Comparison of pulsed‐field gel electrophoresis and three rep‐PCR methods for evaluating the genetic relatedness of Stenotrophomonas maltophilia isolates

Aims: In this study, three facile repetitive‐sequence PCR (rep‐PCR) techniques have been compared with the pulsed‐field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR‐based methods, i.e. enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), BOX‐PCR and repetitive extragenic palindromic‐PCR (REP‐PCR). When compared with PFGE, ERIC‐PCR displayed a much lower discriminatory power, whereas BOX‐PCR and REP‐PCR had a comparable discriminatory power for close genetic‐related isolates. Conclusion: BOX‐PCR and REP‐PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure

Aims: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. Methods and Results: Recovery of Sten. maltophilia‐like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API‐20NE, Vitek‐2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species‐specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. Conclusions: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. Significance and Impact of the Study: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.     

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR‐based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria‐specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro‐organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.     

Molecular characterization of isolates of Beauveria bassiana obtained from overwintering and summer populations of Sunn Pest (Eurygaster integriceps)

Aim:  To examine whether isolates of the entomopathogenic fungus Beauveria bassiana are more closely associated to their summer hosts compared with overwintering hosts, with recently developed molecular tools based on mitochondrial regions. Methods and Results:  Primers for the traditional ITS1‐5·8S‐ITS2 region and two mitochondrial intergenic regions, namely, nad3‐atp9 and atp6‐rns, were used. All amplified products were sequenced, aligned and Neighbour‐Joining (NJ), parsimony and Bayesian phylogenetic inference analyses were performed. The isolates examined were grouped with very good support into three distinct groups, two of them showed geographical correlation, but no clear association to their host. Conclusions:  The mitochondrial intergenic regions used were more informative than the nuclear ITS1‐5·8S‐ITS2 sequences. The sequence variability observed, that allowed the phylogenetic placement of the isolates into distinct groups, depended on the geographical origin of the isolates and can be exploited for designing group‐specific and isolate‐specific primers for their genetic fingerprinting. No clear associations with summer Sunn Pest populations were observed. Significance and Impact of the Study:  Studies on the genetic variability of biocontrol agents like B. bassiana are indispensable for the development of molecular tools for their future monitoring.     

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

Aims: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. Methods and Results: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity‐related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR‐based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus‐PCR and BOX‐PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain‐specific or origin‐specific clades. Conclusions: Biochemical characterization was sensitive for interspecific typing, while PCR‐based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. Significance and Impact of the Study: PCR‐based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.     

The antimicrobial susceptibility of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates using three different methods and their genetic relatedness

Background  

Stenotrophomonas maltophilia is inherently resistant to many antimicrobials. So far, antimicrobial susceptibility tests for S. maltophilia have not been fully standardized. The purpose of the study was to compare the susceptibility of S. maltophilia isolates against seven different antimicrobials using three different methods and to investigate their genetic relatedness.     

Molecular diversity of CTX prophage in Vibrio cholerae

Aims: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results: The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstR^ET ctxB^class and rstR^ET ctxB^ET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstR^ET ctx^class) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions: Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.     

Intra-hospital dissemination of clinical and environmental isolates of Stenotrophomonas maltophilia from Tehran

Stenotrophomonas maltophilia isolates are responsible for various hospital-acquired infections and are particularly increasing in the immunocompromised patients. The aim of this study was to determine the clonal relatedness between S. maltophilia isolates originating from the clinic and environment. A total of 150 S. maltophilia isolates from patients and 1108 environmental samples obtained in three hospitals from Tehran. Following molecular identification targeting 23S rRNA gene, the clonal relatedness of the environmental and clinical isolates was determined using pulsed field gel electrophoresis (PFGE). Of the 150 clinical and 18 environmental isolates identified using phenotypic tests, the speciation of 120 and 15 was confirmed by targeting the 23S rRNA gene. The 24 common pulsotypes (PTs) and 32 single PTs were identified by PFGE. Only a small cluster was shared among the clinic and environment within a hospital; therefore, the intra-hospital dissemination of certain isolates of S. maltophilia among the clinic and environment was demonstrated.     

Modified nitrocefin‐EDTA method to differentially quantify the induced L1 and L2 β‐lactamases in Stenotrophomonas maltophilia

Aim: To estimate the ethylene diamine tetraacetic acid (EDTA) concentration at which the L1 enzyme activity in the cell extracts of Stenotrophomonas maltophilia can be mostly inhibited. Methods and Results: The effective inhibition concentration of EDTA against the L1 enzyme in the cell extracts was firstly evaluated by using the L2 isogenic mutant of S. maltophilia KJ, KJΔL2, as the assayed strain. Approximately 92% L1 activity was inhibited by 10 mmol l^?1 EDTA, which is 100‐fold higher than that from previously reported protocols (0·1 mmol l^?1). Three phylogenetic clusters of L1 proteins were revealed from 11 clinical S. maltophilia isolates, with a L1 protein divergence of 0–11%. The EDTA concentration required to inhibit the L1 enzymes of different phylogenetic clusters was estimated to be 10 mmol l^?1. Conclusion: The previous nitrocefin‐EDTA protocol for differentially quantifying the L1 and L2 activity in the cell extracts has been modified by raising the added EDTA concentration to 10 mmol l^?1. Significance and Impact of the Study: A rapid and accurate method for determination of L1 and L2 activity will provide a convenient tool for enzyme characterization and induction mechanism study of S. maltophilia.     

A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis

Aims: Genetic comparison of Lactococcus garvieae isolated from mammals and fish. Methods and Results: One hundred and ninety‐seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed‐field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0·01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0·71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0·35). Twenty‐one of the 37 swine strains (56·8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio‐respiratory system. Conclusions: Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. Significance and Impact of the Study: The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.     

Molecular cloning of a copper-dependent laccase from the dye-decolorizing strain Stenotrophomonas maltophilia AAP56

Aims: To clone the gene encoding the enzyme with laccase activity expressed by Stenotrophomonas maltophilia AAP56 and to construct an insertional mutation in that gene to determine its effect on dye decolourization and laccase activity in this strain. Methods and Results: Comparative genomics of Sten. maltophilia strains K279a and R551‐3 revealed copA (coding for putative multicopper oxidase) as a candidate gene to encode an enzyme with laccase activity. Stenotrophomonas maltophilia AAP56 copA was amplified by degenerated PCR and cloned. A copA mutant strain, named Stemur, was constructed by homologous recombination. The comparison of wild‐type and mutant strains revealed that CopA shows laccase activity, and it is involved in copper resistance and in vitro dye decolorization. On the contrary, the mutation in copA did not affect the in vivo dye removal by Sten. maltophilia. Conclusions: Stenotrophomonas maltophilia AAP56 shows different mechanisms for dye decolorization. The gene encoding the laccase has been identified, and it has been shown that it is involved in the in vitro decolorization. Significance and Impact of the Study: Stenotrophomonas maltophilia is a micro‐organism of interest in different biotechnological processes including dye removal. This is the first report to address the molecular mechanism of this capacity, what will contribute to further improvements in the process.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Salmonella Virchow isolates from human and avian origins in England--molecular characterization and infection of epithelial cells and poultry

Aims: To characterize 12 Salmonella Virchow isolates from human and avian sources to begin to determine the genetic relationships within the serovar, determine its capacity to invade and induce inflammatory responses in human intestinal epithelial cells and investigate its ability to colonize the chicken gastrointestinal tract. Methods and Results: Multi‐Locus Sequence Typing (MLST) revealed that 11 isolates belonged to sequence type 16 (ST16). Pulsed Field Gel Electrophoresis (PFGE) grouped the isolates into two main clusters. All isolates contained genes associated with virulence determined through PCR virulotyping. All the S. Virchow isolates had the ability to invade human epithelial cells and elicit high levels of production of the pro‐inflammatory chemokine interleukin‐8 (IL‐8). Experimental infection of poultry showed S. Virchow colonizes the caeca and spleen. Conclusions: Isolates within the serovar show high levels of genetic relatedness regardless of the source. The data indicates S. Virchow is an invasive and inflammatory serovar, consistent with its association with invasive salmonellosis in humans. Significance and Impact of the Study: The poultry infection experiment included in this study shows S. Virchow can colonize the gastrointestinal tract rapidly and to high levels with the chickens showing no clinical signs of infection. The asymptomatic colonization of chickens indicates an increased ability of S. Virchow to enter the food chain undetected and cause human salmonellosis which because of the invasive and inflammatory nature of S. Virchow seen during the Caco2 invasion assay and previous studies showing its invasive nature in humans and increasing resistance to antibiotics is a public health concern.     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium

Aims:  To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5‐triphenyltetrazolium chloride (TTC) in MRS medium. Methods and Results:  Single colonies isolated from sediments of a coastal marsh by enrichment in MRS broth were selected from MRS‐TTC plates and classified according to colony phenotype based on TTC reduction. A total of 37 colonies grouped in seven different phenotypes were identified by analysis of its 16S ribosomal gene sequence. Most isolates belonged to the Firmicutes phylum, mainly to orders Bacillales and Lactobacillales. LAB were represented by 20 isolates, 15 of which belong to the genus Weissella. Conclusions:  Enrichment in MRS was highly selective for the isolation of bacteria belonging to phylum Firmicutes. Several different phenotypes were developed by LAB and must be considered during LAB isolation based on TTC reduction. Significance and Impact of the Study:  To our knowledge, this is the first study aimed at determining a relationship between colony phenotype from TTC reduction and a partial identification of isolates based on 16S ribosomal gene sequence similarities. Besides, this is the first report of isolation of W. cibaria from environmental samples.