Encoding Elongation Factor Research Articles

Enterococcus faecalis OG1RF Evolution at Low pH Selects Fusidate-Sensitive Mutants in Elongation Factor G and at High pH Selects Defects in Phosphate Transport.

Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.

First isolation of Arcanobacterium pinnipediorum from a grey seal pup (Halichoerus grypus) in the UK

In the present study, a single Arcanobacterium (A.) pinnipediorum strain isolated from discharge of a jaw swelling of a grey seal pup (Halichoerus grypus) in England, UK, was identified. This strain was further characterized by phenotypical investigations, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), by Fourier transform infrared spectroscopy (FT-IR), and genotypically by sequencing the 16S rRNA gene and the genes gap encoding glyceraldehyde 3-phosphate dehydrogenase, tuf encoding elongation factor tu, and rpoB encoding the β subunit of bacterial RNA polymerase. The present study gives a first detailed characterization of the species A. pinnipediorum from a grey seal in the UK. However, the route of infection of the grey seal with the bacterial pathogen remains unclear.

Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis.

The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and β-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes; thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.

Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius)

BackgroundThe present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).Case presentationThe species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level.ConclusionsBoth strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36g/L L-leucine in shake flask fermentation.

Sequence Analysis of New Tuf Molecular Types of 'Candidatus Phytoplasma Solani' in Iranian Vineyards.

Grapevine Bois noir (BN) is caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) and is one of the most important phytoplasma diseases in the Euro-Mediterranean viticultural areas. The epidemiology of BN can include grapevine as a plant host and is usually transmitted via sap-sucking insects that inhabit herbaceous host plants. Tracking the spread of ‘Ca. P. solani’ strains is of great help for the identification of plant reservoirs and insect vectors involved in local BN outbreaks. The molecular epidemiology of ‘Ca. P. solani’ is primarily based on sequence analysis of the tuf housekeeping gene (which encodes elongation factor Tu). In this study, molecular typing of tuf, through restriction fragment length polymorphism and sequencing, was carried out on grapevine samples from Iranian vineyards. According to the molecular characterization, three molecular types—tuf b1, tuf b5 and tuf b6—were found, with tuf b1 being the most prominent. These data provide further knowledge of tuf gene diversity and question the ecological role of such “minor” tuf types in Iranian vineyards, which have been detected only in grapevines.

Parallel Evolution of Tobramycin Resistance across Species and Environments.

Different species exposed to a common stress may adapt by mutations in shared pathways or in unique systems, depending on how past environments have molded their genomes. Understanding how diverse bacterial pathogens evolve in response to an antimicrobial treatment is a pressing example of this problem, where discovery of molecular parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in environments containing antibiotics, combined with periodic whole-population genome sequencing, can be used to identify many contending routes to antimicrobial resistance. We separately propagated two clinically relevant Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing concentrations of tobramycin in two different environments each: planktonic and biofilm. Independently of the pathogen, the populations adapted to tobramycin selection by parallel evolution of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate phosphotransferase. As neither gene is a direct target of this aminoglycoside, mutations to either are unexpected and underreported causes of resistance. Additionally, both species acquired antibiotic resistance-associated mutations that were more prevalent in the biofilm lifestyle than in the planktonic lifestyle; these mutations were in electron transport chain components in A. baumannii and lipopolysaccharide biosynthesis enzymes in P. aeruginosa populations. Using existing databases, we discovered site-specific parallelism of fusA1 mutations that extends across bacterial phyla and clinical isolates. This study suggests that strong selective pressures, such as antibiotic treatment, may result in high levels of predictability in molecular targets of evolution, despite differences between organisms' genetic backgrounds and environments.IMPORTANCE The rise of antimicrobial resistance is a leading medical threat, motivating efforts to forecast both its evolutionary dynamics and its genetic causes. Aminoglycosides are a major class of antibiotics that disrupt translation, but resistance may occur by a number of mechanisms. Here, we show the repeated evolution of resistance to the aminoglycoside tobramycin in both P. aeruginosa and A. baumannii via mutations in fusA1, encoding elongation factor G, and ptsP, encoding the nitrogen-specific phosphotransferase system. Laboratory evolution and whole-population genome sequencing were used to identify these targets, but mutations at identical amino acid positions were also found in published genomes of diverse bacterial species and clinical isolates. We also identified other resistance mechanisms associated with growth in biofilms that likely interfere with drug binding or uptake. Characterizing the evolution of multiple species in the presence of antibiotics can identify new, repeatable causes of resistance that may be predicted and counteracted by alternative treatment.

Candidate Genes for Freezing and Drought Tolerance Selected on the Basis of Proteome Analysis in Doubled Haploid Lines of Barley.

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, β-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.

Epidemiological analysis of Trueperella abortisuis isolated from cases of pig abortion of a single farm.

The present study was designed to characterize six Trueperella (T.) abortisuis strains, cultured over a period of 5 months from fetus and abortion material of six pigs of a single farm in Mecklenburg-West Pomerania federal state, Germany. It was of interest to investigate the epidemiological relationships of the six strains among each other and whether a single bacterial clone was responsible for the abortion situation of the single farm. All six strains were identified phenotypically, by MALDI-TOF MS analysis and by phylogenetic analysis based on 16S rRNA gene and gap (encoding the glyceraldehyde 3-phosphate dehydrogenase) and tuf (encoding elongation factor tu) gene sequencing. Further genotypic comparison was performed using different genomic DNA fingerprint methods including BOX-PCR, (GTG)5-PCR, and three RAPD-PCRs. The sequence analysis of the genes gap and tuf and the genomic DNA fingerprinting results revealed, as noval findings, that the six T. abortisuis strains cultured from a single farm represent six different bacterial clones showing a genetic variability of this bacterial species in the pig population. All six T. abortisuis strains were isolated in mixed culture with several other bacterial species. However, the T. abortisuis strain, generally found in high numbers, seemed to be responsible for the abortion situation in the farm.

Comparative functional genomics of the acarbose producers reveals potential targets for metabolic engineering

The α-glucosidase inhibitor acarbose is produced in large-scale by strains derived from Actinoplanes sp. SE50 and used widely for the treatment of type-2 diabetes. Compared with the wild-type SE50, a high-yield derivative Actinoplanes sp. SE50/110 shows 2-fold and 3–7-fold improvement of acarbose yield and acb cluster transcription, respectively. The genome of SE50 was fully sequenced and compared with that of SE50/110, and 11 SNVs and 4 InDels, affecting 8 CDSs, were identified in SE50/110. The 8 CDSs were individually inactivated in SE50. Deletions of ACWT_4325 (encoding alcohol dehydrogenase) resulted in increases of acarbose yield by 25% from 1.87 to 2.34 g/L, acetyl-CoA concentration by 52.7%, and PEP concentration by 22.7%. Meanwhile, deletion of ACWT_7629 (encoding elongation factor G) caused improvements of acarbose yield by 36% from 1.87 to 2.54 g/L, transcription of acb cluster, and ppGpp concentration to 2.2 folds. Combined deletions of ACWT_4325 and ACWT_7629 resulted in further improvement of acarbose to 2.83 g/L (i.e. 76% of SE50/110), suggesting that the metabolic perturbation and improved transcription of acb cluster caused by these two mutations contribute substantially to the acarbose overproduction. Enforced application of similar strategies was performed to manipulate SE50/110, resulting in a further increase of acarbose titer from 3.73 to 4.21 g/L. Therefore, the comparative genomics approach combined with functional verification not only revealed the acarbose overproduction mechanisms, but also guided further engineering of its high-yield producers.

Collaboration of tRNA modifications and elongation factor eEF1A in decoding and nonsense suppression

Transfer RNA (tRNA) from all domains of life contains multiple modified nucleosides, the functions of which remain incompletely understood. Genetic interactions between tRNA modification genes in Saccharomyces cerevisiae suggest that different tRNA modifications collaborate to maintain translational efficiency. Here we characterize such collaborative functions in the ochre suppressor tRNA SUP4. We quantified ochre read-through efficiency in mutants lacking either of the 7 known modifications in the extended anticodon stem loop (G26-C48). Absence of U34, U35, A37, U47 and C48 modifications partially impaired SUP4 function. We systematically combined modification defects and scored additive or synergistic negative effects on SUP4 performance. Our data reveal different degrees of functional redundancy between specific modifications, the strongest of which was demonstrated for those occurring at positions U34 and A37. SUP4 activity in the absence of critical modifications, however, can be rescued in a gene dosage dependent fashion by TEF1 which encodes elongation factor eEF1A required for tRNA delivery to the ribosome. Strikingly, the rescue ability of higher-than-normal eEF1A levels extends to tRNA modification defects in natural non-suppressor tRNAs suggesting that elevated eEF1A abundance can partially compensate for functional defects induced by loss of tRNA modifications.

Identification of a New Antimicrobial Resistance Gene Provides Fresh Insights Into Pleuromutilin Resistance in Brachyspira hyodysenteriae, Aetiological Agent of Swine Dysentery.

Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.

Mutations in the fusA Gene Encoding Elongation Factor G in the Coryneform Bacterium Lead to Increased Lysine Production

Resistance to fusidic acid in Corynebacterium glutamicum and Brevibacterium flavum is associated with mutations in the fusA gene, which encodes the elongation factor G (EF-G). Two to ten percent of fusidic acid-resistant clones were shown to produce more lysine than parent strains. Sequencing of the fusA gene in clones with a high level of lysine production made it possible to find two mutations in the gene at position 1383—С1383G and С1383А. These mutations cause amino acid replacement at position 461 in the protein EF-G, namely, histidine is substituted by glutamine (H461Q). The mutation С1383G was introduced in the chromosomal copy of the fusA gene in C. glutamicum and B. flavum strains by homologous recombination. All clones containing the mutant variant of the fusA gene produced 10% more lysine than the parent strains.

Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein

The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year-old inferred ancestral variant of tufB, an essential gene encoding elongation factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a twofold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2000 generations in the laboratory and examined the adaptive response via fitness assays, whole genome sequencing, proteomics, and biochemical assays. Hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by upregulation of protein production. Our results suggest that an ancient–modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.

Mg2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

In Salmonella enterica serovar Typhimurium, Mg2+ limitation induces transcription of the mgtA Mg2+ transport gene, but the mechanism involved is unclear. The 5' leader of the mgtA mRNA contains a 17-codon, proline-rich ORF, mgtL, whose translation regulates the transcription of mgtA [Park S-Y et al. (2010) Cell 142:737-748]. Rapid translation of mgtL promotes formation of a secondary structure in the mgtA mRNA that permits termination of transcription by the Rho protein upstream of mgtA, whereas slow or incomplete translation of mgtL generates a different structure that blocks termination. We identified the following mutations that conferred high-level transcription of mgtA at high [Mg2+]: (i) a base-pair change that introduced an additional proline codon into mgtL, generating three consecutive proline codons; (ii) lesions in rpmA and rpmE, which encode ribosomal proteins L27 and L31, respectively; (iii) deletion of efp, which encodes elongation factor EF-P that assists the translation of proline codons; and (iv) a heat-sensitive mutation in trmD, whose product catalyzes the m1G37 methylation of tRNAPro Furthermore, substitution of three of the four proline codons in mgtL rendered mgtA uninducible. We hypothesize that the proline codons present an impediment to the translation of mgtL, which can be alleviated by high [Mg2+] exerted on component(s) of the translation machinery, such as EF-P, TrmD, or a ribosomal factor. Inadequate [Mg2+] precludes this alleviation, making mgtL translation inefficient and thereby permitting mgtA transcription. These findings are a significant step toward defining the target of Mg2+ in the regulation of mgtA transcription.

Phylogeny of Fomitopsis pinicola: a species complex.

Fungal species with a broad distribution may exhibit considerable genetic variation over their geographic ranges. Variation may develop among populations based on geographic isolation, lack of migration, and genetic drift, though this genetic variation may not always be evident when examining phenotypic characters. Fomitopsis pinicola is an abundant saprotrophic fungus found on decaying logs throughout temperate regions of the Northern Hemisphere. Phylogenetic studies have addressed the relationship of F. pinicola to other wood-rotting fungi, but pan-continental variation within F. pinicola has not been addressed using molecular data. While forms found growing on hardwood and softwood hosts exhibit variation in habit and appearance, it is unknown if these forms are genetically distinct. In this study, we generated DNA sequences of the nuc rDNA internal transcribed spacers (ITS), the TEF1 gene encoding translation elongation factor 1-α, and the RPB2 gene encoding the second largest subunit of RNA polymerase II for collections across all major geographic regions where this fungus occurs, with a primary focus on North America. We used Bayesian and maximum likelihood analyses and evaluated the gene trees within the species tree using coalescent methods to elucidate evolutionarily independent lineages. We find that F. pinicola sensu lato encompasses four well-supported, congruent clades: a European clade, southwestern US clade, and two sympatric northern North American clades. Each clade represents distinct species according to phylogenetic and population-genetic species concepts. Morphological data currently available for F. pinicola do not delimit these species, and three of the species are not specific to either hardwood or softwood trees. Originally described from Europe, F. pinicola appears to be restricted to Eurasia. Based on DNA data obtained from an isotype, one well-defined and widespread clade found only in North America represents the recently described Fomitopsis ochracea The remaining two North American clades represent previously undescribed species.

Мутации в гене fusA, кодирующем фактор элонгации G у коринеформных бактерий, приводят к повышению продукции лизина

Мутации в гене fusA, кодирующем фактор элонгации G у коринеформных бактерий, приводят к повышению продукции лизина

Mutations in the fusA Gene that Encodes Elongation Factor G in Coryneform Bacterium Lead to Increased Lysine Production

Mutations in the fusA Gene that Encodes Elongation Factor G in Coryneform Bacterium Lead to Increased Lysine Production

Virulence and antimicrobial resistance factors of Enterococcusspp. isolated from fecal samples from piggery farms in Eastern Cape, South Africa.

BackgroundEnterococci have emerged as an important opportunistic pathogen causing life-threatening infections in hospitals. The emergence of this pathogen is associated with a remarkable capacity to accumulate resistance to antimicrobials and multidrug-resistance particularly to vancomycin, erythromycin and streptomycin have become a major cause of concern for the infectious diseases community. In this paper, we report the prevalence of Enterococcus in respect to species distribution, their virulence and antibiogram profiles.MethodsFour hundred fecal samples were collected from two piggery farms in the Eastern Cape Province of South Africa. Enterococcus species were isolated and confirmed with generic specific primers targeting the tuf gene (encoding elongation factor). The confirmed isolates were speciated with enterococci species specific primers that aimed at delineating them into six species that are commonly associated with infections in humans. Antibiotic susceptibility testing was performed by disc diffusion method. Six virulence genes and antimicrobial resistance profiles of the isolates were evaluated molecularly.ResultsMolecular identification of the presumptive isolates confirmed 320 isolates as Enterococcus spp. Attempt at speciation of the isolates with primers specific for E. faecalis, E. durans, E. casseliflavus, E. hirae and E. faecium delineated them as follows: E. faecalis (12.5 %), E. hirae (31.25 %), E. durans (18.75 %) and E. faecium (37.5 %) while E. casseliflavus was not detected. All the isolates were resistant to vancomycin, streptomycin and cloxacillin, and to at least two different classes of antibiotics, with 300 (93.8 %) isolates being resistant to five or more antibiotics. Also, three out of the six virulence genes were detected in majority of the isolates and they are Adhesion of collagen in E. faecalis (ace) (96.88 %), gelatinase (gelE) (93.13 %) and surface protein (esp) (67.8 %).ConclusionThere was high prevalence of multi-resistant vancomycin Enterococcus spp. (VREs) in the fecal samples of pigs in the farms studied, and this poses health implications as vancomycin is an important drug in human medicine. Further studies are needed to determine the spread of vancomycin resistance among bacteria of human origin in the communities.

DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin.

Pufferfish accumulate tetrodotoxin (TTX) mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways.