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255300.9008Early-life gut microbiome modulation reduces the abundance of antibiotic-resistant bacteria. BACKGROUND: Antibiotic-resistant (AR) bacteria are a global threat. AR bacteria can be acquired in early life and have long-term sequelae. Limiting the spread of antibiotic resistance without triggering the development of additional resistance mechanisms is of immense clinical value. Here, we show how the infant gut microbiome can be modified, resulting in a significant reduction of AR genes (ARGs) and the potentially pathogenic bacteria that harbor them. METHODS: The gut microbiome was characterized using shotgun metagenomics of fecal samples from two groups of healthy, term breastfed infants. One group was fed B. infantis EVC001 in addition to receiving lactation support (n = 29, EVC001-fed), while the other received lactation support alone (n = 31, controls). Coliforms were isolated from fecal samples and genome sequenced, as well as tested for minimal inhibitory concentrations against clinically relevant antibiotics. RESULTS: Infants fed B. infantis EVC001 exhibited a change to the gut microbiome, resulting in a 90% lower level of ARGs compared to controls. ARGs that differed significantly between groups were predicted to confer resistance to beta lactams, fluoroquinolones, or multiple drug classes, the majority of which belonged to Escherichia, Clostridium, and Staphylococcus. Minimal inhibitory concentration assays confirmed the resistance phenotypes among isolates with these genes. Notably, we found extended-spectrum beta lactamases among healthy, vaginally delivered breastfed infants who had never been exposed to antibiotics. CONCLUSIONS: Colonization of the gut of breastfed infants by a single strain of B. longum subsp. infantis had a profound impact on the fecal metagenome, including a reduction in ARGs. This highlights the importance of developing novel approaches to limit the spread of these genes among clinically relevant bacteria. Future studies are needed to determine whether colonization with B. infantis EVC001 decreases the incidence of AR infections in breastfed infants. TRIAL REGISTRATION: This clinical trial was registered at ClinicalTrials.gov, NCT02457338.201931423298
140010.8982Comparative 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
226820.8945Profile of Bacteria with ARGs Among Real-World Samples from ICU Admission Patients with Pulmonary Infection Revealed by Metagenomic NGS. BACKGROUND: Treatment of pulmonary infections in the intensive care unit (ICU) represents a great challenge, especially infections caused by antibiotic resistance pathogens. A thorough and up-to-date knowledge of the local spectrum of antibiotic resistant bacteria can improve the antibiotic treatment efficiency. In this study, we aimed to reveal the profile of bacteria with antibiotic resistance genes (ARGs) in real-world samples from ICU admission patients with pulmonary infection in Mainland, China, by metagenomic next-generation sequencing (mNGS). METHODS: A total of 504 different types of clinical samples from 452 ICU admission patients with pulmonary infection were detected by mNGS analysis. RESULTS: A total of 485 samples from 434 patients got successful mNGS results. Among 434 patients, one or more bacteria with ARGs were detected in 192 patients (44.24%, 192/434), and ≥2 bacteria with ARGs were detected in 85 (19.59%, 85/434) patients. The predominant detected bacteria were Corynebacterium striatum (C. striatum) (11.76%, 51/434), Acinetobacter baumannii (A. baumannii) (11.52%, 50/434) and Enterococcus faecium (E. faecium) (8.99%, 39/434). ermX conferred resistance to MSL(B) and cmx to phenicol were the only two ARGs detected in C. striatum; in A. baumannii, most of ARGs were resistance-nodulation-division (RND)-type efflux pumps genes, which conferred resistance to multi-drug; ermB conferred resistance to MSL(B) and efmA to multi-drug were the predominant ARGs in E. faecium. Bacteria with ARGs were detected in 50% (140/280) bronchoalveolar lavage fluid (BALF) and 50.5% (48/95) sputum samples, which were significantly higher than in blood and cerebrospinal fluid (CSF) samples. CONCLUSION: High level of bacteria with ARGs was observed in clinical samples, especially BALF and sputum samples from ICU admission patients with pulmonary infection in Mainland, China. And C. striatum resistant to MSL(B) and/or phenicol, multi-drug resistance A. baumannii and E. faecium were the lead bacteria.202134866919
272230.8945Multidrug resistance assessment of indoor air in Portuguese long-term and acute healthcare settings. BACKGROUND: Knowledge about air as a pool of pathogens and multidrug resistance (MDR) in healthcare units apart from hospitals is scarce. AIM: To investigate these features in a Portuguese long-term healthcare unit (LTHU) and a central hospital (CH). METHODS: Air samples were collected and their microbial load (bacteria and fungi) determined. Bacterial isolates were randomly selected for further characterization, particularly identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, antimicrobial susceptibility testing, and polymerase chain reaction screening of extended-spectrum β-lactamases, carbapenemase genes and mecA gene, with RAPD profile assessment of positive results of the latter. FINDINGS: A total of 192 samples were collected (LTHU: 86; CH: 106). LTHU showed a statistically significantly higher bacterial load. CH bacteria and fungi loads in inpatient sites were statistically significantly lower than in outpatients or non-patient sites. A total of 164 bacterial isolates were identified (MALDI-TOF: 78; presumptively: 86), the majority belonging to Staphylococcus genus (LTHU: 42; CH: 57). The highest antimicrobial resistance rate was to erythromycin and vancomycin the least, in both settings. Eighteen isolates (11%) were classified as MDR (LTHU: 9; CH: 9), with 7 MDR Staphylococcus isolates (LTHU: 4; CH: 3) presenting mecA. Nine non-MDR Staphylococcus (LTHU: 5; CH: 4) also presented mecA. CONCLUSION: The current study highlights that healthcare unit indoor air can be an important pool of MDR pathogens and antimicrobial resistance genes. Also, LTHUs appear to have poorer air quality than hospitals, as well as supportive areas compared to curative care areas. This may suggest possible yet unknown routes of infection that need to be explored.202539983920
134840.8942Prevalence and transmission of antimicrobial-resistant Staphylococci and Enterococci from shared bicycles in Chengdu, China. Shared bicycles are prevailing in China but the extent to which they contribute to maintaining and transmitting pathogens and antibiotic-resistant bacteria remain largely unknown. To fill the knowledge gap, herein, swab samples (n = 963) were collected from handlebars of shared bicycles in areas of hospital, school, metro station (n = 887) and riders (n = 76) in Chengdu, China. Staphylococci (n = 241) and Enterococci (n = 69) were widely distributed across sampling locations at a frequency of 2.3%-12.9%, and 0.08%-5.5%, respectively. Bicycle or rider-borne Gram-positive bacteria were frequently resistant to clinically important antibiotics including linezolid, fosfomycin, and vancomycin, and a significant portion of these isolates (3.4%-16.6% for Staphylococci and 0.1%-13.8% for Enterococci) indicated multidrug resistance. Nineteen Staphylococcus aureus isolates were identified in this collection and 52.6% of which were considered as methicillin-resistant S. aureus. Whole genome sequencing further characterized 26 antimicrobial resistance genes (ARGs) including fosB, fusB, and lnu(G) in S. aureus and 21 ARGs including optrA in Enterococci. Leveraging a complementary approach with conventional MLST, whole genome SNP and MLST analyses, we present that genetically closely-related bacteria were found in bicycles and riders across geographical-distinct locations suggesting bacterial transmission. Further, five new ST types 5697-5701 were firstly characterized in S. aureus. ST 942 and ST 1640 are new ST types observed in E. faecalis, and E. faecium, respectively. Our results highlighted the risk of shared bicycle system in disseminating pathogens and antibiotic resistance which warrants effective disinfections.202032531590
306550.8941Species diversity, virulence, and antimicrobial resistance of the nasal staphylococcal and mammaliicoccal biota of reindeer. BACKGROUND: Staphylococcus (S.) spp. and Mammaliicoccus (M.) spp., in addition to their established role as components of the human and animal microbiota, can also cause opportunistic infections. This study aimed to characterize bacteria recovered from nasal cavities of healthy adult reindeer from two farms located in Poland (15 reindeer) and Germany (15 reindeer). The research include bacteria isolation, species identification, detection of selected superantigen (SAg) genes, assessment of biofilm-forming capability in vitro, and evaluation of antimicrobial resistance. RESULTS: Seventy-four staphylococci and mammaliicocci from 14 different species were isolated from 30 nasal swabs, with one to four strains obtained from each reindeer. The most frequently identified species was S. equorum, followed by S. succinus, M. sciuri, S. xylosus, M. lentus, S. chromogenes, S. devriesei, M. vitulinus, S. auricularis, S. agnetis, S. edaphicus, S. petrasii, S. simulans, and S. warneri. A greater species diversity was observed among the reindeer from Poland compared to those from Germany. All isolated bacteria were coagulase negative and clumping factor negative and did not carry any of the 21 analyzed SAg genes. M. sciuri demonstrated the highest antimicrobial resistance (100%), followed by S. succinus (91%) and S. equorum (78%). Resistance to rifampicin was the most common (30% strains). Sixteen strains (22%) exhibited biofilm production at least 10% greater than the strong biofilm-forming S. aureus ATCC 6538. CONCLUSIONS: This study reveals a significant knowledge gap regarding the nasal microbiota of reindeer. It contributes to our understanding of staphylococcal and mammaliicoccal biota of reindeer and underscores the necessity for monitoring of microbial populations to assess their health implications for both animals and humans, particularly concerning the zoonotic transmission of bacteria.202540452044
506760.8938Stepwise Evolution of a Klebsiella pneumoniae Clone within a Host Leading to Increased Multidrug Resistance. Five bla(CTX-M-14)-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.202134817239
236570.8937Vancomycin-resistant enterococci isolates colonizing and infecting haematology patients: clonality, and virulence and resistance profile. BACKGROUND: Vancomycin-resistant enterococci (VRE) are an important agent of colonization and infection in haematology patients. However, the role of virulence on VRE colonization and infection is controversial. AIM: To characterize the lineage, virulence and resistance profile of VRE infection and colonization isolates; as well as their impact on outcome of haematology patients using a regression logistic model. METHODS: Eighty-six isolates (80 Enterococcus faecium and six E. faecalis) from 76 patients were evaluated. Polymerase chain reaction for resistance and virulence genes, and pulsed-field gel electrophoresis and whole genome sequencing of the major clusters, were performed. Bivariate and multivariate analyses were carried out to evaluate the role of virulence genes on outcome. FINDINGS: All isolates harboured the vanA gene. Regarding the virulence genes, 96.5% of isolates were positive for esp, 69.8% for gelE and asa1 genes. VRE infection isolates were more virulent than colonization isolates and harboured more often the gelE gene (P = 0.008). Infections caused by VRE carrying asa1 gene resulted more frequently in death (P = 0.004), but only the predominant clone remained as protector in the multivariate model. The E. faecium strains were assigned to seven STs (ST78, ST412, ST478, ST792, ST896, ST987, ST963) that belonged to CC17. The E. faecalis sequenced belonged to ST9 (CC9). CONCLUSION: E. faecium was predominant, and infection isolates were more virulent than colonization isolates and harboured more often the gene gelE. Infections caused by VRE carrying the asa1 gene appeared to be associated with a fatal outcome.201829066140
226980.8936Genomic detection of Panton-Valentine Leucocidins encoding genes, virulence factors and distribution of antiseptic resistance determinants among Methicillin-resistant S. aureus isolates from patients attending regional referral hospitals in Tanzania. BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a formidable public scourge causing worldwide mild to severe life-threatening infections. The ability of this strain to swiftly spread, evolve, and acquire resistance genes and virulence factors such as pvl genes has further rendered this strain difficult to treat. Of concern, is a recently recognized ability to resist antiseptic/disinfectant agents used as an essential part of treatment and infection control practices. This study aimed at detecting the presence of pvl genes and determining the distribution of antiseptic resistance genes in Methicillin-resistant Staphylococcus aureus isolates through whole genome sequencing technology. MATERIALS AND METHODS: A descriptive cross-sectional study was conducted across six regional referral hospitals-Dodoma, Songea, Kitete-Kigoma, Morogoro, and Tabora on the mainland, and Mnazi Mmoja from Zanzibar islands counterparts using the archived isolates of Staphylococcus aureus bacteria. The isolates were collected from Inpatients and Outpatients who attended these hospitals from January 2020 to Dec 2021. Bacterial analysis was carried out using classical microbiological techniques and whole genome sequencing (WGS) using the Illumina Nextseq 550 sequencer platform. Several bioinformatic tools were used, KmerFinder 3.2 was used for species identification, MLST 2.0 tool was used for Multilocus Sequence Typing and SCCmecFinder 1.2 was used for SCCmec typing. Virulence genes were detected using virulenceFinder 2.0, while resistance genes were detected by ResFinder 4.1, and phylogenetic relatedness was determined by CSI Phylogeny 1.4 tools. RESULTS: Out of the 80 MRSA isolates analyzed, 11 (14%) were found to harbor LukS-PV and LukF-PV, pvl-encoding genes in their genome; therefore pvl-positive MRSA. The majority (82%) of the MRSA isolates bearing pvl genes were also found to exhibit the antiseptic/disinfectant genes in their genome. Moreover, all (80) sequenced MRSA isolates were found to harbor SCCmec type IV subtype 2B&5. The isolates exhibited 4 different sequence types, ST8, ST88, ST789 and ST121. Notably, the predominant sequence type among the isolates was ST8 72 (90%). CONCLUSION: The notably high rate of antiseptic resistance particularly in the Methicillin-resistant S. aureus strains poses a significant challenge to infection control measures. The fact that some of these virulent strains harbor the LukS-PV and LukF-PV, the pvl encoding genes, highlight the importance of developing effective interventions to combat the spreading of these pathogenic bacterial strains. Certainly, strengthening antimicrobial resistance surveillance and stewardship will ultimately reduce the selection pressure, improve the patient's treatment outcome and public health in Tanzania.202539833938
874790.8936An endolysin gene from Candidatus Liberibacter asiaticus confers dual resistance to huanglongbing and citrus canker. The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.202337719271
5142100.8934Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species-specific genomic properties and numerous putative antibiotic resistance determinants. BACKGROUND: Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. RESULTS: The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. CONCLUSION: Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.201627769168
5824110.8934Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis. BACKGROUND: Post-neurosurgical meningitis (PNM) is one of the most severe hospital-acquired infections worldwide, and a large number of pathogens, especially those possessing multi-resistance genes, are related to these infections. Existing methods for detecting bacteria and measuring their response to antibiotics lack sensitivity and stability, and laboratory-based detection methods are inconvenient, requiring at least 24h to complete. Rapid identification of bacteria and the determination of their susceptibility to antibiotics are urgently needed, in order to combat the emergence of multi-resistant bacterial strains. METHODS: This study evaluated a novel, fast, and easy-to-use micro/nanofluidic chip platform (MNCP), which overcomes the difficulties of diagnosing bacterial infections in neurosurgery. This platform can identify 10 genus or species targets and 13 genetic resistance determinants within 1h, and it is very simple to operate. A total of 108 bacterium-containing cerebrospinal fluid (CSF) cultures were tested using the MNCP for the identification of bacteria and determinants of genetic resistance. The results were compared to those obtained with conventional identification and antimicrobial susceptibility testing methods. RESULTS: For the 108 CSF cultures, the concordance rate between the MNCP and the conventional identification method was 94.44%; six species attained 100% consistency. For the production of carbapenemase- and extended-spectrum beta-lactamase (ESBL)-related antibiotic resistance genes, both the sensitivity and specificity of the MNCP tests were high (>90.0%) and could fully meet the requirements of clinical diagnosis. CONCLUSIONS: The MNCP is fast, accurate, and easy to use, and has great clinical potential in the treatment of post-neurosurgical meningitis.201829559366
5065120.8933Locus of Heat Resistance (LHR) in Meat-Borne Escherichia coli: Screening and Genetic Characterization. Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR(+)E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR(+) isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response.IMPORTANCE Currently, a "multiple-hurdle" approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438-1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments.202133483306
2538130.8933Passenger pathogens on physicians. BACKGROUND: Hospital acquired infections pose a significant risk for patients undergoing hematopoietic stem cell transplantation. Horizontal transfer of antimicrobial resistance genes contributes to prevalence of multidrug-resistant infections in this patient population. METHODS: At an academic bone marrow transplantation center, we performed whole genome DNA sequencing (WGS) on commonly used physician items, including badges, stethoscopes, soles of shoes, and smart phones from 6 physicians. Data were analyzed to determine antimicrobial resistance and virulence factor genes. RESULTS: A total of 1,126 unique bacterial species, 495 distinct bacteriophages, 91 unique DNA viruses, and 175 fungal species were observed. Every item contained bacteria with antibiotic and/or antiseptic resistance genes. Stethoscopes contained greatest frequency of antibiotic resistance and more plasmid-carriage of antibiotic resistance. DISCUSSION AND CONCLUSIONS: These data indicate that physician examination tools and personal items possess potentially pathogenic microbes. Infection prevention policies must consider availability of resources to clean physical examination tools as well as provider awareness when enacting hospital policies. Additionally, the prevalence of antimicrobial resistance genes (eg, encoding resistance to aminoglycosides, β-lactams, and quinolones) reinforces need for antimicrobial stewardship, including for immunocompromised patients. Further research is needed to assess whether minute quantities of microbes on physician objects detectable by WGS represents clinically significant inoculums for immunocompromised patients.202336306861
8728140.8933Identification of the defense-related gene VdWRKY53 from the wild grapevine Vitis davidii using RNA sequencing and ectopic expression analysis in Arabidopsis. BACKGROUND: Grapevine is an important fruit crop grown worldwide, and its cultivars are mostly derived from the European species Vitis vinifera, which has genes for high fruit quality and adaptation to a wide variety of climatic conditions. Disease resistance varies substantially across grapevine species; however, the molecular mechanisms underlying such variation remain uncharacterized. RESULTS: The anatomical structure and disease symptoms of grapevine leaves were analyzed for two grapevine species, and the critical period of resistance of grapevine to pathogenic bacteria was determined to be 12 h post inoculation (hpi). Differentially expressed genes (DEGs) were identified from transcriptome analysis of leaf samples obtained at 12 and 36 hpi, and the transcripts in four pathways (cell wall genes, LRR receptor-like genes, WRKY genes, and pathogenesis-related (PR) genes) were classified into four co-expression groups by using weighted correlation network analysis (WGCNA). The gene VdWRKY53, showing the highest transcript level, was introduced into Arabidopsis plants by using a vector containing the CaMV35S promoter. These procedures allowed identifying the key genes contributing to differences in disease resistance between a strongly resistant accession of a wild grapevine species Vitis davidii (VID) and a susceptible cultivar of V. vinifera, 'Manicure Finger' (VIV). Vitis davidii, but not VIV, showed a typical hypersensitive response after infection with a fungal pathogen (Coniella diplodiella) causing white rot disease. Further, 20 defense-related genes were identified, and their differential expression between the two grapevine species was confirmed using quantitative real-time PCR analysis. VdWRKY53, showing the highest transcript level, was selected for functional analysis and therefore over-expressed in Arabidopsis under the control of the CaMV35S promoter. The transgenic plants showed enhanced resistance to C. diplodiella and to two other pathogens, Pseudomonas syringae pv. tomato DC3000 and Golovinomyces cichoracearum. CONCLUSION: The consistency of the results in VID and transgenic Arabidopsis indicated that VdWRKY53 might be involved in the activation of defense-related genes that enhance the resistance of these plants to pathogens. Thus, the over-expression of VdWRKY53 in transgenic grapevines might improve their resistance to pathogens.201931057347
5171150.8932Adaptive laboratory-evolved MRSA with PPEF manifests cross-susceptibility to oxacillin and hypersensitivity to ciprofloxacin. Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved Staphylococcus aureus strains. The growth curve revealed that S. aureus 25923 PPEF-resistant (SA-PR) and methicillin-resistant S. aureus 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that atpD (ATP synthase gene) was downregulated by 2 log(2)-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log(2)-fold downregulation of mecR1 (methicillin resistance-inducing gene), ble (bleomycin resistance-inducing gene), blaZ (beta-lactamase), pbp (penicillin-binding protein gene), ermA (rRNA adenine methyltransferase gene), and kdpB (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log(2)-fold upregulation of comG and a 9 log(2)-fold downregulation of topB, indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including aac(6')-aph(2''), aadD, mecA, and blaZ in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved S. aureus strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how Staphylococcus aureus bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.202540662666
5167160.8932Decreased Antimicrobial Resistance Gene Richness Following Fecal Microbiota, Live-jslm (REBYOTA®) Administration: Post Hoc Analysis of PUNCH CD3. BACKGROUND: The human gastrointestinal microbiome helps maintain vital functions related to overall health, including resistance to pathogen colonization. Disruption of the microbiome, leading to loss of colonization resistance, can be caused by multiple factors, including antimicrobial use. The loss of colonization resistance may lead to establishment or proliferation of opportunistic bacteria that carry genes associated with antimicrobial resistance, potentially increasing the risk of infection by such antimicrobial-resistant bacteria. A potential approach to mitigating this risk involves restoration of healthier microbiota and pathogen colonization resistance. METHODS: A metagenomic sequencing method was used to conduct a post hoc analysis of antibiotic resistance gene richness among fecal samples from participants administered fecal microbiota, live-jslm (REBYOTA; abbreviated as RBL) or placebo in the PUNCH CD3 study (NCT03244644) for the prevention of recurrent Clostridioides difficile infection. RESULTS: At baseline, participants had higher antibiotic resistance gene richness than a representative healthy cohort. Over time, RBL responders had lower antibiotic resistance gene richness at the class, group, and mechanism levels as compared with placebo responders. These differences were evident as early as 1 week after administration and sustained for at least 6 months. RBL responders also had decreased richness of antibiotic resistance genes deemed high risk based on designated bacterial public health threats. CONCLUSIONS: These data support a model in which microbiota-based products, including RBL, may reduce antibiotic resistance gene richness, thereby possibly reducing the risk of antimicrobial-resistant organism infection. TRIAL REGISTRATION: NCT03244644 (https://clinicaltrials.gov/study/NCT03244644; 9 August 2017).202540672762
8472170.8931Genetic 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
5160180.8930Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs. OBJECTIVE: Microbial exposure is critical to neonatal and infant development, growth and immunity. However, whether a microbiome is present in the fetal gut prior to birth remains debated. In this study, lambs delivered by aseptic hysterectomy at full term were used as an animal model to investigate the presence of a microbiome in the prenatal gut using a multiomics approach. DESIGN: Lambs were euthanised immediately after aseptic caesarean section and their cecal content and umbilical cord blood samples were aseptically acquired. Cecal content samples were assessed using metagenomic and metatranscriptomic sequencing to characterise any existing microbiome. Both sample types were analysed using metabolomics in order to detect microbial metabolites. RESULTS: We detected a low-diversity and low-biomass microbiome in the prenatal fetal gut, which was mainly composed of bacteria belonging to the phyla Proteobacteria, Actinobacteria and Firmicutes. Escherichia coli was the most abundant species in the prenatal fetal gut. We also detected multiple microbial metabolites including short chain fatty acids, deoxynojirimycin, mitomycin and tobramycin, further indicating the presence of metabolically active microbiota. Additionally, bacteriophage phiX174 and Orf virus, as well as antibiotic resistance genes, were detected in the fetal gut, suggesting that bacteriophage, viruses and bacteria carrying antibiotic resistance genes can be transmitted from the mother to the fetus during the gestation period. CONCLUSIONS: This study provides strong evidence that the prenatal gut harbours a microbiome and that microbial colonisation of the fetal gut commences in utero.202133589511
2993190.8929Antibiotic resistant zoonotic bacteria in Irrawaddy squirrel (Callosciurus pygerythrus). Irrawaddy squirrel (Callosciurus pygerythrus) may play an important role in the transmission of zoonotic bacteria, but little is known about the carriage of zoonotic bacteria in this common frugivorous rodent in Bangladesh. We aimed to investigate the presence of common zoonotic bacterial pathogens in Irrawaddy squirrel in the southeast part of Bangladesh. A total of 27 rectal and 27 oro-nasal swabs were collected from 27 healthy wild Irrawaddy squirrels. Four common zoonotic bacteria were isolated following routine laboratory procedures, and were identified based on colony morphology, and biochemical and staining properties. The pathogenic potential of the identified bacteria was confirmed by detection of virulence genes by PCR. All isolates were subjected to antimicrobial susceptibility test against seven antibiotics from six generic groups which are commonly used in human and veterinary medicine in Bangladesh. The prevalence of Escherichia coli, Salmonella spp., Yersinia spp. and Staphylococcus spp. was 44.4% (95% CI, 32.0-57.6), 13% (95% CI, 6.1-24.7), 44.4% (95% CI, 32.0-57.6), and 72.2% (95% CI, 59.0-82.5), respectively. We identified potential zoonotic virulence genes in all of these four bacterial species. Antimicrobial susceptibility testing revealed the presence of several multidrug resistant bacterial strains in squirrels. To the best of our knowledge, this is the first report in Bangladesh of the detection of antibiotic resistant zoonotic bacteria in Irrawaddy squirrels. The findings underpin the role of Irrawaddy squirrel as a source of pathogenic antibiotic resistant bacteria, consequently, fruit rejected because of squirrel consumption and squirrel-bites deserve more concern than previously.201930488592