Repetitive Element PCR Fingerprinting Research Articles

Isolation, characterization and antibiotic resistance of Proteus mirabilis from Belgian broiler carcasses at retail and human stool

Proteus mirabilis is an important pathogen involved in human urinary tract infections, and also more isolated from stools of patients with diarrheal disease than from healthy patients. The role of food, especially poultry products as source for human infection and multi-resistant strains remains unclear. As a resident in broilers’ intestines, P. mirabilis can contaminate broiler carcasses due to slaughter practices, and be a risk for human infection.The present study evaluated the performance of five isolation media, and subsequently examined the presence of P. mirabilis on broiler carcasses at retail. Additionally, isolates were characterized by the Dienes’ test, repetitive element PCR fingerprinting and pulsed-field gel electrophoresis, and their antibiotic resistance profile determined.Using a combined isolation protocol on blood agar, xylose lysine deoxycholate agar and violet red bile glucose agar, P. mirabilis was isolated from 29 out of 80 broiler carcasses (36.25%) with a mean contamination level of 2.25 ± 0.50 log10 CFU/g. A high strain heterogeneity was present in isolates from broilers and human stool. The same strains were not shared, but the antibiotic resistance profiling was similar. A role of poultry products as source for human infection should be taken into account.

Molecular Characterization of Pseudomonas syringae pv. coriandricola and Biochemical Changes Attributable to the Pathological Response on Its Hosts Carrot, Parsley, and Parsnip.

Bacterial leaf spot caused by the plant pathogenic bacterium Pseudomonas syringae pv. coriandricola (Psc) was observed on carrot, parsnip, and parsley grown on a vegetable farm in the Vojvodina Province of Serbia. Nonfluorescent bacterial colonies were isolated from diseased leaves and characterized using different molecular techniques. Repetitive element PCR fingerprinting with five oligonucleotide primers (BOX, ERIC, GTG5, REP, and SERE) and the randomly amplified polymorphic DNA-PCR with the M13 primer revealed identical fingerprint patterns for all tested strains. Multilocus sequence analysis of four housekeeping genes (gapA, gltA, gyrB, and rpoD) showed a high degree (99.8 to 100%) of homology with sequences of Psc strains deposited in the Plant-Associated Microbes Database and NCBI database. The tested strains caused bacterial leaf spot symptoms on all three host plants. Host-strain specificity was not found in cross-pathogenicity tests, but the plant response (peroxidase induction and chlorophyll bleaching) was more pronounced in carrot and parsley than in parsnip.

Genotypic diversity of Pasteurella multocida isolates from pigs and poultry in Australia.

To investigate the genotype and diversity of Pasteurella multocida present in pig herds and to determine the extent of overlap with isolates from poultry flocks in Australia. A total of 43 isolates from pigs from different farms and regions of Australia were used in this study. A diverse collection of 41 poultry isolates, with 31 being previously characterised, was also used. The pig isolates and 10 poultry isolates were identified by species-specific PCR assay, serotyped by the Heddleston scheme and genotyped by a multiplex PCR based on the lipopolysaccharide (LPS) outer core biosynthesis locus, repetitive element PCR fingerprinting (rep-PCR) and multilocus sequence typing (MLST), with the latter being used on a subset of the isolates based on the rep-PCR results. Only 4 out of 8 recognised LPS genotypes were found in the pig isolates, with each isolate assigned to an LPS genotype. In contrast, 77% of the isolates were non-typable or cross-reacting in the Heddleston serotyping scheme. The rep-PCR analysis recognised 20 patterns, yet only 16 sequence types (STs) were found and 4 were new STs. There were 5 STs (STs 7, 11, 20, 24 and 58) shared among the pig and poultry isolates. Although only limited numbers of isolates have been examined, there is evidence of a sharing of genotypes between Australian pigs and chickens. These findings have major implication for biosecurity measures with regard to minimising both direct (e.g. animal to animal) and in-direct (e.g. shared staff or cross-visitors) contact between poultry and pigs.

Molecular Genotyping of Acinetobacter baumannii Species Isolated from Patients in Tehran, Iran, by Repetitive Element PCR Fingerprinting

Acinetobacter baumannii is an opportunistic pathogen with high pathogenic and antibiotic-resistance potential and is also considered as one of the main nosocomial agents, specifically in the intensive care units (ICUs). It is highly important to use molecular biology methods in the epidemiological studies, determine the source of infection, and understand the relationships and distributional patterns of pathogens. Therefore, the current study aimed to determining the similar molecular types in the A. baumannii species isolated from patients in Tehran, Iran, by the repetitive element PCR fingerprinting (REP-PCR) method. A total of 350 clinical samples were collected from patients admitted to different hospital in Tehran, assessed to identify Acinetobacter spp., based on the special culture media and biochemical test results. The resistance of isolates was evaluated against 11 different antibiotics. The cefepime and ceftazidime were assessed by the minimum inhibitory concentration (MIC) method, based on serial dilutions. The genome of isolated strains was extracted using the modified boiling method and amplified in REP-PCR technique using specific primers. In the current study, out of 120 isolates of Acinetobacter spp., 100 (76.9%) were identified as A. baumannii, mostly from ICUs and infectious diseases wards. The isolates of A. baumannii in the current study mostly showed antimicrobial resistance against cefepime and ceftazidime, and had the highest sensitivity to polymyxin B. About 70% of A. baumannii isolates in the current study were resistant to 3 or more antibiotics. According to dendrogram analyses, the patterns were classified to AI with the maximum population (36%) of group A. All genotypes of Acinetobacter spp. in the current study showed resistance against carbapenems and aminoglycosides. High similarities between the isolates in the current study indicated the high distribution of A. baumannii species in the hospitals of Tehran.

Antimicrobial potential of actinomycetes isolated from the unexplored hot Merzouga desert and their taxonomic diversity.

ABSTRACTThe absence of new antibiotics is guiding more and more researchers to specific ecosystems. One hundred and sixty-three Actinobacteria isolates were isolated from Merzouga sand and screened for their antibacterial and antifungal activities. To test the antimicrobial effect of isolates, four microorganisms known as human potential pathogens were used. The electrophoretic profiles of isolates obtained by repetitive element PCR fingerprinting (rep-PCR) were compared by clustering. Results showed that among the tested isolates, 59% were active against one or more in testing Gram-positive, Gram-negative and the yeast Candida albicans. The importance of culture media for the activity expression was revealed. Comparative analysis of antimicrobial activity divided isolates into 15 groups. The comparison of the average diameters of inhibition zones using Minitab V.17 allowed subdivision of the 15 groups into 20 subgroups. Dendrograms derived from the BOXA1R-PCR fingerprints showed that 36 isolates were grouped in 16 clusters, containing from two to four isolates while 127 isolates were not grouped. The tested antimicrobial activities showed a high biological diversity with important inhibition of pathogens tested. The rep-PCR revealed a high taxonomic diversity of isolates. The combination of antimicrobial activity and rep-PCR results revealed the diverse pattern of Merzouga sand dune Actinobacteria.

Morphological and Genetic Diversity of Rhizobia Nodulating Cowpea (Vigna unguiculata L.) from Agricultural Soils of Lower Eastern Kenya.

Limited nitrogen (N) content in the soil is a major challenge to sustainable and high crop production in many developing countries. The nitrogen fixing symbiosis of legumes with rhizobia plays an important role in supplying sufficient N for legumes and subsequent nonleguminous crops. To identify rhizobia strains which are suitable for bioinoculant production, characterization of rhizobia is a prerequisite. The objective of this study was to assess the morphological and genetic diversity of rhizobia that nodulates cowpea in agricultural soils of lower eastern Kenya. Twenty-eight rhizobia isolates were recovered from soil samples collected from farmers' fields in Machakos, Makueni, and Kitui counties in lower eastern Kenya and characterized based on morphological characteristics. Thirteen representative isolates were selected and characterized using BOX repetitive element PCR fingerprinting. Based on the dendrogram generated from morphological characteristics, the test isolates were distributed into two major clusters at a similarity of 75%. Phylogenetic tree, based on BOX repetitive element PCR, grouped the isolates into two clusters at 90% similarity level. The clustering of the isolates did not show a relationship to the origin of soil samples, although the isolates were genetically diverse. This study is a prerequisite to the selection of suitable cowpea rhizobia to develop bioinoculants for sustainable crop production in Kenya.

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674(T) and S. equorum subsp. linens DSM 15097(T), respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA-DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.

Study of PROBAC product influence on infant microbiota in a single-chamber colonic fermentation model GIS1

The modulation potential of the PROBAC product on the simulated microbiota of an infant colon was determined by using the GIS1 single-chamber system of simulation of the human colon. The determination of the viability of each group of microorganisms and of the metabolic activity was performed for each simulated compartment. In order to confirm the biochemical tests, at least one colony from each sample was analyzed from a genetic point of view, using the repetitive-element PCR fingerprinting (rep-PCR) technique. Molecular analysis demonstrated the persistence of the two strains representing the basis of the PROBAC product, and the genetic stability of the lactic acid bacteria strains included in the final product after transiting the various parts of the simulated colon. After analyzing the viability of lactic acid, bacteria strains that were introduced in the final product in the presence and absence of the prebiotic while transiting the sections of the colon, produced an increase in lactic and bifidobacteria number, especially in the transverse and descending sections. In vitro approaches to study the human colon microbiota, and intestinal microbial processes offer an excellent experimental set-up to study the mechanism of action.

Characterization of Glycosyltransferase Activity of Wild-Type Leuconostoc mesenteroides Strains from Bulgarian Fermented Vegetables

Glycosyltransferase activity of 13 Leuconostoc mesenteroides strains isolated from Bulgarian fermented vegetables was investigated. All the strains displayed a mucoid phenotype on sucrose-containing agar media. Strains were characterized according to carbohydrate fermentation, species-specific multiple PCR using several primers, repetitive element-PCR fingerprinting using (GTG)(5) primers and glycosyltransferase activity. Level of activity and cellular localization (soluble or cell-associated) were variable among strains. Precipitation of exopolysaccharides produced from sucrose by the soluble fractions from these strains allowed recovery of only glucans and further characterization by (1)H and (13)C NMR analysis and enzymatic digestion with dextranase revealed dextran production. However, levans could be detected in presence of raffinose as fructosyl donor. Both fructosyltransferase and glucosyltransferase encoding genes were detected by PCR and both active enzymes were detected after functional characterization by SDS-PAGE electrophoresis and in situ polymer production after incubation with sucrose. This work therefore showed that concomitant production of glucosyltransferase and fructosyltransferase is widespread in L. mesenteroides strains.

Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)(5) primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase.

Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation

A polyphasic taxonomic study of the two subspecies of Paenibacillus larvae, Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens, supported the reclassification of the subspecies into one species, Paenibacillus larvae, without subspecies separation. Our conclusions are based on the analysis of six reference strains of P. larvae subsp. pulvifaciens and three reference strains and 44 field isolates of P. larvae. subsp. larvae. The latter originated from brood or honey of clinically diseased honey bee colonies or from honey of both clinically diseased and asymptomatic colonies from Sweden, Finland and Germany. Colony and spore morphology, as well as the metabolism of mannitol and salicin, did not allow a clear identification of the two subspecies and SDS-PAGE of whole-cell proteins did not support the subspecies differentiation. For genomic fingerprinting, repetitive element-PCR fingerprinting using ERIC primers and PFGE of bacterial DNA were performed. The latter method is a high-resolution DNA fingerprinting method proven to be superior to most other methods for biochemical and molecular typing and has not previously been used to characterize P. larvae. ERIC-PCR identified four different genotypes, while PFGE revealed two main clusters. One cluster included most of the P. larvae subsp. larvae field isolates, as well as all P. larvae subsp. pulvifaciens reference strains. The other cluster comprised the pigmented variants of P. larvae subsp. larvae. 16S rRNA gene sequences were determined for some strains. Finally, exposure bioassays demonstrated that reference strains of P. larvae subsp. pulvifaciens were pathogenic for honey bee larvae, producing symptoms similar to reference strains of P. larvae subsp. larvae. In comparison with the type strain for P. larvae subsp. larvae, ATCC 9545T, the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT100. An emended description of the species is given.

The use of repetitive element PCR fingerprinting (rep-PCR) for genetic subtyping of German field isolates of Paenibacillus larvae subsp. larvae

Studies using the repetitive element PCR fingerprinting technique (rep-PCR) revealed that BOX A1R-, MBO REP1- and ERIC-primers reproducibly generate distinctive DNA fingerprints from Paenibacillus larvae subsp. larvae. Four different genetic subtypes of P. larvae were identified in Germany using the three primers. Correlating the results from genetic and morphological subtyping, the flat and transparent morphotype could be assigned to one genetic group. Geographic evaluation of our results demonstrated that the different genetic subtypes appeared in clusters correlating with different outbreaks of American foulbrood. Taken together, our results indicated that rep-PCR performed with a combination of BOX A1R- and MBO REP1-primers will be an effective tool for establishing a molecular epidemiology of P. l. larvae. American foulbrood / Paenibacillus larvae larvae / DNA-fingerprinting / genetic subtyping / molecular epidemiology

Diversity of Transconjugants that Acquired Plasmid pJP4 or pEMT1 after Inoculation of a Donor Strain in the A- and B-horizon of an Agricultural Soil and Description of Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov.

We examined the diversity of transconjugants that acquired the catabolic plasmids pJP4 or pEMT1, which encode degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), in microcosms with agricultural soil inoculated with a donor strain (Dejonghe, W., Goris, J., El Fantroussi, S., Höfte, M., De Vos, P., Verstraete, W., and Top, E. M. Appl. Environ. Microbiol. 2000, p. 3297-3304). Using repetitive element PCR fingerprinting, eight different rep-clusters and six separate isolates could be discriminated among 95 transconjugants tested. Representative isolates were identified using 16S rDNA sequencing, cellular fatty acid analysis, whole-cell protein analysis and/or DNA-DNA hybridisations. Plasmids pJP4 and pEMT1 appeared to have a similar transfer and expression range, and were preferably acquired and expressed in soil by indigenous representatives of Ralstonia and Burkholderia. Two rep-clusters were shown to represent novel Burkholderia species, for which the names Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov. are proposed. When easily degradable carbon sources were added together with the plasmid-bearing donor strain, also a significant proportion of Stenotrophomonas maltophilia isolates were found. The transconjugant collections isolated from A- (0-30 cm depth) and B-horizon (30-60 cm depth) soil were similar, except for B. terricola transconjugants, which were only isolated from the B-horizon.

Repetitive element PCR fingerprinting (rep-PCR) using enterobacterial repetitive intergenic consensus (ERIC) primers is not necessarily directed at ERIC elements.

We examined the use of enterobacterial repetitive intergenic consensus (ERIC) sequences in PCR on the DNAs of various bacteria, bacteriophage, invertebrates, fungi, plants and vertebrates and have shown that complex ERIC-PCR patterns can be readily produced from all of these target organisms. A range of annealing temperatures was tested, from 52 degrees C (the commonly used annealing temperature) to 66 degrees C (the approximate Tm of ERIC primers). At the higher temperatures, most bands failed to amplify, the exception being a subset of bands from enterobacterial targets. It was concluded that ERIC-PCR does not necessarily direct amplification from genuine ERIC sequences.