Repetitive Extragenic Palindromic Research Articles

Comparative study on three molecular typing methods of Vibrio parahaemolyticus

To evaluate the correlation among the three molecular typing method of pulsed field gel electrophoresis(PFGE), repetitive extragenic palindromic(REP)-PCR and en-terobacterial repetitive intergenic consensus(ERIC)-PCR, and to explore the genetic relationship among strains, and to further understand the distribution and epidemic trend of Vibrio parahaemolyticus in Liaoning Province by combining Serotype analysis. Serum typing, PFGE, REP-PCR, and ERIC-PCR molecular typing and cluster analysis were performed on 150 VP isolates from Liaoning Province in 2018. 118 isolates could be divided into 14 Serotype, and 32 isolates could not be classified. The main serotypes were O3, O1 and O2. The resolution(DI) of PFGE is 0.969, the resolution(DI) of REP-PCR is 0.948, and the resolution(DI) of ERIC-PCR is 0.927. The Serotype O3 group strains are highly similar to the molecular types of O1 group strains. In 2018, the epidemic Serotype of clinical VP isolates in Liaoning Province is still O3: K6, and the epidemic serotype of food VP isolates is still O2. The result of PFGE, REP-PCR, and ERIC-PCR typing method are consistent, and the resolution and reproducibility of PFGE typing method are superior to the other two method. The Serotype O3 group is closely related to O1 group.

Conformation-based refinement of 18-mer DNA structures.

Nine new crystal structures of CG-rich DNA 18-mers with the sequence 5'-GGTGGGGGC-XZ-GCCCCACC-3', which are related to the bacterial repetitive extragenic palindromes, are reported. 18-mer oligonucleotides with the central XZ dinucleotide systematically mutated to all 16 sequences show complex behavior in solution, but all ten so far successfully crystallized 18-mers crystallized as A-form duplexes. The refinement protocol benefited from the recurrent use of geometries of the dinucleotide conformer (NtC) classes as refinement restraints in regions of poor electron density. The restraints are automatically generated at the dnatco.datmos.org web service and are available for download. This NtC-driven protocol significantly helped to stabilize the structure refinement. The NtC-driven refinement protocol can be adapted to other low-resolution data such as cryo-EM maps. To test the quality of the final structural models, a novel validation method based on comparison of the electron density and conformational similarity to the NtC classes was employed.

Sodium Fluoride Exposure Leads to ATP Depletion and Altered RNA Decay in Escherichia coli under Anaerobic Conditions.

Although fluoride-containing compounds are widely used to inhibit bacterial growth, the reprogramming of gene expression underlying cellular responses to fluoride, especially under anaerobic conditions, is still poorly understood. Here, we compare the genome-wide transcriptomic profiles of E. coli grown in the absence (control) or presence (20 and 70 mM) of sodium fluoride (NaF) under anaerobic conditions and assess the impact of fluoride-dependent ATP depletion on RNA turnover. Tiling array analysis revealed transcripts displaying altered abundance in response to NaF treatments. Quantile-based K-means clustering uncovered a subset of genes that were highly upregulated and then downregulated in response to increased and subsequently decreased fluoride concentrations, many of which (~40%) contained repetitive extragenic palindromic (REP) sequences. Northern blot analysis of some of these highly upregulated REP-containing transcripts (i.e., osmC, proP, efeO and yghA) confirmed their considerably enhanced abundance in response to NaF treatment. An mRNA stability analysis of osmC and yghA transcripts demonstrated that fluoride treatment slows down RNA degradation, thereby enhancing RNA stability and steady-state mRNA levels. Moreover, we demonstrate that turnover of these transcripts depends on RNase E activity and RNA degradosome. Thus, we show that NaF exerts significant effects at the whole-transcriptome level under hypoxic growth (i.e., mimicking the host environment), and fluoride can impact gene expression posttranscriptionally by slowing down ATP-dependent degradation of structured RNAs. IMPORTANCE Gram-negative Escherichia coli is a rod-shaped facultative anaerobic bacterium commonly found in microaerobic/anaerobic environments, including the dental plaques of warm-blooded organisms. These latter can be treated efficiently with fluoride-rich compounds that act as anticaries agents to prevent tooth decay. Although fluoride inhibits microbial growth by affecting metabolic pathways, the molecular mechanisms underlying its activity under anaerobic conditions remain poorly defined. Here, using genome-wide transcriptomics, we explore the impact of fluoride treatments on E. coli gene expression under anaerobic conditions. We reveal key gene clusters associated with cellular responses to fluoride and define its ATP-dependent stabilizing effects on transcripts containing repetitive extragenic palindromic sequences. We demonstrate the mechanisms controlling the RNA stability of these REP-containing mRNAs. Thus, fluoride can affect gene expression posttranscriptionally by stabilizing structured RNAs.

Dynamic Gene Clusters Mediating Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumanni (A. baumannii), a nonfermenting Gram-negative bacterium, has recently been associated with a broad range of nosocomial infections. To gain more meaningful insight into the problem of nosocomial illnesses caused by the multidrug-resistant (MDR) A. baumannii, as well as the factors that increase the risk of catching these infections, this investigation included a total of 86 clinical A. baumannii infections. Repetitive extragenic palindromic (REP)-PCR was used to investigate imipenem-resistant A. baumannii isolates for dynamic gene clusters causing carbapenem resistance. Four distinct A. baumannii lineages were found in the REP-PCR-DNA fingerprints of all isolates, with 95% of the samples coming from two dominant lineages. Imipenem, amikacin, and ciprofloxacin were less effective against genotype (A) isolates because of enhanced antibiotic tolerance. Lastly, to gain more insight into the mode of action of imipenem, we explored the binding affinity of imipenem toward different Acinetobacter baumannii OXA beta-lactamase class enzymes.

A small RNA is functional in Escherichia fergusonii despite containing a large insertion.

Bacterial small RNAs (sRNAs) are important regulators of gene expression; however, the impact of natural mutations on sRNA functions has not been studied extensively. Here we show that the sRNA MgrR contains a unique 53 bp insertion in Escherichia fergusonii, a close relative of Escherichia coli and Salmonella enterica. The insertion is a repetitive extragenic palindromic (REP) sequence that could block transcription, but full-length MgrR is produced in E. fergusonii, showing that the insertion has not affected sRNA production. Additionally, despite containing the large insertion, the sRNA appears to be functional because deletion of mgrR made E. fergusonii more susceptible to H2O2. The molecular details of MgrR's roles in H2O2defence are yet to be defined, but our results suggest that having an alternative function allowed the sRNA to be retained in E. fergusonii despite it sustaining a large, potentially disruptive mutation.

Divergently Transcribed ncRNAs in Escherichia coli: Refinement of the Transcription Starts Assumes Functional Diversification.

Non-coding regulatory RNAs (ncRNAs) comprise specialized group of essential genetically encoded biological molecules involved in the wide variety of cellular metabolic processes. The progressive increase in the number of newly identified ncRNAs and the defining of their genome location indicate their predominant nesting in intergenic regions and expression under the control of their own regulatory elements. At the same time, the regulation of ncRNA’s transcription cannot be considered in isolation from the processes occurring in the immediate genetic environment. A number of experimental data indicate the notable impact of positional regulation of gene expression mediated by dynamic temporal DNA rearrangements accompanying transcription events in the vicinity of neighboring genes. This issue can be perceived as particularly significant for divergently transcribed ncRNAs being actually subjected to double regulatory pressure. Based on available results of RNAseq experiments for Escherichia coli, we screened out divergent ncRNAs and the adjacent genes for the exact positions of transcription start sites (TSSs) and relative efficiency of RNA production. This analysis revealed extension or shortening of some previously annotated ncRNAs resulting in modified secondary structure, confirmed stable expression of four ncRNAs annotated earlier as putative, and approved the possibility of expression of divergently transcribed ncRNAs containing repetitive extragenic palindromic (REP) elements. The biogenesis of secreted ncRNAs from divergently transcribed ffs, chiX, ralA, and ryhB is discussed taking into account positions of TSSs. Refinement of TSSs for the neighboring genes renders some ncRNAs as true antisense overlapping with 5′UTR of divergently transcribed mRNAs.

Structural variability of CG-rich DNA 18-mers accommodating double T-T mismatches.

Solution and crystal data are reported for DNA 18-mers with sequences related to those of bacterial noncoding single-stranded DNA segments called repetitive extragenic palindromes (REPs). Solution CD and melting data showed that the CG-rich, near-palindromic REPs from various bacterial species exhibit dynamic temperature-dependent and concentration-dependent equilibria, including architectures compatible with not only hairpins, which are expected to be biologically relevant, but also antiparallel duplexes and bimolecular tetraplexes. Three 18-mer oligonucleotides named Hpar-18 (PDB entry 6rou), Chom-18 (PDB entry 6ros) and its brominated variant Chom-18Br (PDB entry 6ror) crystallized as isomorphic right-handed A-like duplexes. The low-resolution crystal structures were solved with the help of experimental phases for Chom-18Br. The center of the duplexes is formed by two successive T-T noncanonical base pairs (mismatches). They do not deform the double-helical geometry. The presence of T-T mismatches prompted an analysis of the geometries of these and other noncanonical pairs in other DNA crystals in terms of their fit to the experimental electron densities (RSCC) and their geometric fit to the NtC (dinucleotide conformational) classes (https://dnatco.datmos.org/). Throughout this work, knowledge of the NtC classes was used to refine and validate the crystal structures, and to analyze the mismatches.

High prevalence of mcr-1-encoded colistin resistance in commensal Escherichia coli from broiler chicken in Bangladesh

Colistin is a last-resort antimicrobial used for the treatment of human infections caused by multidrug-resistant Gram-negative bacteria. However, colistin is still widely used in intensive poultry production in Bangladesh. We aimed to investigate the dynamics and genetic diversity of colistin-resistant commensal Escherichia coli from broiler chickens. A total of 1200 E. coli strains were characterized from 20 broiler farms at three-time points along the production period. All strains were screened for mcr-1 to mcr-5 genes by a multiplex PCR, and their genetic diversity was measured by repetitive extragenic palindromic (REP)-PCR fingerprinting. Genomic diversity and characterization were performed by whole genome sequencing (WGS). Twenty-five percent of the commensal E. coli strains harbored mcr-1 genes. Frequency of mcr-1 gene detection correlated positively (odds ratio 1.71; 95% CI 0.96–3.06; p = 0.068) with the use of colistin in poultry flocks. REP-PCR profiles and WGS analysis showed diverse E. coli population carrying multiple antimicrobial resistance genes. Phylogenetic comparison of mcr-1-bearing strains recovered from this study with a global strain collection revealed wide phylogenetic relationship. This study identified a high prevalence of mcr-1 gene among genetically diverse E. coli populations from broiler chickens in Bangladesh suggesting a massive horizontal spread of mcr-1 rather than by clonal expansion.

Occurrence, Diversity of Listeria spp. Isolates from Food and Food-Contact Surfaces and the Presence of Virulence Genes.

This study evaluates the hazards posed by foodborne bacteria of the Listeria genus by analyzing the occurrence, diversity and virulence of Listeria spp.in food and food-manufacturing plants. Seventy-five isolates obtained from the routine analysis of 653 samples taken by three diagnostic laboratories in Northern Italy were genotypically differentiated by Repetitive Extragenic Palindrome (rep) PCR, with the GTG5 primer identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. Within this sample, 76% (n = 57) isolates were identified as L. innocua, 16% (n = 12) as L. monocytogenes, 6.6% (n = 5) as L. welshimeri and 1.3% (n = 1) as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenesinlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin–antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food-contamination-prevention measures against all Listeria species by defining efficiently their environmental distribution.

Molecular characterization of Bacillus thuringiensis strains to control Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) population

Abstract The main objective of this study was to characterize the toxicity and genetic divergence of 18 Bacillus thuringiensis strains in the biological control of Spodoptera eridania. Bacterial suspensions were added to the S. eridania diet. Half of the selected B. thuringiensis strains caused high mortality seven days after infection. The genetic divergence of B. thuringiensis strains was assessed based on Enterobacterial Repetitive Intergenic Consensus (ERIC) and Repetitive Extragenic Palindromic (REP) sequences, and five phylogenetic groups were formed. Despite their genetic diversity B. thuringiensis strains did not show any correlation between the collection sites and toxicity to larvae. Some B. thuringiensis strains are highly toxic to S. eridania, thus highlighting the potential of their endotoxins as biopesticides.

Effect of Biofat on Growing Performance and Health Status of Post-Weaning Piglets

BioFAT is a bioproduct contained PUFAs, Se, Vitamin E and herb (Euphorbia thymifolia Burm (L.)). Data were conducted on experiments of added 2 and 4% of BioFAT into basic diets for post-weaning piglets for 3 weeks of feeding to improve growth performance and health status, 22.45% and 32.86% of daily gain which was a higher than controls. The study also showed that there is reduced feed consumption for kg of growth rate from 1.67 kg of control was down to 1.36; and 1.29 kg of feed consumption (FCR). Furthermore, BioFAT is also enhanced for piglets to resistance E.coli infection and rate of infections was lower in added 2 and 4% of BioFAT from 20% to 6.7 and 0% piglets diarrhea. Especially the practice has shown that diversity of Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting.

Effect of Biofat on Growing Performance and Health Status of Post-Weaning Piglets

BioFAT is a bioproduct contained PUFAs, Se, Vitamin E and herb (Euphorbia thymifolia Burm (L.)). Data were conducted on experiments of added 2 and 4% of BioFAT into basic diets for post-weaning piglets for 3 weeks of feeding to improve growth performance and health status, 22.45% and 32.86% of daily gain which was a higher than controls. The study also showed that there is reduced feed consumption for kg of growth rate from 1.67 kg of control was down to 1.36; and 1.29 kg of feed consumption (FCR). Furthermore, BioFAT is also enhanced for piglets to resistance E.coli infection and rate of infections was lower in added 2 and 4% of BioFAT from 20% to 6.7 and 0% piglets diarrhea. Especially the practice has shown that diversity of Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting.

Molecular Characterization of Escherichia Coli Isolates from Food Animals

The present study was carried out for isolation and molecular characterization of Escherichia coli from faecal and meat samples of food animals viz. cattle, pigs and poultry. A total of 66 E. coli isolates were recovered from 420 samples of different food animals and further confirmed by PCR targeting E. coli specific uidA gene. These isolates were sensitive to chloramphenicol and ciprofloxacin and resistant to ampicillin and cloxacillin. Out of 66 isolates, 42 were typed into 13 different ‘O’ serogroups, 13 untypable and remaining 11 were identified as rough. Serogroup O84, O101, O118, O120 and O147 were predominant and serogroup O118 was found to be common in the samples of all 3 species of food animals. Five (7.57%) and 3 (4.54%) of E. coli isolates were found to harbor virulent genes, stx2 and est, respectively. Twenty representative E. coli isolates selected randomly from 20 different locations were subjected to molecular typing by PCR targeting Repetitive Extragenic Palindromic (REP) sequences. The region specific molecular types of E. coli could not be detected by using REP-PCR based discrimination.

Molecular epidemiological analysis and risk factors for acquisition of carbapenemase-producing Enterobacter cloacae complex in a Japanese university hospital

BackgroundTo clarify the molecular epidemiology of carbapenem-resistant Enterobacter cloacae complex (CREC) and the risk factors for acquisition of carbapenemase-producing E. cloacae complex (CPEC).MethodsUsing clinical CREC isolates detected in a Japanese university hospital over 4 years, carbapenemase production was screened with phenotypic methods. Carbapenemase genes were analysed by PCR and sequencing. Molecular epidemiological analyses were conducted with repetitive extragenic palindromic (REP)-PCR and multilocus sequence typing (MLST). CRECs were identified to the subspecies level by hsp60 sequencing. Whole-genome sequencing of plasmids was conducted. A case-control study was performed to identify risk factors for acquisition of CPEC among patients with CREC.ResultsThirty-nine CRECs including 20 CPECs carrying blaIMP-1 were identified. Patients with CPEC had longer hospital stay before detection (26.5 days vs. 12 days, p = 0.008), a urinary catheter (odds ratio [OR], 5.36; 95% confidence interval [CI], 1.14–30.9; p = 0.023), or intubation (OR, 7.53; 95% CI, 1.47–53.8; p = 0.008) compared to patients without CPEC. Four genetically closely related CPEC clusters were observed, which showed that three of four CPEC clusters corresponded to E. asburiae (ST 53), E. hormaechei subsp. steigerwaltii (ST 113 and ST 1047) and E. cloacae subsp. cloacae (ST 513) by MLST and hsp60 sequencing. Seven representative plasmids shared structures with class I integron containing blaIMP-1 and IncHI2A replicon type.ConclusionsA longer hospital stay, presence of a urinary catheter, and intubation are risk factors for CPEC acquisition. In addition to horizontal transmission of genetically indistinguishable CPECs, IncHI2A plasmid carrying blaIMP-1 appeared to be transferred among genetically different ECs.

Risk factors for endemic Acinetobacter Baumannii colonization: A case–case study

Acinetobacter baumannii causes increasingly resistant nosocomial infections worldwide. Although some patients are already colonized with A baumannii on hospital admission, others become colonized with endemic strains that are more likely to be antibiotic-resistant. Colonization increases risk of infection and transmission to others. This study aimed to identify risk factors for colonization with endemic compared to sporadic A baumannii among hospitalized patients. The study population were patients colonized with A baumannii at a single medical center during a 17-month period of active surveillance. Endemic A baumannii (cases) had a repetitive extragenic palindromic (REP) type that occurred 10 or more times during the surveillance period. Cases carrying 1 of the 5 endemic REP types were matched to comparison cases (controls) carrying sporadic strains by facility and time. There were 69 cases with REP-1, and 64 with REP-2-5. After adjustment, each unit increase in Schmid score was associated with a 70% increase in REP-1 carriage (P = .04) and a 50% increase in REP-2-5 (P = .07). Days in the intensive care unit prior to colonization, longer length of stay, immunosuppression, and the Charlson comorbidity index were not significantly associated with carriage of endemic strains. Following best practices for antibiotic stewardship and hygiene will help minimize the emergence and persistence of A baumannii strains adapted to the health care environment.

Effectiveness of BOX-PCR in Differentiating Genetic Relatedness among Salmonella enterica Serotype 4,[5],12:i:- Isolates from Hospitalized Patients and Minced Pork Samples in Northern Thailand.

Salmonella enterica Serotype 4,[5],12:i:-, a monophasic variant of S. Typhimurium, with high virulence and multidrug resistance is distributed globally causing pathogenicity to both humans and domesticated animals. BOX-A1R-based repetitive extragenic palindromic-PCR (BOX)-PCR proved to be superior to three other repetitive element-based PCR typing methods, namely, enterobacterial repetitive intergenic consensus (ERIC)-, poly-trinucleotide (GTG)5-, and repetitive extragenic palindromic (REP)-PCR (carried out under a single optimized amplification condition), in differentiating genetic relatedness among S. 4,[5],12:i:- isolates from feces of hospitalized patients (n=12) and isolates from minced pork samples of S. 4,[5],12:i:- (n=6), S. Typhimurium (n=6), and Salmonella Serogroup B (n=4) collected from different regions of northern Thailand. Construction of phylogenetic trees from amplicon size patterns allowed allocation of Salmonella isolates into clusters of similar genetic relatedness, with BOX-PCR generating more unique clusters for each serotype than the other three typing methods. BOX-, (GTG)5-, and REP-PCR indicated significant genetic relatedness between S. 4,[5],12:i:- isolates 1 and 9 from hospitalized patients and S. 4,[5],12:i:- isolate en 29 from minced pork, suggesting a possible route of transmission. Thus, BOX-PCR provides a suitable molecular typing method for discriminating genetic relatedness among Salmonella spp. of the same and different serotypes and should be suitable for application in typing and tracking route of transmission in Salmonella outbreaks.

Documentation of virulence and races of Xanthomonas citri pv. malvacearum in India and its correlation with genetic diversity revealed by repetitive elements (REP, ERIC, and BOX) and ISSR markers.

Thirty-four Xanthomonas citri pv. malvacearum (Xcm) isolates collected from three cotton-growing zones of India were subjected for virulence and race documentation and further correlated with genetic diversity as revealed by repetitive elements [repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX elements] and intersimple sequence repeat (ISSR)-PCR analyses. Among the 34 isolates tested for virulence on susceptible cultivar LRA 5166, 7 were recorded as highly virulent (HV), 16 were moderately virulent (MV) and 11 were less virulent (LV). Eight different races were recorded by using ten cotton host differentials. Twenty-two isolates (65%) belonged to race 18. Twelve isolates (35%) pertained to races 3, 5, 6, 7, 8, 11 and 13. REP, ERIC, BOX, combined repetitive elements, and ISSR analyses revealed the presence of 7, 10, 9, 11, and 8 clusters, respectively, at similarity coefficient of 0.70 in dendrograms. Principal coordinate analysis (PCoA) exhibited 76.4% and 77.5% cumulative variability for combined repetitive elements and ISSR analyses. ERIC produced the highest polymorphic information content (PIC) value (0.928). A lot of intra-pathovar variability was observed in virulence and genomic fingerprinting among Xcm isolates. Many of the isolates grouped based on geographical origin irrespective of virulence or race. The spread of the pathogen races in India might be due to the transport of germplasm lines and seed materials from one place to others.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

Genetic diversity of different avian strains of Pasteurella multocida recovered during four outbreaks in India

Pasteurella multocida causes fowl cholera in avian species and type A is predominantly reported from outbreaks of fowl cholera. Biochemical, serological, and molecular methods are employed for its diagnosis and typing. Utilizing rapid molecular tools like repetitive extragenic palindromic (REP) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR, P. multocida isolates from four outbreaks (three from Chennai and one from Ahmadabad, India) were characterized and typed to determine their relationships. A total of 36 isolates were recovered from the outbreaks, including one isolate from a parakeet, which was also subjected to characterization by conventional and molecular analysis. All of the isolates were found to be capsular type A based on PCR assay capsular typing. ERIC- and REP-PCR showed differences in the banding patterns among different outbreak isolates, and also among the geographical regions. Differences were also noticed among different host species, as the banding pattern in the ERIC- and REP-PCR differed; the analysis of results also revealed the same. All of the isolates were found to be sensitive to enrofloxacin and cefotaxime among the antibiotics used in the study. It was found that different strains might have been involved in the different outbreaks reported in the study. The results show that molecular typing methods like ERIC- and REP-PCR are useful epidemiological tools for classifying the strains.

Validation of molecular heterogeneity of Fluorescent Pseudomonas spp. and correlation with their potential biocontrol traits against fusarium wilt disease

Fluorescent Pseudomonas (FPs) are major rhizospheric bacteria with a variety of plant growth promotion attributes having potential field applications. The genomic and outer membrane protein (OMP) diversity were valued of 11 shortlisted FPs exhibiting potential biocontrol activity. The taxonomic diversity of isolates was studied using genomic fingerprinting assays—random amplification of polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX, repetitive extragenic palindromic (REP) and (GTG5), and OMP analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Genomic diversity parameters were calculated: total allelic number, polymorphic information content, expected heterozygosity, dominance, and the Shannon and Simpson indices. All isolates were used to prime tomato seeds and plants were challenge-inoculated with Fusarium oxysporum and the disease incidence was assessed under greenhouse condition. Cluster analyses of RAPD and combined Rep analyses classified isolates into two major clusters. SDS-PAGE analysis of OMP resulted in bands in the range 29–66 kDa. Furthermore, the isolates treated with tomato seeds followed by challenge-inoculation with F. oxysporum under greenhouse conditions showed that the three FPs, M80 (SUB1688209 Seq1 KX570929), M96 (SUB1688209 Seq1 KX570930) and T109 (SUB1688209 Seq1 KX570931) were able to induce systemic resistance. The study examined the possible correlation between strain-specific molecular diversity of FPs and provided an account of their potential biological control activity against F. oxysporum.