Repetitive Element-based Polymerase Chain Reaction Research Articles

Surveillance of Rice Blast Resistance Effectiveness and Emerging Virulent Isolates in Taiwan.

Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.

Research Note: Repetitive element–based polymerase chain reaction genotyping improves efficiency of Salmonella surveillance in a model broiler production system

The genetic relatedness and antimicrobial susceptibility profiles of Salmonella isolated from poultry and their environment were determined. One broiler breeder flock (BBF1) and 2 broiler flocks (BF1 and BF2) were reared over a 1.75-year period on the same poultry research farm. Hatching eggs were obtained from BBF1 to produce BF1 chicks, while BF2 chicks were progeny of a separate, unsampled broiler breeder flock. BF1 and BF2 were reared in the same housing facilities but 6 mo apart. Salmonella isolates were collected via litter sock sampling (BF1), cecal excision (BF1 and BF2), or cloacal swabs (BBF1). Serotyping identified Salmonella enterica subsp. enterica serovar Altona (SA) in BBF1 and S. enterica subsp. enterica serovar Senftenberg (SS) in BF1 and BF2. Genotypic fingerprinting was achieved with Rep-PCR using the (GTG)5 primer and revealed sequence homology among Senftenberg isolates from BF1 and BF2. For each isolate, the minimum inhibitory concentration was determined for 27 antimicrobial agents using Sensititre plates with formularies specific to antimicrobials used in poultry production or those used to control gram negative pathogens. Isolates from the 3 flocks were resistant to clindamycin, erythromycin, novobiocin, penicillin, and tylosin tartrate and demonstrated intermediate resistance to azithromycin, florfenicol, and spectinomycin. These data demonstrated that serovar Altona and Senftenberg were harbored by poultry, the latter appeared to persist in broiler flocks, and both serotypes shared similar patterns of antimicrobial susceptibility in an integrated research operation. In the case of multiple Salmonella isolates, combining genotypic fingerprinting methods with serotyping of representative isolates would reduce the number of samples required for serotyping and more clearly identify relatedness of isolates. These methods facilitate effective surveillance in poultry production systems, thus allowing for implementation of precise Salmonella control measures.

Population diversity and variation of the rice blast fungus under different climate conditions

The natural environment is a critical factor for the epidemiology of rice blast. To demonstrate the population variation of rice blast field isolates in distinct climates of different years, 57 isolates from rice panicle blast samples in Dawei Mountian district of Hunan province were surveyed from 2013 to 2015. In July and August, the key period for the occurrence of rice panicle blast, there was obvious discrepancy among weather conditions over 3 years. The climate in 2013 was the most arid, better in 2014, and the most suitable was in 2015. For isolates purified in different climates, the pathogenicity assay on twenty-four rice monogenic lines (MLs) showed that the effective resistance (R) genes were Pi5 and Pi1 in 2013; Pi1 in 2014; Pita2, Piz5 and Pi9 in 2015. The genomic analysis based on repetitive element-based polymerase chain reaction (Rep-PCR) also confirmed that there was critical discrepancy among isolates in 3 years. The colony morphology performance at 32 °C high temperature showed that isolates in 2013 were smaller, and with more dense and compact mycelium than isolates in 2014 and 2015. These findings revealed the variation and rich population diversity of the rice blast field fungus under different environments, even in a limited location.

Nosocomial outbreak of extended-spectrum β-lactamase-producing Enterobacter cloacae among cardiothoracic surgical patients: causes and consequences

Enterobacteriaceae are recognized as leading pathogens of healthcare-associated infections. To report the investigation of a nosocomial outbreak of extended-spectrum β-lactamase-producing Enterobacter cloacae affecting cardiothoracic surgery patients in a Belgian academic hospital. Cases were defined based on epidemiological and microbiological investigations, including molecular typing using repetitive element-based polymerase chain reaction and multi-locus sequence typing. Case-control studies followed by field evaluations allowed the identification of a possible reservoir, and the retrospective assessment of human and financial consequences. Over a three-month period, 42 patients were infected or colonized by CTX-M-15-producing E.cloacae strains that belonged to the same clonal lineage. Acquisition mainly occurred in the intensive care unit (N= 23) and in the cardiothoracic surgery ward (N= 16). All but one patient had, prior to acquisition, undergone a cardiothoracic surgical procedure, monitored by the same transoesophageal echocardiography (TOE) probe in the operating room. Despite negative microbiological culture results, the exclusion of the suspected probe resulted in rapid termination of the outbreak. Overall, the outbreak was associated with a high mortality rate among infected patients (40%) as well as significant costs (€266,550). The outbreak was indirectly shown to be associated with the contamination of a manually disinfected TOE probe used per-operatively during cardiothoracic surgery procedures, because withdrawal of the putative device led to rapid termination of the outbreak.

Characterisation of Pseudomonas syringae isolates from apricot orchards in north-eastern Italy

A set of 31 isolates were obtained from apricot (Prunus armeniaca L.) orchards showing symptoms of blast and gummosis in orchards located in north-eastern Italy, mostly during three subsequent growing seasons (2014–2016). These isolates were identified by means of their colony morphology, pathogenicity (hypersensitive reaction in tobacco leaves and the detection of both syringomycin and coronatine coding genes), genetic features (BLASTn analysis of the rpoD gene) and physiological tests (GATTa) as Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum race 1 and race 2 strains, respectively. Phylogenetic analysis based on the rpoD gene sequence showed that the two pathovars and two races are well separated and highlighted high intrapathovar/race variation for both P. syringae pv. syringae and P. syringae pv. morsprunorum race 2. Pseudomonas syringae pv. morsprunorum race 1 formed a unique clade that had high sequence homology. Repetitive element-based polymerase chain reaction fingerprinting analysis using the ERIC, REP and BOX primers again showed both high intra-pathovar variation within the P. syringae pv. syringae isolates, which grouped into six distinct clusters, and notable intra-race genetic diversity within both P. syringae pv. morsprunorum races. All 31 strains showed tolerance to copper at 200 ppm in vitro. Among these isolates, 13 were highly resistant (up to 500 ppm) and revealed the presence of the cusCBA gene cluster. All P. syringae pv. syringae strains showed ice nucleation activity, and nine were markedly active (up −3 °C) and characterised by the presence of the INAz gene. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 2 strains from apricot in Italy.

Characterization of Molecular Identity and Pathogenicity of Rice Blast Fungus in Hunan Province of China

The blast (Magnaporthe oryzae) resistance (R) gene is the most economical and environmental method to control rice blast disease. Characterization of molecular identity and pathogenicity of M. oryzae benefits the deployment of effective blast R genes. In order to identify blast R genes that would be effective in Hunan Province,182 M. oryzae strains were analyzed with a Chinese differential system (CDS), repetitive element-based polymerase chain reaction (rep-PCR), and the presence and absence of avirulence (AVR) genes by PCR amplification with gene-specific primers. Identified blast R genes were validated with 24 monogenic lines (ML) carrying 24 major R genes. In total, 28 races (isolates) of M. oryzae was identified with CDS, and classified into 20 distinct groups with rep-PCR. Interestingly, AVR-Pia, AVR-Pik, AVR-Pizt, AVR-Pib, and AVR-Pi9 were detected in more than 86.8% of the isolates; AVR-Pita1 was in 51.3% and AVR-Pii was in only 2.5%. In contrast, pathogenicity assays on 24 ML demonstrated that Pi9, Piz5, Pikh, and Pikm were more effective, with resistant frequencies of 91.6, 91, 87.9, and 87.3%, respectively; Pia, Piks, Pit, Pi12, and Pib were less than 15%. These findings revealed the complexity of a genetic basis of rice blast resistance, and shed light on useful blast R genes in Hunan Province.

Prolonged outbreak of clonal MDR Pseudomonas aeruginosa on an intensive care unit: contaminated sinks and contamination of ultra-filtrate bags as possible route of transmission?

BackgroundWe report on an outbreak in a surgical, interdisciplinary intensive care unit (ICU) of a tertiary care hospital. We detected a cluster of ICU patients colonized or infected with multidrug-resistant Pseudomonas aeruginosa. We established an outbreak investigation team, performed an exploratory epidemiological analysis and initiated an epidemiology-based intervention.MethodsAs part of the outbreak investigation, we performed microbiological examinations of the sinks in the patient rooms and a retrospective case-control study. All patients admitted to the outbreak ICU between January 2012 and February 2014 were included. Cases were patients colonized with the outbreak strain. Controls were patients with a different Pseudomonas aeruginosa strain. Risk factors were evaluated using multivariable conditional logistic regression analysis. Strain typing was performed using the repetitive element-based polymerase chain reaction (rep-PCR) DiversiLab system.ResultsThe outbreak strain was found in the sinks of five (of 16) patient rooms. Altogether 21 cases and 21 (randomly selected) controls were included. In the univariate analysis, there was no significant difference in baseline data of the patients. In the multivariate analysis, stay in a room with a colonized sink (Odds Ratio[OR] 11.2, p = 0.007) and hemofiltration (OR 21.9, p = 0.020) were independently associated with an elevated risk for colonization or infection by the outbreak strain. In a subsequent evaluation of the work procedures associated with hemofiltration, we found that the ultra-filtrate bags had been on average five times per day emptied in the sinks of the patient rooms and were used multiple for the same patient. We exchanged the traps of the contaminated sinks and eliminated work procedures involving sinks in patient rooms by implementation of single use bags, which are emptied outside patient rooms to reduce splash water at the sinks. In the 20 month follow-up period, the outbreak strain was detected only once, which indicated that the outbreak had been ceased (incidence 0.75% vs. 0.04%, p < 0.001) Furthermore, the incidence of Pseudonomas aeruginosa overall was significantly decreased (2.5% vs. 1.5%, p < 0.001).ConclusionIn ICUs, limiting work processes involving sinks results in reduced multidrug-resistant Pseudomonas aeruginosa rates. ICUs with high rates of Pseudomonas aeruginosa should consider eliminating work processes that involve sinks and potentially splash water in close proximity to patients.Trial registrationAll data were surveillance based data which were obtained within the German Law on Protection against Infection (“Infektionsschutzgesetz”). Therefore a trial registration was not required.

Analysis and Forecast for Multidrug-Resistant Acinetobacter baumannii Infections Among Liver Transplant Recipients

BackgroundAcinetobacter baumannii (Ab) has become an important pathogenic bacterium with specific epidemic features in the intensive care unit. We explored the epidemiology of multidrug-resistant Ab infections among liver transplant recipients at the Liver Transplantation Center, 1st Affiliated Hospital of Shanghai Jiao Tong University. MethodsSeventeen multidrug-resistant Ab strains were isolated from the sputum and bronchoalveolar lavage fluid specimens of 249 liver transplant recipients from January 2007 to December 2009. The drug resistance and minimum inhibitory concentration (MIC) for the 17 Ab strains were determined. The Ab strains were genotyped with the use of repetitive element–based polymerase chain reaction. The risk factors were also characterized by single-factor and multifactor analysis to the clinical data of the 249 liver transplant recipients. ResultsThe drug sensitivity results showed that the 17 Ab strains isolated displayed 100% drug resistance rate to aminoglycosides (gentamicin), quinolones (ciprofloxacin), penicillins (piperacillin), cephalosporins (ceftazidime, cefotaxime, and cefepime), and carbapenems (imipenem and meropenem). The 17 Ab strains could be divided into 3 genotypes: 1, 1, and 15 strains for types A, C, and B, respectively. Fungal culture positivity after operation (odds ratio [OR], 5.470) and tracheal intubation twice (OR, 11.538) were the independent risk factors for multidrug-resistant Ab strain infection. ConclusionsType B multidrug-resistant Ab strains are prevalent in the liver transplantation center, and they could be transmitted clonally. Liver transplant recipients with postoperational fungal culture positivity and tracheal intubation twice are prone to multidrug-resistant Ab infections. Therefore, a high degree of vigilance should be paid to those recipients to avoid nosocomial Ab infections.

Analysis of Genetic and Molecular Identity Among Field Isolates of the Rice Blast Fungus with an International Differential System, Rep-PCR, and DNA Sequencing.

The Pi-ta gene deployed in southern U.S. rice germplasm is effective in preventing the infection by strains of Magnaporthe oryzae isolates that carry the avirulence (AVR) gene AVR-Pita1. In the present study, 169 isolates from rice (Oryza sativa) cultivars, with and without Pi-ta, were analyzed for their genetic identity using an international differential system, repetitive element-based polymerase chain reaction (Rep-PCR), and sequence analysis of PCR products of AVR-Pita1. These isolates belong to the races IA1, IB1, IB17, IC1, and IC17 of M. oryzae. These isolates were further classified into 15 distinct groups by Rep-PCR. There was a predominant group within each race. Pathogenicity assays on 'Katy' (Pi-ta) and 'M202' (pi-ta) rice determined that IC1 was virulent to Katy and M202; IB17, IC17, and most of IA1 and IB1 were avirulent to Katy and virulent to M202, suggesting that the Pi-ta gene in Katy is responsible for preventing infection by these isolates. Consistently, AVR-Pita1 was not amplified from 28 virulent isolates. One AVR-Pita1 allele was amplified by AVR-Pita1-specific primers in 78 avirulent isolates. Interestingly, different AVR-Pita1 alleles were found in each of the 12 avirulent isolates, as determined by DNA sequencing. Sequence analysis of 90 PCR products revealed 10 AVR-Pita1 haplotypes, 4 of which were new. In total, 12 amino acid changes were identified in the new variants when compared with the first described AVR-Pita sequence (AF207841). The finding of isolates with altered AVR-Pita1 from rice cultivars with and without Pi-ta suggests that these virulent isolates were adapted to the field environments in the southern United States. Further research will be needed to verify this prediction.

A Real‐Time Polymerase Chain Reaction Assay for Quantification of Edwardsiella ictaluri in Catfish Pond Water and Genetic Homogeneity of Diagnostic Case Isolates from Mississippi

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

Prevalence, mechanisms, and risk factors of carbapenem resistance in bloodstream isolates of Pseudomonas aeruginosa

We examined the prevalence of various carbapenem resistance mechanisms in Pseudomonas aeruginosa bloodstream isolates from a university-affiliated hospital. Isolates obtained in 2003 and 2004 were screened for meropenem/imipenem resistance, and clonality was assessed by repetitive-element–based polymerase chain reaction. The presence of carbapenemase and AmpC overexpression was ascertained by spectrophotometric assays. Outer membrane protein profiles were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and efflux pump overexpression was confirmed by Western blotting. We examined 129 nonrepeat isolates; 21 isolates (from 13 distinct clones) were resistant to meropenem or imipenem (prevalence rate = 16.3%). Nineteen (90.5%) carbapenem-resistant isolates had reduced OprD expression, and 6 (28.6%) isolates had overexpression of MexB. Increased length of hospital stay was identified as a significant risk factor for bacteremia due to carbapenem-resistant P. aeruginosa. Understanding the prevalence and mechanism of carbapenem resistance in P. aeruginosa may guide empiric therapy for nosocomial infections in our hospital.

Genetic Analysis of Pyricularia grisea Population by rep-PCR During Development of Resistance to Scytalone Dehydratase Inhibitors of Melanin Biosynthesis.

In 2001, field isolates of Pyricularia grisea resistant to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D) were reported in Saga prefecture, Kyushu. Among 1,175 isolates collected from six prefectures of Kyushu in 2002 and 2003, 647 were resistant to MBI-D fungicides, each due to a single point mutation of the scytalone dehydratase (SDH) gene. On the basis of repetitive element-based polymerase chain reaction (rep-PCR) fingerprint data, the haplotypes of the resistant isolates showed high genetic diversity, indicating that the resistance existed in a multigenetic background. Three predominant haplotypes mainly contributed to the widespread resistance in Kyushu; haplotype Sa4 was observed frequently in Saga, Sa18 was predominant in Oita and Miyazaki, and Sa5 was widely distributed among all four prefectures. Also, phylogenetic analysis showed that both the resistant and sensitive isolates were clustered together in a closely related group. These results suggest that isolates possessing the SDH mutation would have been selected and then multiplied rapidly in each region of Kyushu as a result of the widespread introduction of MBI-D fungicides in a short period.

Multilocus sequence typing and repetitive element-based polymerase chain reaction analysis of Neisseria meningitidis isolates in Brazil reveal the emergence of 11 new sequence types genetically related to the ST-32 and ST-41/44 complexes and high prevalence of strains related to hypervirulent lineages

Eighty-four strains of Neisseria meningitidis isolated from patients with meningococcal disease in 4 states of Brazil were analyzed by multilocus sequence typing and repetitive element-based polymerase chain reaction (Rep-PCR). The majority of strains analyzed (82%) belonged to 4 hypervirulent lineages, and 11 of 20 new sequence type (STs) characterized were related to hypervirulent lineages. Sequences of fetA and porA genes were analyzed, and the majority were related to profiles present in the ST-32 complex/electrophoretic type (ET)-5 complex. Rep-PCR analysis showed a unique electrophoretic pattern among strains related to hypervirulent lineages. Considering that 81% of the strains were serogroup B and strains belonging to the ST-32/ET-5 complex are genetically related to the Cuban vaccine strain used in a mass vaccination from 1990 to 1994 in Brazil, we believe that this vaccine did not confer effective herd immunity even among the age group within the vaccine showed higher efficacy. Our results once more raise the question about which strains should be used in the development of a new vaccine against N. meningitidis serogroup B.

Diversity Within Streptomyces ipomoeae Based on Inhibitory Interactions, rep-PCR, and Plasmid Profiles

ABSTRACT Streptomyces soil rot is a destructive disease of sweetpotato (Ipomoea batatas) that causes yield loss resulting from decay of the feeder root system and reduced quality due to the presence of necrotic lesions on the storage roots. It is managed by the use of resistant cultivars, but variability of the pathogen has not been previously assessed. This study compared 36 strains of the pathogen Streptomyces ipomoeae from different locations in the United States and Japan. The strains could be separated into three groups on the basis of their ability to inhibit the growth of one another in in vitro assays. Although some strains contained plasmids of approximately 18, 42, or 270 kb in size, plasmid profiles did not correspond to inhibition grouping. Fingerprinting by repetitive element-based polymerase chain reaction (rep-PCR) using outwardly facing primers for the BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) sequences indicated relatively high genomic homogeneity within S. ipomoeae. However, cluster analysis of similarity coefficients among strains using rep-PCR data revealed clusters that correlated with the inhibition grouping. The neotype strain of S. ipomoeae had lower similarity values by rep-PCR than any of the other strains and could not be grouped by inhibitory interactions.

Rapid Population Analysis of Magnaporthe grisea by Using rep-PCR and Endogenous Repetitive DNA Sequences

ABSTRACT DNA samples from Magnaporthe grisea isolates were fingerprinted by using repetitive element-based polymerase chain reaction (rep-PCR) with two outwardly directed primer sequences from Pot2, an element found in approximately 100 copies in the fungal genome. Variable length fragments, defining the sequences lying between these elements, were generated, and fingerprint patterns specific for individual strains were established. "Long PCR" conditions, including higher pH (9.2) and increased extension time (10 min) were used to amplify DNA fragments ranging from 400 bp to longer than 23 kb. Polymorphisms specific to M. grisea strains were generated, allowing inference of their genetic relationships. Segregation analysis was used to confirm single-locus inheritance for the fragments amplified by rep-PCR. Cluster analysis revealed robust groupings that corresponded to previously determined MGR586 restriction fragment length polymorphism lineages of the rice-infecting strains of the pathogen. We have also demonstrated the utility of rep-PCR to differentiate isolates that infect rice from those that infect nonrice hosts. DNA fingerprinting by Pot2 rep-PCR provides an efficient means to monitor the population dynamics of the blast pathogen. Because of the method's low cost and ease in application, it is now feasible to conduct large-scale population studies to understand the impact of host genotypes on pathogen evolution.

Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction genotyping.

Outbreaks of invasive disease caused by Neisseria meningitidis have increased in the United States in the 1990s. Repetitive element-based polymerase chain reaction (rep-PCR), a recently developed genotyping method, was used to evaluate a group of 8 outbreak-related and 35 other meningococcal isolates previously typed by multilocus enzyme electrophoresis (MLEE). All were serogroup B or C. Sets of genotypes were generated using primers based on either of two different repetitive sequences. Genotype sets were analyzed in a blinded fashion. Each set correctly identified outbreak-related isolates. Among the other 35 isolates, rep-PCR delineated 14 and 13 strains, respectively, in the two sets of genotypes. Seventeen electrophoretic types had been delineated by MLEE. Rep-PCR holds promise as a rapid, genome-based typing method for delineation of apparent outbreaks of meningococcal disease.