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973400.9862Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria. Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.202337264114
434910.9862CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.201627303749
996920.9862Molecular Characterization of pBOq-IncQ and pBOq-95LK Plasmids of Escherichia coli BOq 01, a New Isolated Strain from Poultry Farming, Involved in Antibiotic Resistance. The increase in antimicrobial resistance has raised questions about how to use these drugs safely, especially in veterinary medicine, animal nutrition, and agriculture. Escherichia coli is an important human and animal pathogen that frequently contains plasmids carrying antibiotic resistance genes. Extra chromosomal elements are required for various functions or conditions in microorganisms. Several phage-like plasmids have been identified, which are important in antibiotic resistance. In this work, the molecular characterization of the pBOq-IncQ (4.5 kb) and pBOq-95LK (95 kb) plasmids found in the E. coli strain BOq 01, a multidrug resistant bacteria isolated from a poultry farm, are considered. Plasmid pBOq-IncQ belongs to the incQ incompatibility plasmid family and is involved in sulfonamide resistance. Plasmid pBOq-95LK is a lytic phage-like plasmid that is involved in the lysis of the E. coli BOq 01 strain and carries a bleomycin resistance gene and a strain cured of this plasmid shows bleomycin sensitivity. Induction of the lytic cycle indicates that this phage-like plasmid is an active phage. This type of plasmid has been reported to acquire genes such as mcr-1, which codes for colistin resistance and bacterial persistence and is a significant public health threat. A genome comparison, a pangenomic and phylogenomic analysis with other phage-like plasmids reported in the literature were performed to understand better the evolution of this kind of plasmid in bacteria and its potential importance in antibiotic resistance.202235893567
604630.9861Safety 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
20540.9861Antibacterial activity of ethoxzolamide against Helicobacter pylori strains SS1 and 26695. With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium Helicobacter pylori in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one H. pylori strain, P12. We have now performed a susceptibility and resistance study with H. pylori strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10(-8). Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA's potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti-H. pylori drug.202032318117
30950.9860Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost. Plasmids promote adaptation of bacteria by facilitating horizontal transfer of diverse genes, notably those conferring antibiotic resistance. Some plasmids, like those of the incompatibility group IncP-1, are known to replicate and persist in a broad range of bacteria. We investigated a poorly understood exception, the IncP-1β plasmid pBP136 from a clinical Bordetella pertussis isolate, which quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution, we found that the inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid persistence in E. coli. The gene inactivation caused alterations in the plasmid regulatory system, including decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. This is consistent with our findings that Upf31 represses its own transcription. It also caused secondary transcriptional changes in many chromosomal genes. In silico analyses predicted that Upf31 interacts with the plasmid regulator KorB at its C-terminal dimerization domain (CTD). We showed experimentally that adding the CTD of upf31/pBP136 to the naturally truncated upf31 allele of the stable IncP-1β archetype R751 results in plasmid destabilization in E. coli. Moreover, mutagenesis showed that upf31 alleles encoded on nearly half of the sequenced IncP-1β plasmids also possess this destabilization phenotype. While Upf31 might be beneficial in many hosts, we show that in E. coli some alleles have harmful effects that can be rapidly alleviated with a single mutation. Thus, broad-host-range plasmid adaptation to new hosts can involve fine-tuning their transcriptional circuitry through evolutionary changes in a single gene.202540138356
819360.9859Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation. Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.202134311575
623270.9859Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes. The inability of Hyphomicrobium X to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (PDH) complex. Further support for this was sought by studying the effect of the introduction of the Escherichia coli pdh genes in Hyphomicrobium X on the pattern of substrate utilization by the latter organism. These genes were cloned by in vivo techniques using the broad-host range conjugative plasmid RP4::Mucts. Plasmid RP4 derivatives containing pdh genes were selected by their ability to complement a pyruvate dehydrogenase deletion mutant of E. coli, strain JRG746 recA (ace-1pd) delta 18. The plasmids thus obtained could be transferred through an intermediary host (C600 recA), selecting only for an antibiotic resistance coded for by RP4 and back into JRG746 or other E. coli pdh mutants, upon which they still conferred the wild type phenotype. Enzyme assays showed that the latter strains, when carrying plasmid RP4'pdh1 also possessed PDH complex activity. Conjugation between the auxotrophic E. coli JRG746 (RP4'pdh1) strain and Hyphomicrobium X on pyruvate minimal agar gave rise to progeny which, on the basis of its morphology (stalked bacteria), their ability to grow on C1-compounds and to denitrify (now also with pyruvate) were identified as hyphomicrobia. This Hyphomicrobium X transconjugant was also able to grow in minimal medium with succinate, but no other novel growth substrates have been identified so far.(ABSTRACT TRUNCATED AT 250 WORDS)19846393893
836980.9859Phage-resistant Pseudomonas aeruginosa against a novel lytic phage JJ01 exhibits hypersensitivity to colistin and reduces biofilm production. Pseudomonas aeruginosa, a major cause of nosocomial infections, has been categorized by World Health Organization as a critical pathogen urgently in need of effective therapies. Bacteriophages or phages, which are viruses that specifically kill bacteria, have been considered as alternative agents for the treatment of bacterial infections. Here, we discovered a lytic phage targeting P. aeruginosa, designated as JJ01, which was classified as a member of the Myoviridae family due to the presence of an icosahedral capsid and a contractile tail under TEM. Phage JJ01 requires at least 10 min for 90% of its particles to be adsorbed to the host cells and has a latent period of 30 min inside the host cell for its replication. JJ01 has a relatively large burst size, which releases approximately 109 particles/cell at the end of its lytic life cycle. The phage can withstand a wide range of pH values (3-10) and temperatures (4-60°C). Genome analysis showed that JJ01 possesses a complete genome of 66,346 base pairs with 55.7% of GC content, phylogenetically belonging to the genus Pbunavirus. Genome annotation further revealed that the genome encodes 92 open reading frames (ORFs) with 38 functionally predictable genes, and it contains neither tRNA nor toxin genes, such as drug-resistant or lysogenic-associated genes. Phage JJ01 is highly effective in suppressing bacterial cell growth for 12 h and eradicating biofilms established by the bacteria. Even though JJ01-resistant bacteria have emerged, the ability of phage resistance comes with the expense of the bacterial fitness cost. Some resistant strains were found to produce less biofilm and grow slower than the wild-type strain. Among the resistant isolates, the resistant strain W10 which notably loses its physiological fitness becomes eight times more susceptible to colistin and has its cell membrane compromised, compared to the wild type. Altogether, our data revealed the potential of phage JJ01 as a candidate for phage therapy against P. aeruginosa and further supports that even though the use of phages would subsequently lead to the emergence of phage-resistant bacteria, an evolutionary trade-off would make them more sensitive to antibiotics.202236274728
627190.9858Mechanisms of methicillin resistance in staphylococci. The continuously high prevalence of methicillin-resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin-binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for beta-lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating beta-lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid-borne blaR1-bla1 inducer-repressor system and the corresponding genomic mecRl-mecl system. The blaRl-blal products are important both for the regulation of beta-lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem-genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to beta-lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include beta-lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.19979164468
299100.9858Breaking barriers: pCF10 type 4 secretion system relies on a self-regulating muramidase to modulate the cell wall. Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria.202438940556
9990110.9857Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium. Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.200312603745
8863120.9857Resistance and tolerance to tropodithietic acid, an antimicrobial in aquaculture, is hard to select. The antibacterial compound tropodithietic acid (TDA) is produced by bacteria of the marine Roseobacter clade and is thought to explain the fish probiotic properties of some roseobacters. The aim of the present study was to determine the antibacterial spectrum of TDA and the likelihood of development of TDA resistance. A bacterial extract containing 95% TDA was effective against a range of human-pathogenic bacteria, including both Gram-negative and Gram-positive bacteria. TDA was bactericidal against Salmonella enterica serovar Typhimurium SL1344 and Staphylococcus aureus NCTC 12493 and killed both growing and nongrowing cells. Several experimental approaches were used to select mutants resistant to TDA or subpopulations of strains with enhanced tolerance to TDA. No approach (single exposures to TDA extract administered via different methods, screening of a transposon library for resistant mutants, or prolonged exposure to incremental concentrations of TDA) resulted in resistant or tolerant strains. After more than 300 generations exposed to sub-MIC and MIC concentrations of a TDA-containing extract, strains tolerant to 2× the MIC of TDA for wild-type strains were selected, but the tolerance disappeared after one passage in medium without TDA extract. S. Typhimurium mutants with nonfunctional efflux pump and porin genes had the same TDA susceptibility as wild-type strains, suggesting that efflux pumps and porins are not involved in innate tolerance to TDA. TDA is a promising broad-spectrum antimicrobial in part due to the fact that enhanced tolerance is difficult to gain and that the TDA-tolerant phenotype appears to confer only low-level resistance and is very unstable.201121263047
6023130.9857Bile-inducible efflux transporter from Bifidobacterium longum NCC2705, conferring bile resistance. Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.200919304838
307140.9856Escape from Lethal Bacterial Competition through Coupled Activation of Antibiotic Resistance and a Mobilized Subpopulation. Bacteria have diverse mechanisms for competition that include biosynthesis of extracellular enzymes and antibiotic metabolites, as well as changes in community physiology, such as biofilm formation or motility. Considered collectively, networks of competitive functions for any organism determine success or failure in competition. How bacteria integrate different mechanisms to optimize competitive fitness is not well studied. Here we study a model competitive interaction between two soil bacteria: Bacillus subtilis and Streptomyces sp. Mg1 (S. Mg1). On an agar surface, colonies of B. subtilis suffer cellular lysis and progressive degradation caused by S. Mg1 cultured at a distance. We identify the lytic and degradative activity (LDA) as linearmycins, which are produced by S. Mg1 and are sufficient to cause lysis of B. subtilis. We obtained B. subtilis mutants spontaneously resistant to LDA (LDAR) that have visibly distinctive morphology and spread across the agar surface. Every LDAR mutant identified had a missense mutation in yfiJK, which encodes a previously uncharacterized two-component signaling system. We confirmed that gain-of-function alleles in yfiJK cause a combination of LDAR, changes in colony morphology, and motility. Downstream of yfiJK are the yfiLMN genes, which encode an ATP-binding cassette transporter. We show that yfiLMN genes are necessary for LDA resistance. The developmental phenotypes of LDAR mutants are genetically separable from LDA resistance, suggesting that the two competitive functions are distinct, but regulated by a single two-component system. Our findings suggest that a subpopulation of B. subtilis activate an array of defensive responses to counter lytic stress imposed by competition. Coordinated regulation of development and antibiotic resistance is a streamlined mechanism to promote competitive fitness of bacteria.201526647299
8372150.9856A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis. Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.202031757821
5143160.9856Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles. Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.202133737921
114170.9856A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific. Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1 x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.200616641084
414180.9856A plasmid-encoded papB paralogue modulates autoaggregation of Escherichia coli transconjugants. OBJECTIVE: Plasmids are key to antimicrobial resistance transmission among enteric bacteria. It is becoming increasingly clear that resistance genes alone do not account for the selective advantage of plasmids and bacterial strains that harbor them. Deletion of a 32 Kb fitness-conferring region of pMB2, a conjugative resistance plasmid, produced a hyper-autoaggregation phenotype in laboratory Escherichia coli. This study sought to determine the genetic basis for hyper-autoaggregation conferred by the pMB2-derived mini-plasmid. RESULTS: The 32 Kb fragment deleted from pMB2 included previously characterized nutrient acquisition genes as well as putative transposase and integrase genes, a 272 bp papB/ pefB-like gene, and several open-reading frames of unknown function. We cloned the papB/ pefB paralogue and found it sufficient to temper the hyper-autoaggregation phenotype. Hyper-autoaggregation conferred by the mini-plasmid did not occur in a fim-negative background. This study has identified and characterized a gene capable of down-regulating host adhesins and has shown that trans-acting papB/pefB paralogues can occur outside the context of an adhesin cluster. This plasmid-mediated modification of a bacterial host's colonization program may optimize horizontal transfer of the mobile element bearing the genes.202033317611
446190.9856Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.200312676681