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616500.9976Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora. The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial concentration 25 μg ml(-1)) resulted in the selection of Ks resistance (at 150 μg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.201120923369
624510.9974Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii. Penicillin resistance in Streptococcus spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal Streptococcus gordonii. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 microg/ml). The major MIC increase (> or = 35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 microg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (> or = 2 microg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.200617000741
481020.9974Salmonella carrier-state in hens: study of host resistance by a gene expression approach. Salmonellosis is one of the main causes of food-borne poisoning due to the consumption of contaminated poultry products. In the flocks, Salmonella is able to persist in the digestive tract of birds for weeks without triggering any symptom. In order to identify molecules and genes involved in the mechanism of host resistance to intestinal carrier-state, two different inbred lines of laying hens were orally inoculated with Salmonella Enteritidis. Bacterial colonization and host gene expression were measured in the caecum and its sentinel lymphoid tissue, respectively. Significantly increased expression of chemokine, anti-infectious cytokine, bacterial receptor, antimicrobial mediator and particularly, defensin genes was observed in the line carrying a lower level of bacteria in the caecum. These innate immunity molecules were either constitutively or inductively highly expressed in resistant adult birds and thus present candidate genes to play an important role in the host defence against Salmonella colonization.200616702014
893730.9974Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis. The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.202337065137
70040.9974The extracytoplasmic function sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis. BACKGROUND: Enterococcus faecalis is one of the leading agents of nosocomial infections. To cause diseases, pathogens or opportunistic bacteria have to adapt and survive to the defense systems encountered in the host. One of the most important compounds of the host innate defense response against invading microorganisms is lysozyme. It is found in a wide variety of body fluids, as well as in cells of the innate immune system. Lysozyme could act either as a muramidase and/or as a cationic antimicrobial peptide. Like Staphylococcus aureus, E. faecalis is one of the few bacteria that are completely lysozyme resistant. RESULTS: This study revealed that oatA (O-acetyl transferase) and dlt (D-Alanylation of lipoteicoic acids) genes contribute only partly to the lysozyme resistance of E. faecalis and that a specific transcriptional regulator, the extracytoplasmic function SigV sigma factor plays a key role in this event. Indeed, the sigV single mutant is as sensitive as the oatA/dltA double mutant, and the sigV/oatA/dltA triple mutant displays the highest level of lysozyme sensitivity suggesting synergistic effects of these genes. In S. aureus, mutation of both oatA and dlt genes abolishes completely the lysozyme resistance, whereas this is not the case in E. faecalis. Interestingly SigV does not control neither oatA nor dlt genes. Moreover, the sigV mutants clearly showed a reduced capacity to colonize host tissues, as they are significantly less recovered than the parental JH2-2 strain from organs of mice subjected to intravenous or urinary tract infections. CONCLUSIONS: This work led to the discovery of an original model of lysozyme resistance mechanism which is obviously more complex than those described for other Gram positive pathogens. Moreover, our data provide evidences for a direct link between lysozyme resistance and virulence of E. faecalis.201020300180
893650.9973Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene. Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.202235756037
839160.9973The Analysis of Field Strains Isolated From Food, Animal and Clinical Sources Uncovers Natural Mutations in Listeria monocytogenes Nisin Resistance Genes. Nisin is a commonly used bacteriocin for controlling spoilage and pathogenic bacteria in food products. Strains possessing high natural nisin resistance that reduce or increase the potency of this bacteriocin against Listeria monocytogenes have been described. Our study sought to gather more insights into nisin resistance mechanisms in natural L. monocytogenes populations by examining a collection of 356 field strains that were isolated from different foods, food production environments, animals and human infections. A growth curve analysis-based approach was used to access nisin inhibition levels and assign the L. monocytogenes strains into three nisin response phenotypic categories; resistant (66%), intermediate (26%), and sensitive (8%). Using this categorization isolation source, serotype, genetic lineage, clonal complex (CC) and strain-dependent natural variation in nisin phenotypic resistance among L. monocytogenes field strains was revealed. Whole genome sequence analysis and comparison of high nisin resistant and sensitive strains led to the identification of new naturally occurring mutations in nisin response genes associated with increased nisin resistance and sensitivity in this bacterium. Increased nisin resistance was detected in strains harboring RsbU(G77S) and PBPB3(V240F) amino acid substitution mutations, which also showed increased detergent stress resistance as well as increased virulence in a zebra fish infection model. On the other hand, increased natural nisin sensitivity was detected among strains with mutations in sigB, vir, and dlt operons that also showed increased lysozyme sensitivity and lower virulence. Overall, our study identified naturally selected mutations involving pbpB3 (lm0441) as well as sigB, vir, and dlt operon genes that are associated with intrinsic nisin resistance in L. monocytogenes field strains recovered from various food and human associated sources. Finally, we show that combining growth parameter-based phenotypic analysis and genome sequencing is an effective approach that can be useful for the identification of novel nisin response associated genetic variants among L. monocytogenes field strains.202033123101
66270.9973Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress. Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.201323687271
441580.9973Staphylococcal resistance to streptogramins and related antibiotics. Streptogramin and related antibiotics are mixtures of two compounds, A and B (e.g. Dalfopristin and Quinupristin), particularly against Gram-positive bacteria. Staphylococci resistant to these mixtures are always resistant to the A compounds but are not necessarily resistant to the B compounds. Resistance to A compounds and to the mixtures is conferred by acetyltransferases or ATP-binding proteins via unknown mechanisms. Several genes encoding each of the two categories of protein have been characterized and regularly detected on plasmids. Genes encoding lactonases, which inactivate B compounds, have been occasionally detected on these plasmids. Staphylococci which harbour plasmids conferring resistance to A compounds should not be treated with the mixtures even if they appear susceptible in vitro. Indeed, susceptibility to the mixtures of staphylococci carrying resistance to A compounds has often been attributed to partial loss of the plasmids conferring this resistance. When staphylococci are constitutively resistant to B compounds, the in vitro activities of the mixtures should be evaluated, because they are better correlated than MICs with their efficacy in therapy.199817092802
624490.9973Whole-genome sequencing reveals a link between β-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus. The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to β-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 μg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 μg/ml) was also present with a frequency of ∼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of β-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.201323659600
4385100.9973Genes Contributing to the Unique Biology and Intrinsic Antibiotic Resistance of Enterococcus faecalis. The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci.IMPORTANCE Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci.202033234689
4812110.9973Production of the Bsa lantibiotic by community-acquired Staphylococcus aureus strains. Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.201020023032
6234120.9972Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli. BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.200717386106
6168130.9972SdiA aids enterohemorrhagic Escherichia coli carriage by cattle fed a forage or grain diet. Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activated gad gene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.201323836826
636140.9972Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes. Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.201425157076
6271150.9972Mechanisms 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
8863160.9972Resistance 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
8926170.9972Evolution of honey resistance in experimental populations of bacteria depends on the type of honey and has no major side effects for antibiotic susceptibility. With rising antibiotic resistance, alternative treatments for communicable diseases are increasingly relevant. One possible alternative for some types of infections is honey, used in wound care since before 2000 BCE and more recently in licensed, medical-grade products. However, it is unclear whether medical application of honey results in the evolution of bacterial honey resistance and whether this has collateral effects on other bacterial traits such as antibiotic resistance. Here, we used single-step screening assays and serial transfer at increasing concentrations to isolate honey-resistant mutants of Escherichia coli. We only detected bacteria with consistently increased resistance to the honey they evolved in for two of the four tested honey products, and the observed increases were small (maximum twofold increase in IC(90)). Genomic sequencing and experiments with single-gene knockouts showed a key mechanism by which bacteria increased their honey resistance was by mutating genes involved in detoxifying methylglyoxal, which contributes to the antibacterial activity of Leptospermum honeys. Crucially, we found no evidence that honey adaptation conferred cross-resistance or collateral sensitivity against nine antibiotics from six different classes. These results reveal constraints on bacterial adaptation to different types of honey, improving our ability to predict downstream consequences of wider honey application in medicine.202134025770
211180.9971Preclinical pharmacology of GAR-936, a novel glycylcycline antibacterial agent. GAR-936 is an analog of minocycline, a semisynthetic derivative of tetracycline. It has broad-spectrum antibacterial activity in vitro and in vivo. The new class of tetracyclines to which GAR-936 belongs is named the glycylcyclines. Tetracyclines act by inhibiting protein translation in bacteria, presumably by binding to the 30S ribosomal subunit and blocking entry of amino-acyl transfer RNA molecules into the A site of the ribosome. This prevents incorporation of amino acid residues into elongating peptide chains. In general, tetracyclines are considered bacteriostatic and the critical therapeutic parameter is the area under the concentration-time curve. GAR-936 has bactericidal activity; at 4 times the minimum inhibitory concentration, a 2- to 3-log reduction in colony counts was seen against Streptococcus pneumoniae, Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, and Staphylococcus aureus. GAR-936 is active against the antibiotic-resistant gram-positive bacteria methicillin-resistant Staphylococcus aureus, penicillin-resistant S. pneumoniae, and vancomycin-resistant enterococci. It is most significant that GAR-936 and other glycylcyclines are active against bacterial strains carrying either or both of the two major forms of tetracycline resistance: efflux and ribosomal protection. Using isogenic panels of bacteria carrying various tetracycline-resistance determinants, a series of more than 300 analogs was tested for antibacterial activity, which allowed for structure-activity relationships to be determined. Results indicated that certain substituents at the 9 position of the tetracycline molecule restored activity against bacteria harboring genes encoding either or both efflux and ribosomal protection. A single chemical modification overcame the two molecularly distinct forms of resistance while maintaining activity against susceptible gram-positive, gram-negative, aerobic, and anaerobic bacteria. Although mutants can be generated that are less susceptible to previously studied glycylcyclines, only marginal differences in susceptibility to GAR-936 were noted. Therefore, whereas emergence of resistance to any widely administered antibiotic is a foregone conclusion, resistance to GAR-936 will not readily arise by trivial mutations in existing resistance genes.200011001329
4809190.9971Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria. Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.200616888344