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613400.9856Complete genome and gene expression analyses of Asaia bogorensis reveal unique responses to culture with mammalian cells as a potential opportunistic human pathogen. Asaia bogorensis, a member of acetic acid bacteria (AAB), is an aerobic bacterium isolated from flowers and fruits, as well as an opportunistic pathogen that causes human peritonitis and bacteraemia. Here, we determined the complete genomic sequence of the As. bogorensis type strain NBRC 16594, and conducted comparative analyses of gene expression under different conditions of co-culture with mammalian cells and standard AAB culture. The genome of As. bogorensis contained 2,758 protein-coding genes within a circular chromosome of 3,198,265 bp. There were two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases: cyoABCD-1 and cyoABCD-2. The cyoABCD-1 operon was phylogenetically common to AAB genomes, whereas the cyoABCD-2 operon belonged to a lineage distinctive from the cyoABCD-1 operon. Interestingly, cyoABCD-1 was less expressed under co-culture conditions than under the AAB culture conditions, whereas the converse was true for cyoABCD-2. Asaia bogorensis shared pathogenesis-related genes with another pathogenic AAB, Granulibacter bethesdensis, including a gene coding pathogen-specific large bacterial adhesin and additional genes for the inhibition of oxidation and antibiotic resistance. Expression alteration of the respiratory chain and unique hypothetical genes may be key traits that enable the bacterium to survive under the co-culture conditions.201526358298
843810.9856Virulence 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
617320.9838Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae. BACKGROUND: RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants. METHODS: A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression. RESULTS: Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance. CONCLUSION: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.202337331490
506430.9837Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli. The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1(GI), yfdX2, hdeD(GI), orf11, trx(GI), kefB, and psiE(GI) by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript "GI" [genomic island] if an ortholog of the same gene is present in genomes of E. coli) LHR-encoded heat shock proteins sHSP20, ClpK(GI), and sHSP(GI) are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx(GI), kefB, and psiE(GI) from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food.IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.201728802266
41440.9835A 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
24350.9834Phylogenetic distribution of translational GTPases in bacteria. BACKGROUND: Translational GTPases are a family of proteins in which GTPase activity is stimulated by the large ribosomal subunit. Conserved sequence features allow members of this family to be identified. RESULTS: To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies. Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes.Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation--SelB--was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species. Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other groups of bacteria. CONCLUSION: Four of the translational GTPase subfamilies (IF2, EF-Tu, EF-G and LepA) are represented by at least one member in each bacterium studied, with one exception in LepA. This defines the set of translational GTPases essential for basic cell functions.200717214893
82460.9834Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola. Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3. 0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based Plac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.19989726857
619470.9834Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome. In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes. A 4.4-kb fragment encoding the S. typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC. This gene organization is conserved between E. coli and S. typhimurium. We determined that the S. typhimurium sdiA gene was able to weakly complement the E. coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele. To better understand the function of sdiA in S. typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S. typhimurium virulence plasmid. The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.19989495757
514380.9833Genomic 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
903390.9831Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde. Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H(2)O(2)) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [P(adj) ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.201830217837
6182100.9831An RND-type multidrug efflux pump SdeXY from Serratia marcescens. OBJECTIVES: Serratia marcescens, an important cause of nosocomial infections, shows intrinsic resistance to a wide variety of antimicrobial agents (multidrug resistance). Multidrug efflux pumps are often involved in the multidrug resistance in many bacteria. A study was undertaken to characterize the multidrug efflux pumps in S. marcescens. METHODS: The genes responsible for the multidrug resistance phenotype in S. marcescens were cloned into Escherichia coli KAM32, a drug-hypersusceptible strain, for further analysis. RESULTS: We cloned sdeXY genes and determined the nucleotide sequence. Clones that carried the sdeXY genes displayed reduced susceptibility to several antimicrobial agents including erythromycin, tetracycline, norfloxacin, benzalkonium chloride, ethidium bromide, acriflavine and rhodamine 6G. A protein similarity search using GenBank revealed that SdeY is a member of the resistance nodulation cell-division (RND) family of multidrug efflux proteins and SdeX is a member of the membrane fusion proteins. Introduction of sdeXY into E. coli cells possessing tolC, but not in cells lacking tolC, resulted in multidrug resistance. We observed energy-dependent ethidium efflux in cells of E. coli KAM32 possessing sdeXY and tolC. CONCLUSIONS: SdeXY is the first RND-type multidrug efflux pump to be characterized in multidrug-resistant S. marcescens.200312837741
9040110.9830Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum. BACKGROUND: Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria. METHODS: A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out. RESULTS: A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition. CONCLUSION: Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.200818801206
5162120.9830Genomic identification and characterization of Streptococcus oralis group that causes intraamniotic infection. BACKGROUND: Intraamniotic infection is a cause of spontaneous preterm labor. Streptococcus mitis is a common pathogen identified in intraamniotic infection, with the possible route of hematogenous dissemination from the oral cavity or migration from the vaginal canal. However, there are a few reports on Streptococcus oralis, a member of the S. mitis group, as a cause of pathogen in intraamniotic infection. We reported herein whole genome sequencing and comparative genomic analysis of S. oralis strain RAOG5826 that causes intraamniotic infection. RESULTS: Streptococcus mitis was initially identified from amniotic fluid, vaginal swab, and fetal blood of a patient presenting with preterm prelabor rupture of membranes with intraamniotic infection by the use of conventional microbiological methods (biochemical phenotype, MALDI-ToF, 16 S rRNA). Subsequently, this strain was later identified as S. oralis RAOG5826 by whole-genome hybrid sequencing. Genes involved in macrolide and tetracycline resistance, namely ermB and tet(M), and mutations in penicillin-binding protein were present in the genome. Moreover, potential virulence genes were predicted and compared with other Streptococcal species. CONCLUSION: We reported a comprehensive genomic analysis of S. oralis, which causes intraamniotic infection. S. mitis was initially identified by conventional microbiological identification. However, whole-genome hybrid sequencing demonstrates S. oralis with complete profiles of antimicrobial resistance genes and potential virulence factors. This study highlights the limitations of traditional techniques and underscores the importance of genomic sequencing for accurate diagnosis and tailored antimicrobial treatment. The study also suggests that S. oralis may be an underestimated pathogen in intraamniotic infection.202541023353
6195130.9829Differential gene expression analysis shows that cephalosporin resistance is intrinsic to Clostridioides difficile strain 630. Clostridioides difficile infection (CDI) is the most common nosocomial infection in the US. CDI has become a growing concern due to C. difficile's resistance to several antibiotics, including cephalosporins. Furthermore, patients administered cephalosporins are at higher risk of contracting CDI. Cephalosporins are β-lactam antibiotics, which prevent bacterial cell wall synthesis by inhibiting penicillin-binding proteins (PBPs). β-lactam-resistant bacteria evade these antibiotics by producing β-lactamases or by harboring low-affinity PBPs. A genomic analysis of C. difficile strain 630 identified 31 putative β-lactam resistance genes. Upon cefoxitin exposure, few C. difficile strain 630 putative antibiotic-resistant genes were overexpressed. Most notably, the β-lactamase blaCDD gene was upregulated approximately 600-fold, as previously reported. Deletion of the blaCDD locus did not change in cephalosporin susceptibility. Deletion of the second most upregulated gene, the PBP vanY, was also ineffective at decreasing cephalosporin resistance. Cefoxitin exposure of the C. difficile strain 630ΔblaCDD mutant did not increase upregulation of other putative antibiotic resistance genes compared to wildtype C. difficile strain 630. Transcriptomic analyses of wildtype C. difficile strain 630 exposed to cephradine, cefoxitin, ceftazidime, or cefepime revealed the shared upregulation of a putative heterodimeric ABC transporter encoded by loci CD630_04590 (ABC transporter ATP-binding protein) and CD630_04600 (ABC transporter permease). These genes are genomically located directly downstream of blaCDD (CD630_04580). The deletion mutant CD630_04600 remained resistant to a number of antibiotics. Thus, even though blaCDD, CD630_04590, and CD630_04600 are all upregulated when exposed to cephalosporins, they do not seem to be involved in antibiotic resistance in C. difficile strain 630.202539672901
9041140.9828Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis. BACKGROUND: Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. RESULTS: Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. CONCLUSIONS: Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.201121781329
5959150.9827High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7. The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR(+) open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10-100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.201424361397
9038160.9827Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms. The high tolerance of biofilm-grown Burkholderia cepacia complex bacteria against antimicrobial agents presents considerable problems for the treatment of infected cystic fibrosis patients and the implementation of infection control guidelines. In the present study, we analyzed the tolerance of planktonic and sessile Burkholderia cenocepacia J2315 cultures and examined the transcriptional response of sessile cells to treatment with chlorhexidine. At low (0.0005%) and high (0.05%) concentrations, chlorhexidine had a similar effect on both populations, but at intermediate concentrations (0.015%) the antimicrobial activity was more pronounced in planktonic cultures. The exposure of sessile cells to chlorhexidine resulted in an upregulation of the transcription of 469 (6.56%) and the downregulation of 257 (3.59%) protein-coding genes. A major group of upregulated genes in the treated biofilms encoded membrane-related and regulatory proteins. In addition, several genes coding for drug resistance determinants also were upregulated. The phenotypic analysis of RND (resistance-nodulation-division) efflux pump mutants suggests the presence of lifestyle-specific chlorhexidine tolerance mechanisms; efflux system RND-4 (BCAL2820-BCAL2822) was more responsible for chlorhexidine tolerance in planktonic cells, while other systems (RND-3 [BCAL1672-BCAL1676] and RND-9 [BCAM1945-BCAM1947]) were linked to resistance in sessile cells. After sessile cell exposure, multiple genes encoding chemotaxis and motility-related proteins were upregulated in concert with the downregulation of an adhesin-encoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carrying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.201121357299
8843170.9826Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space. Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease.IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.201931409659
9019180.9826Deleting qseC downregulates virulence and promotes cross-protection in Pasteurella multocida. QseC, a histidine sensor kinase of the QseBC two-component system, acts as a global regulator of bacterial stress resistance, biofilm formation, and virulence. The function of QseC in some bacteria is well understood, but not in Pasteurella multocida. We found that deleting qseC in P. multocida serotype A:L3 significantly down-regulated bacterial virulence. The mutant had significantly reduced capsule production but increased resistance to oxidative stress and osmotic pressure. Deleting qseC led to a significant increase in qseB expression. Transcriptome sequencing analysis showed that 1245 genes were regulated by qseC, primarily those genes involved in capsule and LPS biosynthesis and export, biofilm formation, and iron uptake/utilization, as well as several immuno-protection related genes including ompA, ptfA, plpB, vacJ, and sodA. In addition to presenting strong immune protection against P. multocida serotypes A:L1 and A:L3 infection, live ΔqseC also exhibited protection against P. multocida serotype B:L2 and serotype F:L3 infection in a mouse model. The results indicate that QseC regulates capsular production and virulence in P. multocida. Furthermore, the qseC mutant can be used as an attenuated vaccine against P. multocida strains of multiple serotypes.202134801081
8372190.9826A 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