Repetitive Element PCR 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.

Distribution and Characteristics of Bacteria Isolated from Cystic Fibrosis Patients with Pulmonary Exacerbation.

Cystic fibrosis (CF) is an inherited recessive disorder characterized by recurrent and persistent pulmonary infections, resulting in lung function deterioration and early mortality. A cross-sectional study was conducted on the bacterial profile and antibiotic resistance pattern of 103 respiratory specimens from CF patients with signs of pulmonary exacerbation. Antibiotic susceptibility testing and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa isolates were performed by the Kirby-Bauer disc diffusion method and microtiter plate assay, respectively. Molecular typing of S. aureus and P. aeruginosa isolates was carried out by spa typing and repetitive extragenic palindromic element PCR. In a total of 129 isolates, the most prevalent organisms were S. aureus (55.3%) and P. aeruginosa (41.7%). Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. The highest rate of resistance for S. aureus was observed to azithromycin and erythromycin (80%), ciprofloxacin (52.3%), clindamycin (44.6%) and tetracycline (43%). Twenty percent of S. aureus isolates were methicillin-resistant S. aureus (MRSA) and 47.6% were MDR S. aureus. For P. aeruginosa isolates the highest resistance was to cefepime (38.3%) and levofloxacin (33.3%) and 20% showed MDR phenotype. Our study demonstrated a significant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa showed a shift in prevalence toward older ages. Molecular typing methods showed great diversity between isolates.

Real-time Screening of Foods Using Repetitive Element PCR Reveals a DNA Marker Characteristic for Enterotoxigenic Bacillus Species

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.

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.

Isolation and Characterization of Potential Starter Cultures from the Nigerian Fermented Milk Product nono.

Nono, an important traditional fermented dairy food produced from cow’s milk in Nigeria, was studied for microbial diversity and for starter culture development for industrial production. On the basis of a polyphasic approach, including phenotypic and genotypic methods such as 16S rRNA gene sequencing, repetitive element PCR (rep-PCR) fingerprinting metagenomics, and whole genome sequencing, we identified Lactobacillus (Lb.) helveticus, Limosilactobacillus (L.) fermentum, Lb. delbrueckii, and Streptococcus (S.) thermophilus as predominant bacterial species involved with milk fermentation during traditional nono production in Nigeria, while the predominant yeast species in nono was identified as Saccharomyces cerevisiae. Using metagenomics, Shigella and potential pathogens such as enterobacteria were detected at low levels of abundance. Strains of the predominant lactic acid bacteria (LAB) were selected for starter cultures combination on the basis of their capacities for rapid growth in milk and reduction of pH below 4.5 and their gelling characteristic, which was demonstrated noticeably only by the S. thermophilus strains. Whole genome sequence analysis of selected bacterial strains showed the largest assembled genome size to be 2,169,635 bp in Lb. helveticus 314, while the smallest genome size was 1,785,639 bp in Lb. delbrueckii 328M. Genes encoding bacteriocins were not detected in all the strains, but all the LAB possessed genes potentially involved in diacetyl production and citrate metabolism. These bacteria isolated from nono can thus be used to improve the microbial safety quality of nono in Nigeria, in addition to improving technological parameters such as gelling viscosity, palatability, and product consistency.

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.

Genotoxicity effects of medicinal plants extracts against bacterial species, Mycoplasma hominis

Aim: To assess the antimicrobial activity and genotoxicity of three medicinal plants used by Saudi Arabian people as traditional medicine against Mycoplasma hominis. Methodology: Different concentrations of Syzygium aromaticum (clove), Vachellia nilotica (acacia), and Thyme vulgaris (thyme) extracts were used as antimicrobial agents against M. hominis, and their lethal effects on Mycoplasma genome DNA were analyzed using repetitive element PCR(Rep-PCR). Results: The aqueous extracts of clove and Acacia at 3.125 mg ml-1 were found to be active antimicrobials against three tested Mycoplasm. Thyme extract exhibited antimicrobial activity at 12.5 mg ml-1. Moreover, this extract revealed potent lethal activities as growth turbidity decreased with increasing concentration or exposure time as compared to untreated Mycoplasma. The results of Rep-PCR clearly indicate that changes occured in the number of genetic bands in treated Mycoplasma at certain concentrations as compared to untreated Mycoplasma. Interpretation: These results indicate the possibility of using these extracts as a source of antibacterial compounds for treating infections caused by Mycoplasma. Key words: Antimicrobial activity, Genotoxicity, Mycoplasma hominis, Medicinal plants, S. aromaticum

Occurrence and Identification of Pectobacterium carotovorum subsp. brasiliensis and Dickeya dianthicola Causing Blackleg in Some Potato Fields in Serbia.

Blackleg outbreaks were noticed on three fields (about 100 ha total) in 2 consecutive years (2018, 2019) in one of the main potato growing areas in Serbia (Bačka region, Vojvodina). The percentage of infected plants reached 40 to 70%, with 10.5 to 44.7% yield reductions. From the three fields, out of 90 samples Pectobacterium carotovorum subsp. brasiliensis was most frequently identified and diagnosed as causal agent of potato blackleg in Serbia for the first time (29 isolates). Dickeya dianthicola was a less frequently causative bacterium, which was also noticed for the first time (nine isolates). A total of 38 isolates were characterized based on their phenotypic and genetic features, including a pathogenicity test on potato. The repetitive element PCR (rep-PCR) using BOX, REP, and ERIC primer pairs differentiated five genetic profiles among 38 tested isolates. Multilocus sequence analysis (MLSA) of four housekeeping genes, acnA, gapA, icdA, and mdh, revealed the presence of three so far unknown P. c. subsp. brasiliensis multilocus genotypes and confirmed clustering into two main genetic clades as determined in other studies. MLSA also revealed the presence of a new genotype of D. dianthicola in Serbia.

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.

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.

Genotypic diversity in multi-drug-resistant E. coli isolated from animal feces and Yamuna River water, India, using rep-PCR fingerprinting.

Genotypic diversity among multi-drug-resistant (MDR) aquatic E. coli isolated from different sites of Yamuna River was analyzed using repetitive element PCR (rep-PCR) methods viz. ERIC-PCR and (GTG)5-PCR and compared with the MDR animal fecal isolates. The 97 E. coli isolates belonging to different serotypes, phylogroups, and multi-drug resistance patterns were analyzed. High genetic diversity was observed by both the methods; however, (GTG)5 typing showed higher discriminating potential. Combination of ERIC types (E1-E32) and (GTG)5 types (G1-G46) generated 77 genotypes. The frequency of genotypes ranged from 0.013 to 0.065. The genotype composition of E. coli isolates was highly diverse at all the sampling sites across Yamuna River except at its entry site in Delhi. The sampling sites under the influence of high anthropogenic activities showed an increase in number of unique genotype isolates. These sites also exhibited high multiple antibiotic resistance (MAR) indexes (above 0.25) suggesting high risk of contamination. Principal coordinate analysis (PCoA) showed limited clustering of genotypes based on the sampling sites. The most frequent genotypes were grouped in the positive zone of both the principal coordinates (PC1 and PC2). The genotypes of most of the animal fecal isolates were unique and occupied a common space in the negative PC1 area forming a separate cluster. High genotypic diversity among the aquatic E. coli and the drain isolates, discharging the untreated municipal waste in the river, was observed, suggesting that the sewage effluents contribute substantially to contamination of this river system than animal feces. The presence of such a high diversity among the MDR E. coli isolates in the natural river systems is of great public health significance and highlights the need of an efficient surveillance system for better management of Indian natural water bodies.

Antibacterial Activity And Genotoxicity Effect of Ethanolic Leaves Extract of Rosmarinus Officinalis

The recent increasing use of artificial antibiotics has prompted an expansion in resistant strains and high site reactions. Medicinal plants have for quite some time been utilized as traditional medicine to treat pathogenic bacteria. In such manner, consistently numerous scientists are sending a range of plant’s secondary compounds to the customer advertise for the treatment of human illnesses. Accordingly, the distinguishing proof of plant spices with antimicrobial impacts can assist with delivering new medications with a wide range of impacts. The aim of the present research was to examine the ability of ethanolic leaves extracts of Rosmarinus officinalis plant as antibacterial agent against the Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. The zone of inhibition increased with increase in concentration of the test solution. Higher activity of ethanolic extract was found against S. aureus (2.4 cm) than E. coli (1.8 cm). In addition, the repetitive element PCR (Rep-PCR) significantly showed that several genetic numbers of polymorphic bands were observed in S. aureus and E. coli treated bacteria with leaves extracts and not observed in the control. These results indicate that these extracts have a genotoxicity effect on the two bacterial genomes. The obtained results demonstrate that R. officinalis can be used as a potential source of antibacterial and genotoxicity factors.

Molecular Mechanism of Multi-Drug Resistance in Mycobacterium tuberculosis

In this study, we demonstrate the clinical applicability of molecular assays for the differential identification of M. tuberculosis isolates by Double Repetitive Element-PCR (DRE-PCR), Duplex PCR (DPCR), Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and IS6110 flanking PCR and for the detection of specific codon mutations in antibiotic-resistant genes, rpoB and katG in 55 MDR-TB and 25 drug-susceptible clinical isolates by Multiplex PCR assays. The MAS-PCR assay was identified as the most prevalent rpoB gene mutations at codon 531 (83.6%), followed by codon 526 (12.7%), and no mutation was found in codon 516. Among the 55 MDR-TB isolates, 49 (89%) isolates had S315T, 5 (9%) had S315N mutations. DRE-PCR and RAPD-PCR generated similar banding of cluster III strains and suggested that MDR-TB strain genotype C may be responsible for the transmission of TB infection among the study population. PCR based differential identification of mtp40 and rpoB DPCR procedures identified two NTM strains among the isolates studied. Genotypic method DRE-PCR was found highly reproducible, followed by RAPD-PCR and mtp40, and rpoB DPCR methods effectively-identified NTM infection in this region. The presence of S315T mutation in katG gene and S531L, H526Y mutations in rpoB gene in MDR-TB isolates proved resistant phenotype. The simplicity of the MAS-PCR assay permits its implementation for the detection of resistance to INH and RIF in clinical laboratories in regions where this mutation is predominant among MDR-TB strains.

High‐throughput screening reveals small molecule modulators inhibitory to Acidovorax citrulli

AbstractAcidovorax citrulli is a seed‐transmitted gram‐negative bacterium that can cause substantial economic yield loss in watermelon and melon production worldwide. Four small‐molecule libraries containing 4,952 compounds were selected for high‐throughput screening against A. citrulli wild‐type strain Xu3‐14 by evaluation of growth inhibition. One hundred and twenty‐seven molecules (2.5% hit rate) were identified as bactericidal or bacteriostatic against A. citrulli at 100 μM. Secondary screens indicated that 27 candidate compounds were more effective against A. citrulli Group II strains than Group I strains (classified using repetitive element PCR). Several compounds were inhibitory to other pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Xanthomonas campestris pv. campestris, but did not affect the growth of plant beneficial bacteria Pseudomonas fluorescens and Bacillus subtilis. More than half of the compounds did not inhibit germination of Arabidopsis or watermelon seeds. The effect of small molecules on A. citrulli seed‐to‐seedling transmission was evaluated by applying each compound to inoculated watermelon seeds and assessing seedling infection. Nine compounds were chosen for further investigation based on their reduction of percentage seedling infection and compiling scores on their specificity, sensitivity, and phytotoxicity obtained in the secondary screens. The five best compounds were selected (thiamphenicol, nadifloxacin, pipemidic acid, ciclopirox, and zinc pyrithione) for greenhouse tests and were found to effectively reduce the seed‐to‐seedling transmission of A. citrulli in both artificially and naturally infested seeds. These top five compounds provide a basis for future development of an A. citrulli‐specific bactericide.

Molecular detection and genotypic characterisation of Streptococcus pneumoniae isolated from children in Malaysia

ABSTRACTStreptococcus pneumoniae (S. pneumoniae) is one of the main causative agents of pneumococcal diseases. To date, more than 90 distinct serotypes have been identified. Implementation of vaccines has caused a drastic reduction in vaccine-serotype pneumococcal diseases but increase in cases due to non-vaccine serotype has been observed in Malaysia. However, further investigation on different serotype incidence in Malaysia is needed and the rate of pneumococcal vaccination for new-born babies in Malaysia remains low. The recent emergence of drug-resistant S. pneumoniae (DRSP) has also been a global concern, especially penicillin resistance. This study determined the serotypes of S. pneumoniae strains (n = 95) isolated from nasopharyngeal specimens from children admitted to UMMC from 2013 to 2015. In accordance with previous studies, PCR result showed 40% of NT isolates were successfully typed as 3 less common serotypes, namely 9N/L, 17A, and 23B. The repetitive-element PCR (REP-PCR) result revealed genetic variations among the strains whereby five major clusters were observed at the similarity of 80% by clustering analysis based on fingerprint data. Penicillin-binding proteins (pbps) of selected isolates were studied by PCR and sequencing. Three strains with ≤19-mm diameter zone for Oxacillin Disc Diffusion (ODD) test previously were recorded to have mutation on all pbp1a, pbp2b, and pbp2x with MIC of 4 µg/ml, which were penicillin-intermediate resistance according to the CLSI breakpoints.

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.

Phenotypic and Genetic Diversity of Xanthomonas perforans Populations from Tomato in North Carolina.

Bacterial spot caused by Xanthomonas spp. is one of the most devastating diseases of tomato in North Carolina (NC). In total, 290 strains of Xanthomonas spp. from tomato in NC collected over 2 years (2015 and 2016) were analyzed for phenotypic and genetic diversity. In vitro copper and streptomycin sensitivity assays revealed that >95% (n = 290) of the strains were copper tolerant in both years, whereas 25% (n = 127) and 46% (n = 163) were streptomycin tolerant in 2016 and 2015, respectively. Using BOX repetitive element PCR assay, fingerprint patterns showed four haplotypes (H1, H2, H3, and H4) among the strains analyzed. The multiplex real-time quantitative PCR on a subset of representative strains (n = 45) targeting the highly conserved hrcN gene identified Xanthomonas strains from tomato in NC that belonged to X. perforans. Race profiling of the representative strains (n = 45) on tomato and pepper differentials confirmed that ∼9 and 91% of strains are tomato races T3 and T4, respectively. Additionally, PCR assays and sequence alignments confirmed that the copL, copA, copB (copLAB copper tolerance gene cluster), and avrXv4 genes are present in the strains analyzed. Phylogenetic and comparative sequence analyses of six genomic regions (elongation factor G [fusA], glyceraldehyde-3-phosphate dehydrogenase A [gapA], citrate synthase [gltA], gyrase subunit B [gyrB], ABC transporter sugar permease [lacF], and GTP binding protein [lepA]) suggested that 13 and 74% of X. perforans strains from NC were genetically similar to races T3 and T4 from Florida, respectively. Our results provide insights that bacterial spot management practices in tomato should focus on deploying resistance genes to combat emerging pathogenic races of X. perforans and overcome the challenges currently posed by intense use of copper-based bactericides.

Phenotypic and Molecular Analyses of Rhizoctonia spp. Associated with Rice and Other Hosts.

Forty-two Rhizoctonia isolates were collected from rice, mung bean, and grasses from Laguna, Philippines. Sixteen isolates were binucleate Rhizoctonia (BNR), while 26 were multinucleate Rhizoctonia (MNR). BNR isolates produced white to brown, small sclerotia (<1.0 mm) except for mung bean isolates. Twenty MNR isolates produced big (>1.0 mm), light to dark brown sclerotia, three produced salmon-colored masses in the medium, and three did not produce sclerotia. Twenty-three MNR isolates were identified as R. solani AG1-IA using specific primers. Deduced Internal Transcribed Spacer (ITS) sequences of BNR isolates D1FL, NVL, and ScNL shared 100, 97, and 100% identity with R. oryzae-sativae, respectively, while MNR isolates BMgL, IbMgL, and MaSL that produced salmon-colored masses shared 100, 90, and 100% identity with R. oryzae, respectively. Preliminary analysis of the DNA fingerprint patterns generated by repetitive-element PCR (rep-PCR) clustered the 42 isolates into three: R. solani, R. oryzae-sativae, and R. oryzae, together with Ceratobasidium sp. R. solani isolates were pathogenic on rice (TN1), barnyard grass, mungbean (Pagasa 3), and tomato (Athena), while R. oryzae and R. oryzae-sativae isolates were only pathogenic on rice, Echinochloa crus-galli, and tomato. R. solani and R. oryzae were found to be more virulent than R. oryzae-sativae.