Repetitive Element Polymerase Chain Research Articles

Comparative Evaluation of RLEP-PCR and Conventional Laboratory Methods for Confirmation of Clinically Diagnosed Early-stage Leprosy in Eastern India

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae). Diagnosis of leprosy is confirmed based on cardinal signs and symptoms along with supportive laboratory investigations. Early diagnosis and treatment of new cases are essential to prevent disease burden and spread in community. Aim: To evaluate the efficacy of currently available laboratory tests like M.leprae-specific repetitive element Polymerase Chain Reaction (RLEP-PCR) and conventional methods like, Slit Skin Smears (SSS), Histopathology (HP) and Fite-Faraco (FF) staining for detection of M. leprae in clinically diagnosed cases of early- stage leprosy. Materials and Methods: A cross-sectional study of 82 newly diagnosed cases of leprosy was carried out in S.C.B Medical College, a tertiary care hospital of Odisha, India from September 2018 to August 2020. For every case, skin tissue was punched out to get biopsy for HP and FF staining and incised for SSS. At least two SSS slides were prepared per case. One SSS slide was stained by modified Ziehl-Neelsen (ZN) staining to demonstrate Acid Fast Bacilli (AFB). The other SSS slide was air dried and the dried material was scraped off to perform PCR. Data were analysed by Statistics software IBM Statistical Package for the Social Sciences (SPSS), version 24.0. Association between categorical variables was studied by using Chi-square test. Comparison of mean±SD and median (IQR) were done by using independent sample‘t’ test. Agreement on different types of laboratory methods was done by using Kappa test. Results: Among 82 cases, 68.3% were RLEP-PCR positive, 56% had relevant chronic granulomatous features histopathologically, 39% were FF stain positive, and only 29% were ZN stain positive. Improvement in case detection of 39%, 29.3%, and 12.2% by PCR over SSS, FF Stain and HP respectively proved superiority of PCR over other procedures. Conclusion: PCR was most sensitive to detect leprosy both in pauci and multibacillary groups, thus considered as investigation of choice for diagnosis of early-stage leprosy.

Antimicrobial Activity of Ca-Alginate/Chitosan Nanocomposite Loaded with Camptothecin.

The main aim of this study was to prepare antimicrobial nanocomposites consisting of alginate, chitosan, and camptothecin (CPT). CPT-loaded calcium alginate (Ca-Alg2) and calcium alginate/chitosan (Ca-Alg2-CH) nanomaterials were synthesized and characterized using infrared (IR) spectroscopy, X-ray diffraction (XRD), UV-Vis spectroscopy, and scanning electron microscopy (SEM). The antimicrobial activity and the genetic effects of Ca-Alg2/CPT and Ca-Alg2-CH/CPT nanomaterials on Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia were studied. The repetitive element polymerase chain reaction analysis technique was used to assess the changes in the bacterial genetic material due to the processing of the nanomaterials. The results showed the presence of a strong chemical interaction between alginate and chitosan, and CPT was loaded successfully in both Ca-Alg2/CPT and Ca-Alg2-CH/CPT nanomaterials. Furthermore, the antimicrobial test showed that the Ca-Alg2/CPT nanocomposite was susceptible to S. aureus, E. coli, and K. pneumonia; on the other hand, Ca-Alg2-CH/CPT nanocomposite was more susceptible to E. coli and K. pneumonia and was resistant to S. aureus. The results showed that the Ca-Alg2/CPT nanocomposite was less efficient than Ca-Alg2-CH/CPT nanocomposite in killing Gram-negative treated bacteria. Moreover, results revealed that the PCR analysis revealed a polymorphic banding pattern. This observation provides an excellent guide to the ability of some polymers to induce point mutations in DNA.

Comparison of superantigens and attachment factors genes of Staphylococcus aureus in clinical isolates and nasal colonizers in the same patients

BackgroundStaphylococcus aureus (S. aureus) is a bacterial pathogen can cause a wide range of nosocomial infections. Nasal colonization by S.aureus plays important role both in the epidemiology and pathogenesis of infection. ObjectivesThe purpose of this study was to investigate the association of clinical isolates and nasal colonizers of S. aureus in the same patients by molecular methods, and their antibiotic susceptibility pattern. MethodsA total of 181 S. aureus isolates were collected from 100 patients admitted that including 100 clinical isolates and 81 nasal swabs from the same patients (19 cases were found as noncarriers). Superantigens and adhesion genes were identified by PCR. Molecular typing of the isolates was performed by repetitive element polymerase chain reaction (Rep-PCR). Antimicrobial susceptibility pattern of the isolates was conducted by disk diffusion. MIC of the isolates to vancomycin was determined by microbroth dilution. The ability of S. aureus isolates to form biofilm was determined by microtiter plate assay. ResultsThe most frequent adhesion gene in both clinical isolates and nasal colonizer was clfA with 93% and 76%, respectively. Staphylococcal enterotoxin A (SEA) was the most commonly superantigen (68%) in both nasal colonizers (71.6%) and clinical isolates (65%). The highest resistance rate was to erythromycin (45.3%) with 36% and 56.8% in clinical and nasal colonizer isolates, respectively. All S. aureus isolates were susceptible to linezolid and vancomycin. Multiple drug resistance (MDR) was detected in 36% (n = 65) of the isolates. Biofilm formation was identified in 160 (88.4%) isolates with 87% and 90% in clinical isolates and nasal colonizers, respectively. Repetitive element polymerase chain reaction (Rep-PCR) typing divided 181 S. aureus isolates into six clusters. Twelve isolates from clinical isolates and nasal carriers were closely related. ConclusionThere is a high concordance rate between colonizing and clinical isolates of S. aureus in terms of adhesion factors and superantigen genes. It is suggested that nasal decolonization could be effective in the preventing of S. aureus infections.

Proteolytic Lactococcus lactis and Lipolytic Enterococcus durans of Dairy Origin as Meat Functional Starter Cultures.

SUMMARYResearch backgroundAs fermentation is an integral feature of both, dry sausage and cheese production, this has led to the evaluation of bacterial cultures Lactococcus lactis ssp. cremoris (LL8307) and Enterococcus durans (ED0207) originally isolated from artisanal Croatian hard type cheese to diversify the range of flavours of dry fermented sausages and to increase their microbiological safety. Both strains were chosen for their high or medium acidifying, proteolytic and/or lipolytic activity and bioprotective potential after step-by-step selection of wild isolates. Therefore, this study aims to evaluate the survival rate of selected starter cultures in wild boar meat sausages during the ripening period of 40 days at a local small-scale facility under artisanal conditions as well as their influence on sausage quality parameters.Experimental approachSafety, biotechnological and probiotic properties of twenty-three enterococcal and lactococcal isolates of dairy origin were studied. Based on the results, two best candidates were selected and added to the meat batter during the artisanal wild boar meat sausage preparation where their survival rate, effect on physicochemical, microbiological and sensorial properties and histamine content were evaluated.Results and conclusionsAs revealed by repetitive element-polymerase chain reaction (rep-PCR), native starter cultures survived up to 15 days of ripening and were either absent from (LL8307) or reduced by 80% (ED0207) in final products. The application of native starter cultures rapidly decreased pH (p<0.05), leading to the significantly lower load of E. coli, coliforms and Enterobacteriaceae in ready-to-eat sausages prepared by the addition of starter cultures (3.04-3.94 log CFU/g) than in the control (3.88-5.00 log CFU/g). Analysis of hedonic test data revealed that some of the sensory traits (odour, flavour, juiciness) of treatments with starter cultures were highly liked by the higher percentage of consumers. The results suggest that these starter cultures would represent a valuable tool to improve the homogeneity of artisanal manufacture and hygienic quality of fermented sausages and can be safely used for food application.Novelty and ccientific contributionThis is the first study to explore in depth the biotechnological potential of bacterial cultures isolated from artisanal Croatian cheese as functional starter cultures for high-quality game meat sausage production.

Whole-Genome Sequencing for Investigating a Health Care-Associated Outbreak of Carbapenem-Resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAB) outbreaks in hospital settings challenge the treatment of patients and infection control. Understanding the relatedness of clinical isolates is important in distinguishing outbreak isolates from sporadic cases. This study investigated 11 CRAB isolates from a hospital outbreak by whole-genome sequencing (WGS), utilizing various bioinformatics tools for outbreak analysis. The results of multilocus sequence typing (MLST), single nucleotide polymorphism (SNP) analysis, and phylogenetic tree analysis by WGS through web-based tools were compared, and repetitive element polymerase chain reaction (rep-PCR) typing was performed. Through the WGS of 11 A. baumannii isolates, three clonal lineages were identified from the outbreak. The coexistence of blaOXA-23, blaOXA-66, blaADC-25, and armA with additional aminoglycoside-inactivating enzymes, predicted to confer multidrug resistance, was identified in all isolates. The MLST Oxford scheme identified three types (ST191, ST369, and ST451), and, through whole-genome MLST and whole-genome SNP analyses, different clones were found to exist within the MLST types. wgSNP showed the highest discriminatory power with the lowest similarities among the isolates. Using the various bioinformatics tools for WGS, CRAB outbreak analysis was applicable and identified three discrete clusters differentiating the separate epidemiologic relationships among the isolates.

Diagnosis of leaf bacterial diseases of coffee reveals the prevalence of halo blight

ABSTRACT The diagnosis of foliar bacterial diseases in coffee (Coffea arabica), such as halo blight (Pseudomonas syringae pv. garcae), bacterial leaf spot (P. syringae pv. tabaci), bacterial blight (P. cichorii), and dark leaf spot (Robbsia andropogonis), is considered a challenge for plant pathologists. The misidentification has been occurring when the diagnosis is solely based on symptoms and biochemical properties. Thus, the objective of this study was to identify and differentiate species and pathovars of Pseudomonas pathogenic to coffee plants, enabling a survey of the occurrence of these bacteria in the main producing regions of Minas Gerais state, Brazil. Firstly, the pathogenicity of the isolates was confirmed by inoculation in C. arabica cv. Catuaí Vermelho IAC 99. Then, biochemical analyses, combined with, repetitive element-polymerase chain reaction (rep-PCR) and phylogeny based on rpoD gene sequences were used to characterize 84 Pseudomonas isolates from coffee crops and nurseries. Based on rpoD-phylogeny, 73 isolates were identified as P. syringae pv. garcae, five as P. syringae pv. tabaci and six as P. cichorii. The rep-PCR results suggest a high genetic variability in populations of Pseudomonas syringae pv. garcae and P. cichorii. This is the first report of the occurrence of bacterial leaf spot (P. syringae pv. tabaci) in the coffee-producing filed in Minas Gerais State. The findings confirmed the prevalence of P. syringae pv. garcae in coffee production fields in the State and the generated knowledge will contribute for the development of species-specific primers for the identification and detection of this pathogen.

Molecular typing of Lactobacillus brevis isolates from Korean food using repetitive element-polymerase chain reaction.

Lactobacillus brevis is a part of a large family of lactic acid bacteria that are present in cheese, sauerkraut, sourdough, silage, cow manure, feces, and the intestinal tract of humans and rats. It finds its use in food fermentation, and so is considered a "generally regarded as safe" organism. L. brevis strains are extensively used as probiotics and hence, there is a need for identifying and characterizing these strains. For identification and discrimination of the bacterial species at the subspecific level, repetitive element-polymerase chain reaction method is a reliable genomic fingerprinting tool. The objective of the present study was to characterize 13 strains of L. brevis isolated from various fermented foods using repetitive element-polymerase chain reaction. Repetitive element-polymerase chain reaction was performed using three primer sets, REP, Enterobacterial Repetitive Intergenic Consensus (ERIC), and (GTG)5, which produced different fingerprinting patterns that enable us to distinguish between the closely related strains. Fingerprinting patterns generated band range in between 150 and 5000 bp with REP, 200-7500 bp with ERIC, and 250-2000 bp with (GTG)5 primers, respectively. The Jaccard's dissimilarity matrices were used to obtain dendrograms by the unweighted neighbor-joining method using genetic dissimilarities based on repetitive element-polymerase chain reaction fingerprinting data. Repetitive element-polymerase chain reaction proved to be a rapid and easy method that can produce reliable results in L. brevis species.

Analysis of the Molecular Diversity of Common Bacterial Blight (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscansi) Strains from Ethiopia Revealed by Rep-PCR Genomic Fingerprinting

Common bacterial blight (CBB) disease of the common bean (Phaseolus vulgaris) caused by Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, is one of the most damaging foliar diseases of common bean production in Ethiopia. CBB causes economic losses due to reduction in seed quality and yield in common bean producing regions of Ethiopia. Currently, information on the genetic diversity of CBB strains in Ethiopia has been lacking. Here, for this specific study common bean bacterial blight strains were obtained from infected leaves collected from diverse bean growing areas. The collected strains of CBB were characterized to study the genetic diversity and relatedness of the CBB strains using repetitive extragenic elements polymerase chain reaction (rep-PCR) genomic fingerprinting technique. Analysis of Molecular Variance (AMOVA) revealed the existence of genetic diversity among bacterial strains and confirmed the presence of genetically distinct strains in Ethiopia. CBB pathogens are seed-borne so the lack of geographic differentiation among the six-different common bean growing localities could be the result of the distribution of one or some limited bacterial genotypes. Common bean improvement programs that develop CBB-resistant bean varieties for higher production should consider this information to determine the relevance and extent of resistance of improved bean cultivars.

DNA Profiling of Leuconostoc citreum Strains in Fermented Foods by Repetitive Element Polymerase Chain Reaction.

To identify and discriminate the bacterial species at the subspecific level, rep-PCR is a reliable genomic fingerprinting tool. Fourteen strains of bacteria were isolated from different food sources, identified as Leuconostoc citreum using 16S rRNA gene sequencing, and amplified using rep-primers (REP, ERIC, and (GTG)5). Fingerprinting patterns generated bands in the range of 300-6,000 bp with REP, 150-6,000 bp with ERIC, and 200-1,700 bp with (GTG)5 primers. In UPGMA dendrogram analysis, 14 strains were clustered into three clades (I, II, and III) with all the primers, thus differentiating them at the molecular level. The present study revealed the differentiation of L. citreum strains using rep-PCR.

Occurrence of Staphylococcus aureus in cattle, sheep, goat, and pig rearing in the Czech Republic

The study focused on the prevalence ofStaphylococcus aureusand resistant strains in livestock. In this study, 114 different samples from three cattle farms (84 from two farms of dairy cows and 30 from one farm of suckler cows), 132 samples from one sheep farm, 120 samples from one goat farm, and 82 samples from three pig farms were examined. Strains identified asStaphylococcus aureuswere further analysed by the polymerase chain reaction method for detection of themecA gene and for confirmation of the sequence type 398. Positive incidence ofStaphylococcus aureuswas confirmed in farms of suckler cows, sheep, goats and pigs. The incidence of methicilin-resistantStaphylococcus aureuswas confirmed at a goat farm, with all strains belonging to the sequence type 398. Repetitive element palindromic-polymerase chain reaction analysis was performed to compare the relatedness of selected human and animalS. aureusstrains at the goat and sheep farms. The obtained data from repetitive element-polymerase chain reaction analysis showed significant clonal similarity among the tested isolates and indicated the possibility of mutual transmission between animals or animal and human and possible transfer in the food chain.

Prevalence of Major Methicillin-Resistant Staphylococcus aureus Clones in Korea Between 2001 and 2008.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) are important pathogens causing nosocomial infections in Korean hospitals. This study aimed to investigate the epidemiological and genetic diversity of clinical S. aureus isolates in healthcare settings from 2001 to 2008.MethodsSamples and data were obtained from 986 individuals as part of the National Antimicrobial Surveillance Project, involving 10 regions nationwide. Molecular typing studies, including multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were performed, and a representative clone of Korean MRSA was classified by combinational grouping using a DiversiLab (DL; bioMérieux, France) repetitive element polymerase chain reaction (rep-PCR) system.ResultsNine Korean MRSA clones (KMRSA-1 to -9) were identified by analysis of genetic backgrounds and molecular characteristics. KMRSA-1 to -3, expressing clonal complex (CC) 5 (carrying SCCmec II), CC8 (carrying SCCmec III), and CC72 (carrying SCCmec IV) were spread nationwide. In contrast, KMRSA-6 was highly prevalent in Gyeongsangnam-do, and KMRSA-4 was highly prevalent in Jeollanam-do and Jeollabuk-do.ConclusionsEpidemic KMRSA clones were genetically similar to major clones identified from the USA, with the exception of KMRSA-2, which had the SCCmec III type. Our results provide important insights into the distribution and molecular genetics of MRSA strains in Korea and may aid in the monitoring of MRSA spread throughout the country.

Clinical and microbiological features of infections caused by Pseudomonas aeruginosa in patients hospitalized in intensive care units.

The spread of multidrug-resistant Pseudomonas aeruginosa in Brazilian hospitals has greatly impacted upon the morbidity and mortality of individuals in intensive care units. Given the lack of information regarding the dynamics of multidrug resistance in northern Brazil, we analyzed the clinical and microbiological features of nosocomial infections caused by P. aeruginosa. Between January 2010 and March 2012, we conducted a retrospective cohort study of P. aeruginosa isolates from 54 patients who were hospitalized in intensive care units. The clinical and epidemiologic variables were analyzed, including the patients' demographic data and comorbidities, and the lengths of the intensive care unit stays, the classification of the infections as nosocomial, the use of invasive procedures, antimicrobial therapy, and the patients' outcomes. We undertook susceptibility tests, molecular detection of the metallo-β-lactamase genes, and genotypic analyses of the isolates using the repetitive element-polymerase chain reaction. Multidrug resistance occurred most frequently among isolates from adults who had been hospitalized for an average of 87.1 days. The use of mechanical ventilation and urinary catheters were risk factors for infection. The four isolates that harbored the blaSPM-1-like gene showed >95% genetic similarity. This study's findings show that P. aeruginosa has a high death rate, and that inadequate treatment and invasive procedures are risk factors for infection. This is the first report describing the detection of the blaSPM-1-like gene in northern Brazil. These results highlight the need for better monitoring and a greater understanding of nosocomial infections and their public health impacts.

Diversity of Xanthomonas campestris Isolates from Symptomatic Crucifers in New York State.

To assess the diversity of Xanthomonas campestris spp. infecting crucifers in New York, 154 isolates were collected over 10 years across the state. The goal was to determine if isolates of the pathogen were overwintering in New York and serving as primary inoculum in subsequent years, or if novel isolates were entering the state each year. Pure cultures of isolates were characterized using multilocus sequence analysis (MLSA), a greenhouse pathogenicity assay, repetitive element-polymerase chain reaction (Rep-PCR) using the BOX-A1R primer, and enzyme-linked immunosorbent assay. The MLSA scheme proved to be more efficient than Rep-PCR for a large sample population and for comparison with global isolates. X. campestris isolated from crucifers in New York comprised of X. campestris pv. campestris and X. campestris pv. raphani, with X. campestris pv. raphani being predominately isolated from transplants. Evidence for unique haplotypes persisting on the same farm for several years due to improper seedbed rotations was documented in addition to novel haplotypes being spread throughout states through infected transplants and seed. Rep-PCR confirmed the high diversity of X. campestris and was used to generate 15 unique fingerprint patterns from isolates collected in the first 5 years. A worldwide comparison of isolates suggests that the X. campestris pv. campestris population appears to be very homogenous with dominant haplotypes persisting for extended periods and being globally disseminated.

Evaluation of repetitive element polymerase chain reaction for surveillance of methicillin-resistant Staphylococcus aureus at a large academic medical center and community hospitals

Repetitive element polymerase chain reaction (rep-PCR) typing has been used for methicillin-resistant Staphylococcus aureus (MRSA) strain characterization. The goal of this study was to determine if a rapid commercial rep-PCR system, DiversiLab™ (DL; bioMérieux, Durham, NC, USA), could be used for MRSA surveillance at a large medical center and community hospitals. A total of 1286 MRSA isolates genotyped by the DL system were distributed into 84 distinct rep-PCR patterns: 737/1286 (57%) were clustered into 6 major rep-PCR patterns. A subset of 220 isolates was further typed by pulsed-field gel electrophoresis (PFGE), spa typing, and SCCmec typing. The 220 isolates were distributed into 80 rep-PCR patterns, 94 PFGE pulsotypes, 27 spa, and 3 SCCmec types. The DL rep-PCR system is sufficient for surveillance, but the DL system alone cannot be used to compare data to other institutions until a standardized nomenclature is established and the DL MRSA reference library is expanded.

Outbreak of Bacteremia Due toBurkholderia contaminansLinked to Intravenous Fentanyl From an Institutional Compounding Pharmacy

IMPORTANCE Many health care facilities compound medications on site to fulfill local demands when customized formulations are needed, national supply is critically low, or costs for manufactured pharmaceuticals are excessive. Small, institutional compounding facilities may perform the same high-risk procedures as large distributors of compounded medications. OBJECTIVES To investigate an outbreak related to contamination of compounded sterile preparations and to determine processes to prevent future outbreaks. DESIGN, SETTING, AND PARTICIPANTS We performed an outbreak investigation of inpatients at Duke University Hospital from August 31 through September 6, 2012. The investigation included a case-control study, compounding facility inspection and environmental sampling, observation of a mock compounding demonstration, and microbiologic and molecular testing of sequestered medication. EXPOSURES Intravenous fentanyl prepared by an institutional compounding pharmacy. MAIN OUTCOMES AND MEASURES Microbiologic and molecular evidence of contamination of a compounded sterile preparation and failure of routine sterility testing. RESULTS Blood cultures of 7 patients during a 7-day period at Duke University Hospital yielded pan-susceptible Burkholderia cepacia complex bacteria. The risk factor common to all patients was receipt of continuous fentanyl infusion prepared by our institutional compounding pharmacy (odds ratio, 11.22; 95% CI, 2.09-∞; P = .01). The outbreak was terminated after sequestration of compounded fentanyl. An intensive evaluation of the compounding facility, its practice, and its procedures was completed. Investigators evaluated the clean room, collected targeted microbiologic samples within the compounding pharmacy environment, and observed a mock demonstration of compounding practice. The B cepacia complex was found in the anteroom sink drain and pH probe calibration fluid from the compounding clean room. Multiple microbiologic analyses of sequestered fentanyl initially failed. Ultimately, a batched, vacuum-assisted filtration method produced B cepacia complex from a single lot. Molecular analyses using repetitive element polymerase chain reaction and pulsed-field gel electrophoresis confirmed a clonal Burkholderia contaminans strain from patients, fentanyl, and environmental samples. CONCLUSIONS AND RELEVANCE An outbreak of B contaminans bacteremia was linked to contamination of locally compounded intravenous fentanyl. Health care facilities that house institutional compounding facilities must be vigilant in efforts to prevent, recognize, and terminate medication-related outbreaks.

Bulgaristanda Paenibacillus larvae Repetitive Element Polymerase Chain Reaction (rep-PCR) Genotiplerinin Biyokimyasal Profili

Bulgaristanda Paenibacillus larvae Repetitive Element Polymerase Chain Reaction (rep-PCR) Genotiplerinin Biyokimyasal Profili

Co‐invasion of South African ecosystems by an Australian legume and its rhizobial symbionts

AbstractAimTo determine and compare the taxonomic identity and diversity of root nodule and rhizospheric microbial symbionts associated with Acacia pycnantha Benth. in its native (Australian) and invasive (South African) ranges, and to establish whether these associations are general or host specific.LocationThe native range of A. pycnantha in Australia and invasive ranges in South Africa and Western Australia.MethodsBacteria were isolated from root nodules collected from 18 populations of A. pycnantha. Repetitive element polymerase chain reaction (REP‐PCR) fingerprinting was used to assess overall bacterial diversity and clustering. Molecular phylogenies for a subset of isolates representing major REP‐PCR clades were reconstructed using maximum parsimony and Bayesian phylogenetic analyses of the nuclear 16S–23S rRNA intergenic spacer (IGS), 16S rRNA, and the symbiotic nodA genes.ResultsTwelve clusters were identified from the REP‐PCR analysis; 11 included isolates from both the native range in Australia and invasive range in South Africa, while one cluster comprised only Australian isolates. Six rhizobial species were found in association with A. pycnantha: Bradyrhizobium japonicum, Rhizobium gallicum, R. lusitanum, R. miluonense, R. multihospitium and R. tropici. We also identified three plant‐growth promoting bacteria isolated from root nodules of A. pycnantha: Burkholderia caledonica, B. graminis and B. phytofirmans. Phylogenetic analysis of the IGS gene retrieved clades containing symbionts from both Australia and South Africa while others comprised only South African taxa, suggesting the introduction of bacterial lineages from Australia to South Africa. Our phylogeographic analysis of the nodA gene confirmed that A. pycnantha was co‐introduced with its symbionts to South Africa.Main conclusionsAcacia pycnantha is a promiscuous legume, associated with at least six different rhizobial symbionts, and forms associations with plant‐growth promoting rhizosphere bacteria from the genus Burkholderia. In the invasive range of A. pycnantha in South Africa, nodules contained some symbionts of South African origin while other symbionts appear to have been co‐introduced from Australia. Acacia pycnantha is associated with a wider suite of symbionts in its invasive than native range.

Molecular Typing of Paenibacillus larvae Strains Isolated from Bulgarian Apiaries Based on Repetitive Element Polymerase Chain Reaction (Rep-PCR)

The aim of the present study was to perform molecular typing of Paenibacillus larvae (P. larvae) isolates from Bulgarian apiaries with repetitive element polymerase chain reaction (rep-PCR) using BOX A1R, MBO REP1, and ERIC primers. A total of 96 isolates collected from brood combs with clinical symptoms of American foulbrood originating from apiaries located in different geographical regions of Bulgaria, a reference strain P. larvae NBIMCC 8478 and 30 commercial honey samples with Bulgarian origin were included in the study. Rep-PCR fingerprinting analysis revealed two genotypes ab and AB of P. larvae isolates from brood combs and honey samples. A combination of genotypes ab/AB was detected in one apiary and honey sample. The prevailing genotype ab was found in 78.1 % of brood combs isolates as well as in the reference strain whereas genotype AB was determined in 21.9 % of isolates. The examination of honey samples confirmed the preponderance of ab genotype which was demonstrated in 20 of 30 samples analyzed. In conclusion, the genetic epidemiology of P. larvae revealed two genotypes--ab and AB for Bulgarian strains. Developed protocols for molecular typing of P. larvae are reliable and may be used to trace the source of infection.

Genetic diversity of resident soil rhizobia isolated from nodules of distinct hairy vetch (Vicia villosa Roth) genotypes

Hairy vetch (Vicia villosa Roth, HV) is widely grown as a legume cover crop throughout the U.S.A., with biological nitrogen fixation (BNF) through symbiosis with Rhizobium leguminosarum biovar viciae (Rlv) being one of the most sought after benefits of its cultivation. This study determined if HV cultivation history and plant genotype affect genetic diversity of resident Rlv. Soil samples were collected from within farmers’ fields at Graham, Cedar Grove and Ivanhoe sites in North Carolina and pairs of genetically similar hairy vetch genotypes used as trap hosts. A total of 519 Rlv strains were isolated from six paired field soils, three with and three without histories of HV cultivation. A total of 46 strains failed to PCR-amplify the nifH gene; however nodC PCR amplification of these nifH-negative strains resulted in amplification of 22 of the strains. Repetitive element polymerase chain reaction (rep-PCR) with BOX-A1R primer and redundancy analysis showed rhizobial diversity to vary greatly within and between fields, with over 30 BOX banding patterns obtained across the six fields. Cluster analysis of BOX-PCR banding patterns resulted in 36 genetic groups of Rlv at a similarity level of 70%, with 15 of the isolates from fields with HV history not belonging to any of the clusters. Site was found to be the main driver of isolate diversity overall, explaining 57%, of the total variation among rhizobia occupying HV nodules, followed by history of hairy vetch cultivation. Evidence of a HV host genotype influence on the populations of rhizobia that infect hairy vetch was also observed, with plant genotype explaining 12.7% of the variation among all isolates. Our results show that second to site, HV cultivation history was the most important driver of rhizobial nodule community structure and increases the genetic diversity of resident Rlv in soils.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.