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604600.9059Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
10610.9056Genomic evidence of the illumination response mechanism and evolutionary history of magnetotactic bacteria within the Rhodospirillaceae family. BACKGROUND: Magnetotactic bacteria (MTB) are ubiquitous in natural aquatic environments. MTB can produce intracellular magnetic particles, navigate along geomagnetic field, and respond to light. However, the potential mechanism by which MTB respond to illumination and their evolutionary relationship with photosynthetic bacteria remain elusive. RESULTS: We utilized genomes of the well-sequenced genus Magnetospirillum, including the newly sequenced MTB strain Magnetospirillum sp. XM-1 to perform a comprehensive genomic comparison with phototrophic bacteria within the family Rhodospirillaceae regarding the illumination response mechanism. First, photoreceptor genes were identified in the genomes of both MTB and phototrophic bacteria in the Rhodospirillaceae family, but no photosynthesis genes were found in the MTB genomes. Most of the photoreceptor genes in the MTB genomes from this family encode phytochrome-domain photoreceptors that likely induce red/far-red light phototaxis. Second, illumination also causes damage within the cell, and in Rhodospirillaceae, both MTB and phototrophic bacteria possess complex but similar sets of response and repair genes, such as oxidative stress response, iron homeostasis and DNA repair system genes. Lastly, phylogenomic analysis showed that MTB cluster closely with phototrophic bacteria in this family. One photoheterotrophic genus, Phaeospirillum, clustered within and displays high genomic similarity with Magnetospirillum. Moreover, the phylogenetic tree topologies of magnetosome synthesis genes in MTB and photosynthesis genes in phototrophic bacteria from the Rhodospirillaceae family were reasonably congruent with the phylogenomic tree, suggesting that these two traits were most likely vertically transferred during the evolution of their lineages. CONCLUSION: Our new genomic data indicate that MTB and phototrophic bacteria within the family Rhodospirillaceae possess diversified photoreceptors that may be responsible for phototaxis. Their genomes also contain comprehensive stress response genes to mediate the negative effects caused by illumination. Based on phylogenetic studies, most of MTB and phototrophic bacteria in the Rhodospirillaceae family evolved vertically with magnetosome synthesis and photosynthesis genes. The ancestor of Rhodospirillaceae was likely a magnetotactic phototrophic bacteria, however, gain or loss of magnetotaxis and phototrophic abilities might have occurred during the evolution of ancestral Rhodospirillaceae lineages.201931117953
523720.9045Phenotypic and genomic analysis of Enterococcus avium MC09 pathogenicity isolated from Scylla spp. (mud crab) in a Thai market. Enterococcus avium is a Gram-positive pathogenic bacterium classified under the Enterococcaceae family. E. avium has been isolated from diverse environmental sources, raising concerns about its potential role in the spread of antibiotic resistance. E. avium MC09, isolated from a mud crab in a Thai market, was analyzed for its antibiotic resistance and pathogenic potential in this study. The isolation of E. avium from mud crab is significant as it highlights the potential role of seafood as a reservoir for antibiotic-resistant bacteria, which may pose risks to public health throughout the food chain. Antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method revealed that E. avium MC09 is resistant to clindamycin, erythromycin, streptomycin, and tetracycline, and exhibits alpha hemolysis on blood agar, indicating its potential virulence. Genomic DNA was extracted and sequenced using the Oxford Nanopore Technologies (ONT) platform, revealing the presence of resistance genes for macrolides (ermB) and tetracyclines (tetL and tetM). Furthermore, several virulence-associated genes were detected, such as srtC, ecbA, efaA, dltA, cpsA/uppS, cpsB/cdsA, cylR2, icps4I, cpsY, epsE, vctC, mgtB, ndk, lisR, and lgt suggesting a pathogenic potential. Additionally, the study identified several insertion sequences (ISs), including (IS1216, IS1216E, IS1216V, IS6770, ISEfa7, ISEfa8, and ISS1W which are commonly found in pathogenic Enterococcus strains. The presence of these IS elements further emphasizes the strain's potential for virulence and genetic adaptability. This study provides comprehensive insights into both the phenotypic and genotypic characteristics of E. avium MC09, highlighting its antimicrobial resistance and pathogenic mechanisms, and underlines the importance of monitoring antibiotic resistance in seafood-associated bacteria.202540015576
468230.9032Revealing antimicrobial resistance profile of the novel probiotic candidate Faecalibacterium prausnitzii DSM 17677. Faecalibacterium prausnitzii, a resident anaerobic bacterium commonly found in healthy gut microbiota, has been proposed as a next generation probiotic with high potential for application in food matrices and pharmaceutical formulations. Despite its recognized health benefits, detailed information regarding its antimicrobial susceptibility profile is still lacking. However, this information is crucial to determine its safety, since the absence of acquired antimicrobial resistance is required to qualify a probiotic candidate as safe for human and animal consumption. Herein, the antimicrobial susceptibility profile of F. prausnitzii DSM 17677 strain was evaluated by integrating both phenotypic and in silico data. Phenotypic antimicrobial susceptibility was evaluated by determining minimum inhibitory concentrations of 9 antimicrobials using broth microdilution and E-test® methods. Also, the whole genome of F. prausnitzii DSM 17677 was analysed, using several databases and bioinformatics tools, to identify possible antibiotic resistance genes (ARG), genomic islands (GI) and mobile genetic elements (MGE). With exception of erythromycin, the same classification (susceptible or resistant) was obtained in both broth microdilution and E-test® methods. Phenotypic resistance to ampicillin, gentamycin, kanamycin and streptomycin were detected, which was supported by the genomic context. Other ARG were also identified but they seem not to be expressed under the tested conditions. F. prausnitzii DSM 17677 genome contains 24 annotated genes putatively involved in resistance against the following classes of antimicrobials: aminoglycosides (such as gentamycin, kanamycin and streptomycin), macrolides (such as erythromycin), tetracyclines and lincosamides. The presence of putative ARG conferring resistance to β-lactams could only be detected using a broader homology search. The majority of these genes are not encoded within GI or MGE and no plasmids were reported for this strain. Despite the fact that most genes are related with general resistance mechanisms, a streptomycin-specific ARG poses the only potential concern identified. This specific ARG is encoded within a GI and a MGE, meaning that it could have been laterally acquired and might be transferred to other bacteria present in the same environment. Thus, our findings provide relevant insights regarding the phenotypic and genotypic antimicrobial resistance profiles of the probiotic candidate F. prausnitzii DSM 17677.202234953344
514240.9032Comparative 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
61250.9026Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus. The thick peptidoglycan layers of Gram-positive bacteria are connected to polyanionic glycopolymers called wall teichoic acids (WTA). Pathogens such as Staphylococcus aureus, Listeria monocytogenes, or Enterococcus faecalis produce WTA with diverse, usually strain-specific structure. Extensive studies on S. aureus WTA mutants revealed important functions of WTA in cell division, growth, morphogenesis, resistance to antimicrobials, and interaction with host or phages. While most of the S. aureus WTA-biosynthetic genes have been identified it remained unclear for long how and why S. aureus glycosylates WTA with α- or β-linked N-acetylglucosamine (GlcNAc). Only recently the discovery of two WTA glycosyltransferases, TarM and TarS, yielded fundamental insights into the roles of S. aureus WTA glycosylation. Mutants lacking WTA GlcNAc are resistant towards most of the S. aureus phages and, surprisingly, TarS-mediated WTA β-O-GlcNAc modification is essential for β-lactam resistance in methicillin-resistant S. aureus. Notably, S. aureus WTA GlcNAc residues are major antigens and activate the complement system contributing to opsonophagocytosis. WTA glycosylation with a variety of sugars and corresponding glycosyltransferases were also identified in other Gram-positive bacteria, which paves the way for detailed investigations on the diverse roles of WTA modification with sugar residues.201424365646
843960.9024Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens. BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.202235761183
902870.9021Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model. Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.201931324628
813380.9019Symbiotic bacteria confer insecticide resistance by metabolizing buprofezin in the brown planthopper, Nilaparvata lugens (Stål). Buprofezin, a chitin synthesis inhibitor, is widely used to control several economically important insect crop pests. However, the overuse of buprofezin has led to the evolution of resistance and exposed off-target organisms present in agri-environments to this compound. As many as six different strains of bacteria isolated from these environments have been shown to degrade buprofezin. However, whether insects can acquire these buprofezin-degrading bacteria from soil and enhance their own resistance to buprofezin remains unknown. Here we show that field strains of the brown planthopper, Nilaparvata lugens, have acquired a symbiotic bacteria, occurring naturally in soil and water, that provides them with resistance to buprofezin. We isolated a symbiotic bacterium, Serratia marcescens (Bup_Serratia), from buprofezin-resistant N. lugens and showed it has the capacity to degrade buprofezin. Buprofezin-susceptible N. lugens inoculated with Bup_Serratia became resistant to buprofezin, while antibiotic-treated N. lugens became susceptible to this insecticide, confirming the important role of Bup_Serratia in resistance. Sequencing of the Bup_Serratia genome identified a suite of candidate genes involved in the degradation of buprofezin, that were upregulated upon exposure to buprofezin. Our findings demonstrate that S. marcescens, an opportunistic pathogen of humans, can metabolize the insecticide buprofezin and form a mutualistic relationship with N. lugens to enhance host resistance to buprofezin. These results provide new insight into the mechanisms underlying insecticide resistance and the interactions between bacteria, insects and insecticides in the environment. From an applied perspective they also have implications for the control of highly damaging crop pests.202338091367
601690.9014Investigating human-derived lactic acid bacteria for alcohol resistance. BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.202438659044
3763100.9011Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands. Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates.IMPORTANCES. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.202032071265
8462110.9008Comparative Genomics of Lactiplantibacillus plantarum: Insights Into Probiotic Markers in Strains Isolated From the Human Gastrointestinal Tract and Fermented Foods. Lactiplantibacillus (Lpb.) plantarum is a versatile species commonly found in a wide variety of ecological niches including dairy products and vegetables, while it may also occur as a natural inhabitant of the human gastrointestinal tract. Although Lpb. plantarum strains have been suggested to exert beneficial properties on their host, the precise mechanisms underlying these microbe-host interactions are still obscure. In this context, the genome-scale in silico analysis of putative probiotic bacteria represents a bottom-up approach to identify probiotic biomarkers, predict desirable functional properties, and identify potentially detrimental antibiotic resistance genes. In this study, we characterized the bacterial genomes of three Lpb. plantarum strains isolated from three distinct environments [strain IMC513 (from the human GIT), C904 (from table olives), and LT52 (from raw-milk cheese)]. A whole-genome sequencing was performed combining Illumina short reads with Oxford Nanopore long reads. The phylogenomic analyses suggested the highest relatedness between IMC513 and C904 strains which were both clade 4 strains, with LT52 positioned within clade 5 within the Lpb. plantarum species. The comparative genome analysis performed across several Lpb. plantarum representatives highlighted the genes involved in the key metabolic pathways as well as those encoding potential probiotic features in these new isolates. In particular, our strains varied significantly in genes encoding exopolysaccharide biosynthesis and in contrast to strains IMC513 and C904, the LT52 strain does not encode a Mannose-binding adhesion protein. The LT52 strain is also deficient in genes encoding complete pentose phosphate and the Embden-Meyerhof pathways. Finally, analyses using the CARD and ResFinder databases revealed that none of the strains encode known antibiotic resistance loci. Ultimately, the results provide better insights into the probiotic potential and safety of these three strains and indicate avenues for further mechanistic studies using these isolates.202235663852
8438120.9006Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs. BACKGROUND: We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. METHODS: We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. RESULTS: The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. CONCLUSIONS: These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.201626431369
5235130.9004Draft genome sequences of rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South Africa. OBJECTIVES: Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogens is a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 in Africa. METHODS: The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees. RESULTS: Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (bla(ACT-9)), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin. CONCLUSION: The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobei in Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance.202336948496
9876140.9003The Facts and Family Secrets of Plasmids That Replicate via the Rolling-Circle Mechanism. Plasmids are self-replicative DNA elements that are transferred between bacteria. Plasmids encode not only antibiotic resistance genes but also adaptive genes that allow their hosts to colonize new niches. Plasmid transfer is achieved by conjugation (or mobilization), phage-mediated transduction, and natural transformation. Thousands of plasmids use the rolling-circle mechanism for their propagation (RCR plasmids). They are ubiquitous, have a high copy number, exhibit a broad host range, and often can be mobilized among bacterial species. Based upon the replicon, RCR plasmids have been grouped into several families, the best known of them being pC194 and pUB110 (Rep_1 family), pMV158 and pE194 (Rep_2 family), and pT181 and pC221 (Rep_trans family). Genetic traits of RCR plasmids are analyzed concerning (i) replication mediated by a DNA-relaxing initiator protein and its interactions with the cognate DNA origin, (ii) lagging-strand origins of replication, (iii) antibiotic resistance genes, (iv) mobilization functions, (v) replication control, performed by proteins and/or antisense RNAs, and (vi) the participating host-encoded functions. The mobilization functions include a relaxase initiator of transfer (Mob), an origin of transfer, and one or two small auxiliary proteins. There is a family of relaxases, the MOB(V) family represented by plasmid pMV158, which has been revisited and updated. Family secrets, like a putative open reading frame of unknown function, are reported. We conclude that basic research on RCR plasmids is of importance, and our perspectives contemplate the concept of One Earth because we should incorporate bacteria into our daily life by diminishing their virulence and, at the same time, respecting their genetic diversity.202234878299
5912150.9003Antibiotic Resistance-Susceptibility Profiles of Enterococcus faecalis and Streptococcus spp. From the Human Vagina, and Genome Analysis of the Genetic Basis of Intrinsic and Acquired Resistances. The spread of antibiotic resistance is a major public health concern worldwide. Commensal bacteria from the human genitourinary tract can act as reservoirs of resistance genes playing a role in their transfer to pathogens. In this study, the minimum inhibitory concentration of 16 antibiotics to 15 isolates from the human vagina, identified as Enterococcus faecalis, Streptococcus anginosus, and Streptococcus salivarius, was determined. Eight isolates were considered resistant to tetracycline, five to clindamycin and quinupristin-dalfopristin, and four to rifampicin. To investigate the presence of antimicrobial resistance genes, PCR analysis was performed in all isolates, and five were subjected to whole-genome sequencing analysis. PCR reactions identified tet(M) in all tetracycline-resistant E. faecalis isolates, while both tet(M) and tet(L) were found in tetracycline-resistant S. anginosus isolates. The tet(M) gene in E. faecalis VA02-2 was carried within an entire copy of the transposon Tn916. In S. anginosus VA01-10AN and VA01-14AN, the tet(M) and tet(L) genes were found contiguous with one another and flanked by genes encoding DNA mobilization and plasmid replication proteins. Amplification and sequencing suggested the lsaA gene to be complete in all E. faecalis isolates resistant to clindamycin and quinupristin-dalfopristin, while the gene contain mutations rendering to a non-functional LsaA in susceptible isolates. These results were subsequently confirmed by genome analysis of clindamycin and quinupristin-dalfopristin resistant and susceptible E. faecalis strains. Although a clinical breakpoint to kanamycin for S. salivarius has yet to be established, S. salivarius VA08-2AN showed an MIC to this antibiotic of 128 μg mL(-1). However, genes involved in kanamycin resistance were not identified. Under the assayed conditions, neither tet(L) nor tet(M) from either E. faecalis or S. anginosus was transferred by conjugation to recipient strains of E. faecalis, Lactococcus lactis, or Lactobacillus plantarum. Nonetheless, the tet(L) gene from S. anginosus VA01-10AN was amplified by PCR, and cloned and expressed in Escherichia coli, to which it provided a resistance of 48-64 μg mL(-1) to tetracycline. Our results expand the knowledge of the antibiotic resistance-susceptibility profiles of vaginal bacteria and provide the genetic basis of their intrinsic and acquired resistance.202032695087
5438160.9002Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility. Daptomycin is one of the last therapeutic resources for multidrug-resistant gram-positive bacteria. Despite its structural similarities with glycopeptides, its mechanisms of action and resistance are different and in some respects are not completely understood. Mutations in several genes have been associated with daptomycin resistance, especially in mprF, walkR, rpoB and rpoC, but their role and importance remain to be elucidated. We have studied mutations in 11 genes, which have been previously associated with daptomycin non-susceptibility, in nine daptomycin-non-susceptible Staphylococcus aureus clinical isolates (daptomycin MIC: >1 mg/L). Susceptibility to daptomycin, vancomycin, linezolid, oxacillin, telavancin and dalbavancin was studied. walkR, agrA, cls1, cls2, fakA, pnpA, clpP, prs, rpoB, rpoC and mprF were amplified by PCR and sequenced. The sequences were compared with the S. aureus ATCC 25923 complete genome (GenBank gi: 685631213) by using BLAST(®) software. We did not find any changes in walkR, pnpA, prs and clpP. All isolates excepting isolate MSa5 showed a high number of significant mutations (between 13 and 25 amino acid changes) in mprF. Most isolates also showed mutations in the rpo genes, the cls genes and fakA. Daptomycin non-susceptibility in S. aureus clinical isolates seems to be reached through different mutation combinations when compared to S. aureus ATCC 25293. Especially mprF and cls1 showed very high polymorphism in most isolates. Meanwhile, one isolate, MSa5, showed only single mutation in mprF (P314T).202438535549
3001170.9001IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers. SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.202438436262
1535180.9001Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
3004190.9000IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit. We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE: Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.201526646012