Similar Restriction Fragment Length Polymorphism Research Articles

Characteristics of Carbapenem-Resistant Enterobacteriaceae in Ready-to-Eat Vegetables in China.

Vegetables harboring bacteria resistant to antibiotics are a growing food safety issue. However, data concerning carbapenem-resistant Enterobacteriaceae (CRE) in ready-to-eat fresh vegetables is still rare. In this study, 411 vegetable samples from 36 supermarkets or farmer's markets in 18 cities in China, were analyzed for CRE. Carbapenemase-encoding genes and other resistance genes were analyzed among the CRE isolates. Plasmids carrying carbapenemase genes were studied by conjugation, replicon typing, S1-PFGE southern blot, restriction fragment length polymorphism (RFLP), and sequencing. CRE isolates were also analyzed by pulsed-field gel electrophoresis (PFGE). Ten vegetable samples yielded one or more CRE isolates. The highest detection rate of CRE (14.3%, 4/28) was found in curly endive. Twelve CRE isolates were obtained and all showed multidrug resistance: Escherichia coli, 5; Citrobacter freundii, 5; and Klebsiella pneumoniae, 2. All E. coli and C. freundii carried blaNDM, while K. pneumoniae harbored blaKPC−2. Notably, E. coli with blaNDM and ST23 hypervirulent Klebsiella pneumoniae (hvKP) carrying blaKPC−2 were found in the same cucumber sample and clonal spread of E. coli, C. freundii, and K. pneumoniae isolates were all observed between vegetable types and/or cities. IncX3 plasmids carrying blaNDM from E. coli and C. freundii showed identical or highly similar RFLP patterns, and the sequenced IncX3 plasmid from cucumber was also identical or highly similar (99%) to the IncX3 plasmids from clinical patients reported in other countries, while blaKPC−2 in K. pneumoniae was mediated by similar F35:A-:B1 plasmids. Our results suggest that both clonal expansion and horizontal transmission of IncX3- or F35:A-:B1-type plasmids may mediate the spread of CRE in ready-to-eat vegetables in China. The presence of CRE in ready-to-eat vegetables is alarming and constitutes a food safety issue. To our knowledge, this is the first report of either the C. freundii carrying blaNDM, or K. pneumoniae harboring blaKPC−2 in vegetables. This is also the first report of ST23 carbapenem-resistant hvKP strain in vegetables.

Migration and tuberculosis transmission in a middle-income country: a cross-sectional study in a central area of S\xe3o Paulo, Brazil

BackgroundLittle is known about the impact of growing migration on the pattern of tuberculosis (TB) transmission in middle-income countries. We estimated TB recent transmission and its associated factors and investigated the presence of cross-transmission between South American migrants and Brazilians.MethodsWe studied a convenient sample of cases of people with pulmonary TB in a central area of São Paulo, Brazil, diagnosed between 2013 and 2014. Cases with similar restriction fragment length polymorphism (IS6110-RFLP) patterns of their Mycobacterium tuberculosis complex isolates were grouped in clusters (recent transmission). Clusters with both Brazilian and South American migrants were considered mixed (cross-transmission). Risk factors for recent transmission were studied using logistic regression.ResultsIsolates from 347 cases were included, 76.7% from Brazilians and 23.3% from South American migrants. Fifty clusters were identified, which included 43% South American migrants and 60.2% Brazilians (odds ratio = 0.50, 95% confidence interval = 0.30–0.83). Twelve cross-transmission clusters were identified, involving 24.6% of all clustered cases and 13.8% of all genotyped cases, with migrants accounting for either an equal part or fewer cases in 11/12 mixed clusters.ConclusionsOur results suggest that TB disease following recent transmission is more common among Brazilians, especially among those belonging to high-risk groups, such as drug users. Cross-transmission between migrants and Brazilians was present, but we found limited contributions from migrants to Brazilians in central areas of São Paulo and vice versa.

Investigation ofMannheimia haemolyticabacteriophages relative to host diversity

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle. Strictly lytic phages were not detected from bovine nasopharyngeal (n=689) or water trough (n=30) samples, but Myoviridae- or Siphoviridae-like phages were induced from 54 of 72M.haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P<0·0001). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70-79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2-like phage φMhaA1-PHL101. Prophages are a significant component of the genome of M.haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M.haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen. This study demonstrated that prophages are widespread within the genome of M.haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.

Antibiotic resistance and genetic diversity in water-borne Enterobacteriaceae isolates from recreational and drinking water sources

A total of 240 water-borne bacteria including 72 Escherichia coli, 83 Enterobacter, 30 Klebsiella, 36 Salmonella and 19 Shigella spp. isolates from drinking and recreational water sources were assessed for antibiotic resistance and genetic diversity. Escherichia coli (88.89 %) and species of Enterobacter (86.75 %), Klebsiella (83.33 %) and Salmonella (100 %) were resistant to cefadroxil, while >94 % Shigella spp. were resistant to cefaclor and cefuroxime. Ofloxacin was the most effective antibiotic against isolates of all the genera. Multiple antibiotic resistance index identified dug well, pond and piped water supplies as high risk sources of enteric pathogens. Random amplified polymorphic DNA analysis and restriction fragment length polymorphism of amplified 16S rRNA gene were studied for genetic relatedness of Enterobacteriaceae isolates. Primer P1254 identified 10, 16, 4, 4 and 1 distinct random amplified polymorphic DNA group(s) of E. coli, Enterobacter, Klebsiella, Salmonella and Shigella species, respectively. Unlike random amplified polymorphic DNA, restriction fragment length polymorphism using AluI and HaeIII could not segregate isolates in different genetic profiles. 16S rRNA gene of three Enterobacter spp. strains from different sources with similar restriction fragment length polymorphism but different random amplified polymorphic DNA patterns was sequenced, and identified as Enterobacter hormaechei strains skg0061, 0062 and 0063. The sequence information has been submitted to GenBank (HQ322393-95). Biochemically similar but genetically diverse Enterobacteriaceae members from drinking and recreational water sources exhibited varying antibiotic sensitivity. Contamination of water sources with such multiple antibiotic-resistant enteric pathogens poses threat to human health.

First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia.

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable.

Metallo-β-lactamases among Enterobacteriaceae from routine samples in an Italian tertiary-care hospital and long-term care facilities during 2008

The emergence of metallo-β-lactamase (MBL)-producing Enterobacteriaceae is a serious public health concern. Producers have been repeatedly isolated from patients and long-term care facility (LTCF) residents around Bolzano, and we sought to assess their prevalence and clinical impact. All routine Enterobacteriaceae isolates from a Bolzano tertiary-care hospital and associated long-term care facilities in 2008 (n = 5500) were screened for MBLs, with case details reviewed for the source patients. In total, 36 producers were obtained from 29 patients, comprising 14 Escherichia coli, six Klebsiella pneumoniae, four Klebsiella oxytoca, four Citrobacter freundii, two Enterobacter cloacae and two Morganella morganii, as well as single Citrobacter amalonaticus, Enterobacter aerogenes, Providencia stuartii and Proteus mirabilis isolates. All were PCR-positive for blaVIM and 25 were PCR-positive for qnrS; 19 non-K. pneumoniae had blaSHV and one had blaCTX-M-group1; 13 were from 12 LTCF residents and 23 were from 17 acute-care patients. All these patients had serious underlying diseases with prolonged hospitalization or LTCF stay; only seven had infections due to the MBL producers, comprising four urinary tract infections, two catheter-related bloodstream infections and one patient with both a surgical site infection and pneumonia. Five patients had more than one MBL-producing organism. Pulsed-field gel electrophoresis identified a cluster of six related E. coli, whereas pairs of K. pneumoniae and C. freundii isolates had >85% profile similarity. Transformants prepared from two isolates were shown to be PCR-positive for blaVIM, qnrS and blaSHV; their plasmids gave similar restriction fragment length polymorphism patterns, and blaVIM-1, qnrSI and blaSHV-12 were detected by sequencing.

Genotypic and phenotypic diversity of Bacillus spp. isolated from Freshwater Ecosystems

The efficacy of the restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified rrs (16S ribosomal ribonucleic acid [rRNA] gene) for the identification of Bacillus spp. isolated from freshwater pond ecosystems was evaluated. Results showed that no restriction site differences occur between rrs operons within the bacteria belonging to the same species and as a result members of the same species yield very similar RFLP patterns at 100% similarity level. Cluster analysis of PCR-RFLP patterns grouped all the isolates into three clusters; each cluster represented a single species except one for which no placement was obtained. Phenotypic characteristics of the isolates were found to be well concordance with the results of PCR-RFLP. Our findings suggested that PCR- RFLP is suitable tool for discrimination and identification of endoglucanase positive Bacillus spp. isolated from freshwater ecosystems.

Metallo-β-lactamases among Enterobacteriaceae from routine samples in an Italian tertiary care hospital and long-term care facilities during 2008

The emergence of metallo-β-lactamase (MBL)-producing Enterobacteriaceae is a serious public health concern. Producers have been repeatedly isolated from patients and long-term care facility (LTCF) residents around Bolzano, and we sought to assess their prevalence and clinical impact. All routine Enterobacteriaceae isolates from a Bolzano tertiary-care hospital and associated long-term care facilities in 2008 (n = 5500) were screened for MBLs, with case details reviewed for the source patients. In total, 36 producers were obtained from 29 patients, comprising 14 Escherichia coli, six Klebsiella pneumoniae, four Klebsiella oxytoca, four Citrobacter freundii, two Enterobacter cloacae and two Morganella morganii, as well as single Citrobacter amalonaticus, Enterobacter aerogenes, Providencia stuartii and Proteus mirabilis isolates. All were PCR-positive for bla(VIM) and 25 were PCR-positive for qnrS; 19 non-K. pneumoniae had bla(SHV) and one had bla(CTX-M-group1); 13 were from 12 LTCF residents and 23 were from 17 acute-care patients. All these patients had serious underlying diseases with prolonged hospitalization or LTCF stay; only seven had infections due to the MBL producers, comprising four urinary tract infections, two catheter-related bloodstream infections and one patient with both a surgical site infection and pneumonia. Five patients had more than one MBL-producing organism. Pulsed-field gel electrophoresis identified a cluster of six related E. coli, whereas pairs of K. pneumoniae and C. freundii isolates had >85% profile similarity. Transformants prepared from two isolates were shown to be PCR-positive for bla(VIM), qnrS and bla(SHV); their plasmids gave similar restriction fragment length polymorphism patterns, and bla(VIM-1), qnrS1 and bla(SHV-12) were detected by sequencing.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 μl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia.

Transmission of Mycobacterium tuberculosis in a Family Proved by Genotyping

Molecular genetic methods have been applied in various epidemiologic studies including investigations of disease acquisition by contact. This report describes the use of various molecular genetic methods in tracing possible household transmission of tuberculosis by contact. Four Mycobacterium tuberculosis strains, each from four members of a family, were first isolated and identified in the clinical laboratory of the Chest Hospital and were submitted to the National Reference Laboratory of Mycobacteriology for further confirmation and genotyping. In this study, IS6110 restriction fragment length polymorphism (RFLP), spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), and rpoB gene sequencing were used for genotyping. All four strains were found to have identical spoligotypes, MIRU-VNTR patterns, and similar IS6110 RFLP profiles. The results of the drug susceptibility test and of rpoB sequencing showed that all four strains were rifampicin resistant. Household transmission through close contact was thus proved by genotyping. We conclude that all four family members were infected with the same lineage of M. tuberculosis.

Clinical Significance of Azole Antifungal Drug Cross-Resistance in Candida glabrata

Candida glabrata, which can become resistant to fluconazole, is a common cause of bloodstream infection. This study was performed to determine the significance of cross-resistance to new azole drugs among C. glabrata isolates recovered as a cause of infection in azole-treated hematopoietic stem cell transplant (HSCT) recipients. Seven cases of invasive candidiasis caused by C. glabrata occurred in HSCT recipients who were receiving azole therapy between January 2000 and December 2004 in our institution. Case characteristics were ascertained. Sequential colonizing and invasive isolates were examined to determine susceptibilities to fluconazole, itraconazole, and voriconazole, and molecular relatedness by restriction fragment length polymorphism (RFLP) analysis. Twenty-three C. glabrata isolates were recovered from 4 patients who developed candidemia while receiving fluconazole and three patients who developed candidemia while receiving voriconazole. The mode MICs of fluconazole, itraconazole, and voriconazole for these isolates were > or =64 microg/ml (range, 4 to > or =64 microg/ml), 2 microg/ml (range, 0.25 to > or =16 microg/ml), and 1 microg/ml (range, 0.03 to > or =16 microg/ml), respectively. Kendall tau b correlation coefficients demonstrated significant associations between the MICs of voriconazole with fluconazole (P = 0.005) and itraconazole (P = 0.008). Colonizing and invasive isolates exhibiting variable susceptibilities had similar RFLP patterns. These observations suggest that C. glabrata exhibits considerable clinically significant cross-resistance between older azole drugs (fluconazole and itraconazole) and voriconazole. Caution is advised when considering voriconazole therapy for C. glabrata candidemia that occurs in patients with extensive prior azole drug exposure.

Data from 11 Years of Molecular Typing Infectious Bronchitis Virus Field Isolates

In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.

Restriction Fragment Length Polymorphism of 16S–23S rDNA Intergenic Spacer of Aeromonas spp.

We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species. A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study. ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI. The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species. The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA). The isolates were allocated into 15 groups, three strains were unclustered. The strains belonging to the following genomic species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. schubertii, A. allosaccharophila, A. popoffii, and A. culicicola formed distinct clusters. Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group. Similarly, the strains of A. jandaei (HG 9) and the type strain of A. trota were allocated into one cluster. Two isolates of HG 14 formed distinct cluster. We noticed a genetic diversity among A. veronii isolates, the strains were clustered in two groups. Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas.

Genetic diversity of indigenous common bean ( Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean ( Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean ( Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli , including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii . Other strains showed higher similarity of bases with R. tropici , R. leguminosarum and Mesorhizobium plurifarium , but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena .

The genetic relatedness of Porphyromonas gingivalis clinical and laboratory strains assessed by analysis of insertion sequence (IS) element distribution.

Porphyromonas gingivalis is frequently found in periodontitis lesions. This organism contains a large number of insertion sequence (IS) elements. We sought to determine the distribution of seven IS elements from strain W83 among nine P. gingivalis laboratory strains and nine clinical isolates and to use these findings to determine strain relationships. Southern blots of BamHI digested genomic DNA digests were probed with insertion sequence elements ISPg1-7. The restriction fragment length polymorphism (RFLP) patterns revealed that five of the nine laboratory strains, including strain W83, were nearly identical for all seven IS elements. Two of nine clinical isolates were similar to the five laboratory strains. Two of the four remaining laboratory strains had similar or identical RFLP patterns. The remaining two laboratory strains had limited similarity to clinical strains. Four of the clinical isolates had identical RFLP patterns for all seven IS elements. The three remaining clinical isolates were unique in their RFLP patterns. Several strains lacked from one to four of the IS elements. Similar strain relationships were suggested regardless of the IS element examined. Transposition and recombination between IS elements are not sufficiently pervasive to obscure strain relationships, though this does not preclude the possibility that such events play an important role in allowing P. gingivalis to adapt to new environments. Given the level of genetic diversity observed, it may be especially important to examine genetically diverse strains when drawing conclusions based on the W83 P. gingivalis genomic database.

Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanB phenotype.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR(B) and -S(B), and the vancomycin-inducible promoter P(YB). Expression of the resistance genes vanH(B), -B, and -X(B) was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

Multidrug-resistant tuberculosis in prison inmates, Azerbaijan.

In a tuberculosis (TB) program in the Central Penitentiary Hospital of Azerbaijan, we analyzed 65 isolates of Mycobacterium tuberculosis by IS6110-based restriction fragment-length polymorphism (RFLP) and spoligotyping. From 11 clusters associated with 33 patients, 31 isolates had an IS6110-based banding pattern characteristic of the Beijing genotype of M. tuberculosis. In addition, 15 M. tuberculosis isolates with similar RFLP patterns constituted a single group by spoligotyping, matching the Beijing genotype. Multidrug resistance, always involving isoniazid and rifampin, was seen in 34 (52.3%) of 65 isolates, with 28 belonging to the Beijing genotype.

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3[ratio ]K6 and O4[ratio ]K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

A multidrug-resistant tuberculosis microepidemic caused by genetically closely related Mycobacterium tuberculosis strains.

IS6110 DNA fingerprinting was used to characterize an outbreak of multidrug-resistant tuberculosis in 21 individuals (17 males and 4 females) living in or roaming among four distantly separated areas in the Czech Republic. The restriction fragment length polymorphism (RFLP) analysis separated the collected Mycobacterium tuberculosis strains into group A, including 14 patients with six IS6110 copies, and group B, with 7 patients displaying highly similar RFLP patterns but with two additional IS6110 bands. A switch from pattern A to pattern B was observed in one patient, and the subsequent detection of subclone B in seven more individuals has been explained by the instability of DNA genotypes caused by transposition of IS6110 elements.