were determined using broth microdilution and E-test strips. Overall, 19/718 (2.6%) gram-negative rods (GNRs) harboring mcr were identified, particularly in pus (p = 0.01) and tracheal secretions (p = 0.03). Molecular epidemiology data confirmed 18/19 (95%) mcr-1 and 1/19 (5%) mcr-2 genes. Integron detection revealed 15/17 (88%) Int-1 and 2/17 (12%) Int-2. Common co-expressing drug-resistant β-lactamase genes included 8/16 (50%) bla(CTM-1), 3/16 (19%) bla(CTM-15), 3/3 (100%) bla(CMY-2), 2/8 (25%) bla(NDM-1), and 2/8 (25%) bla(NDM-5). The MIC(50) and MIC(90) values (µg/mL) were as follows: Escherichia coli, 12 and 24; Klebsiella pneumoniae, 12 and 32; Acinetobacter baumannii, 8 and 12; and Pseudomonas aeruginosa, 32 and 64, respectively. Treatment of XDR strains has become challenging owing to the co-expression of mcr-1, mcr-2, multifarious β-lactamase genes, and integrons.202133923991
808170.9326Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H(2)O(2)) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.202438305908
6158180.9326Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase. Porphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 (PG0893ermF) and FLL456 (PG2213ermF) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.201222247513
1218190.9325Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania. We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.202031999747