Enterobacterial Repetitive Intergenic Consensus Fingerprinting Research Articles

Prevalence, genetic diversity, and antimicrobial susceptibility of Vibrio spp. infected gilthead sea breams from coastal farms at Damietta, Egypt

BackgroundVibriosis is one of the most serious bacterial diseases and causes high morbidity and mortality among cultured sea breams. This study was undertaken to track the surveillance of Vibrio infection and its correlation to environmental factors. A total of 115 gilthead sea breams were collected seasonally from a private earthen pond fish farm in the Shatta area of Damietta, Egypt from September 2022 to July 2023. Physicochemical parameters of water were analyzed, and heavy metal levels were measured. The fish samples were subjected to clinical, bacteriological, Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting, and hematoxylin and Eosin histopathological staining.ResultsThe results revealed significant variations in the water quality parameters over different seasons, in addition to an increase in heavy metals. Naturally infected fish showed external signs and postmortem lesions that were relevant to bacterial infection. Two dominant Vibrio subspecies of bacteria were identified: V. alginolyticus (205 isolates) and V. fluvialis (87 isolates). PCR confirmed the presence of V. alginolyticus using the species-specific primer collagenase at 737 bp. The highest prevalence of V. alginolyticus was detected during the summer season (57.72%), and the lowest prevalence was observed in autumn (39.75%). The correlation analysis revealed a positive relationship between V. alginolyticus and water temperature (r = 0.69). On the other hand, V. fluvialis showed a high prevalence during the autumn season (25.30%) and the lowest prevalence during the summer season (10.56%), where it was negatively correlated with water temperatures (r =—0.03). ERIC fingerprinting showed genetic variation within the Vibrio isolates. Antimicrobial susceptibility testing revealed sensitivity to ciprofloxacin and doxycycline, and resistance to amoxicillin and erythromycin. The multiple antibiotic resistance (MAR) index values for V. alginolyticus and V. fluvialis ranged from 0.3 to 0.7, with a multi-drug resistance pattern to at least three antibiotics. Histopathological alterations in the affected tissues revealed marked hemorrhage, vascular congestion, and hemosiderosis infiltration.ConclusionThis study provides insights into the potential propagation of waterborne diseases and antibiotic resistance in the environment. Ensuring that the environment does not serve as a reservoir for virulent and contagious Vibrio species is a critical concern for regional aquaculture industries. Therefore, we recommend implementing environmental context-specific monitoring and surveillance tools for microbial resistance.

Isolation, diversity and antimicrobial activity of planctomycetes from the Tejo river estuary (Portugal).

The discovery of new bioactive compounds is an invaluable aid to the development of new drugs. Strategies for finding novel molecules can focus on the exploitation of less studied organisms and ecosystems such as planctomycetes and brackish habitats. The unique cell biology of the underexplored Planctomycetota mean it is of particular interest. In this study, we aimed to isolate planctomycetes from the estuary of the Tejo river (Portugal). To reach this goal, macroalgae, water and sediments were sampled and diverse media and isolation techniques applied. Sixty-nine planctomycetal strains were brought into pure culture. An analysis of the 16S rRNA genes found that the majority of the isolates were affiliated to the genus Rhodopirellula. Putative novel taxa belonging to genera Stieleria and Rhodopirellula were also isolated and characterized morphologically. Enterobacterial repetitive intergenic consensus fingerprinting analyses showed higher diversity and different genotypes within close strains. Relevant biosynthetic gene clusters were found in most isolates and acetone extracts from representative strains exhibited mild antimicrobial activities against Escherichia coli and Staphylococcus aureus. Our work has not only enlarged the number and diversity of cultured planctomycetes but has also shown the potential for the discovery of bioactive compounds from the novel taxa.

Genetic diversity, virulence and distribution of antimicrobial resistance among Listeria monocytogenes isolated from milk, beef, and bovine farm environment.

Listeria monocytogenes is an opportunistic intracellular foodborne pathogen and is ubiquitous in nature. The occurrence of L. monocytogenes in animal production units coupled with their presence in milk, faeces, feed, water, sewage, and soil is a contributory factor for foodborne listeriosis in humans and animals. The study was aimed to characterize genotype and serogroup of L. monocytogenes recovered from different types of samples and also to study antimicrobial patterns by phenotypic and genotypic methods. Multiplex polymerase chain reaction (PCR) was used for the confirmation of L. monocytogenes, the identification of its serogroup and lineage, and the detection of virulence markers. Enterobacterial repetitive intergenic consensus (ERIC), and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize those isolates, and antimicrobial patterns were studied phenotypically by Kirby-Bauer method and genotypically by PCR. Out of the screened 474 samples (274 milk and 50 each of soil, feed, sewage, and beef), ten L. monocytogenes isolates (milk=8, soil=1, and beef=1) were confirmed by PCR targeting the hlyA gene and found to belong to the 1/2a, 3a serogroup and fall under type II lineage. Virulence potential assessment revealed that all the ten isolates harbored the iap gene while the presence of plcA and plcB genes were noticed in seven and eight isolates respectively. Six isolates from milk were found to group in the same cluster by ERIC and RAPD fingerprinting, suggesting both methods to be efficient molecular typing tools for L. monocytogenes. Genotypic characterization of antimicrobial resistance (AMR) genes revealed that seven isolates were positive for tetM, five for mefA, four for msrA, and one for lnuA genes while none of the isolates showed tetK, ermA, ermB, and lnuB genes. The presence of L. monocytogenes in bovine farm environments coupled with virulence markers, and multidrug resistance from the study area suggest a possible transmission from the environment to humans and animals which needs to be monitored regularly to ensure food safety and the well-being of animals and humans.

Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Genetic diversity and antimicrobial resistance of Escherichia coli as microbial source tracking tools of Karaj River, Iran

The aim of this study was to analyze the enterobacterial repetitive intergenic consensus (ERIC)-types, phylo-groups and antimicrobial resistance (AMR) patterns of Escherichia coli and to investigate if these approaches are suitable for microbial source tracking (MST). E. coli strains were isolated from cattle faeces and Karaj River. For genetic diversity, AMR profile, and phylo-grouping, we applied ERIC-PCR, disk diffusion, and multiplex-PCR, respectively. Fifty isolates from each sample group were used in the study. ERIC fingerprinting produced ten different bands, demonstrating 64 unique and 36 repetitive profiles. Six isolates from the river showed the same ERIC pattern of the cattle, of which four expressed the same AMR profile. E. coli isolates from water were represented in A, B1, C, and D phylo-groups. Phylo-groups A, B1, and E were more prevalent in the cattle isolates and B2 was absent in both sources. Three of the water isolates with the same ERIC-type and AMR to cattle isolates showed the same phylo-groups. Genetic characteristics, AMR, and phylo-groups of the isolates from the river are diverse and complex. For accurate MST, complementary approaches should be applied together and a comprehensive library should be provided.

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province, South Africa.

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.

Comparison of five molecular subtyping methods for differentiation of Salmonella Kentucky isolates in Tunisia

Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16%) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1%) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.

Diversity and Genetic Population Structure of Fungal Pathogens Infecting White Grub Larvae in Agricultural Soils

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.

ERIC-PCR-Generated Genomic Fingerprints and Their Relationship with Pathogenic Variability of Xanthomonas campestris pv. punicae, the Incitant of Bacterial Blight of Pomegranate

Bacterial blight caused by Xanthomonas campestris pv. punicae (Xcp) has emerged as a potential threat in pomegranate (Punica granatum) cultivation in India. Here, we report the genomic fingerprints and their correlation with virulence pattern of Xcp isolates from Maharashtra and Delhi. The genomic fingerprints of Xcp isolates were generated using enterobacterial repetitive intergenic consensus (ERIC) sequence-based primers, and virulence level was based on their reaction upon infiltration to susceptible pomegranate cultivar. Maharashtra isolate PGM1 showed only 50% similarity with Delhi isolate PGD8 forming a distinct genotype, whereas the Delhi isolates PGD5 and PGD6 form a cluster with Maharashtra isolates PGM2 and PGM4. The isolates PGM2, PGM4, PGD5, and PGD6 showing mean disease score of 7.47 were marked as group A or highly virulent. The moderately virulent or group B isolates PGM3 and PGD7 produced mean disease score of 4.19, whereas less virulent or group C isolates PGD8 and PGM1 gave mean disease intensity of 1.91. A correlation between genotypic groups based on ERIC fingerprints and pathogenicity of the isolates was established. The highly virulent isolates PGM2, PGM4, PGD5, and PGD6 formed a single cluster. A unique 900 bp amplicon present in all highly virulent isolates has been identified that can be used as genetic marker to screen isolates for virulence. The less virulent isolates PGD8 and PGM1 formed single cluster at 50% similarity coefficient. This seems to be the first report to establish a correlation between ERIC-PCR fingerprints and their corresponding virulence pattern of the pomegranate bacterial blight pathogen.

Comparison of four molecular typing methods for the differentiation of Salmonella spp.

A total of 57 strains of Salmonella spp. were differentiated by random amplification of polymorphic DNA (RAPD) fingerprinting using three different primers (OPL-03, primer 1, and primer A); by Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting; by ribotyping-PCR; and by Single Strand Conformation Polymorphism (SSCP). From the 57 strains, RAPD fingerprinting with primers OPL-03, 1, and A produced 42, 51, and 54 fingerprint patterns respectively. ERIC fingerprinting produced 50 patterns; ribotyping-PCR produced four patterns, and SSCP produced 11 patterns. Combinations of two different typing methods generally increased the discrimination of Salmonella strains. A combination of two different RAPDs or a combination of RAPD and ERIC was better than the other combinations. Discrimination using a combination of RAPD (primer 1 or primer A) and ERIC, which could differentiate all 57 Salmonella strains, was better than the combination of two RAPDs. This study indicated that the use of a combination of RAPD (primer 1 or primer A) and ERIC should be useful for the differentiation of field-isolated Salmonella strains and epidemiological studies.

Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans.

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.

리스테리아균의 특성분석을 위한 Molecular Typing 방법의 상호보완

리스테리아를 보다 효과적으로 typing할 수 있는 방법을 찾기 위해 표준균주 13종을 대상으로 하여 RAPD, ERIC (Enterobacterial repetitive intergenic consensus) fingerprinting, ribotyping-PCR의 분리력을 비교해 보았다. DG107 (primer 6) 또는 DG122 (Lis 11) primer를 이용한 RAPD의 경우 11가지의 유형으로 분류되는 반면, ERIC fingerprinting은 9가지, ribotyping-PCR은 7가지씩의 유형을 보였다. 그러나 2가지 primer를 이용하여 각각 행한 RAPD 결과를 종합하거나, DG122를 이용한 RAPD와 ribotyping-PCR의 결과를 종합할 경우 13가지의 유형으로 모두 분리할 수 있었다. The results typed by random amplification of polymorphic DNA (RAPD) were compared with those obtained by Enterobacterial repititive intergenic consensus (ERIC) fingerprinting and ribotyping-PCR. The discriminatory power of RAPD typing was the best among the methods tested. RAPD typing with two different primers for 13 Listeria spp. reference strains produced 11 patterns each. In contrast, ERIC fingerprinting produced 9 patterns and ribotyping-PCR produced 7 patterns each. Composite of two separate RAPD (Lis 11 and primer 6) results or RAPD (Lis11)/ ribotyping-PCR differentiated all 13 Listeria spp. reference strains. Therefore, composite of 2 separate RAPD (Lis11 and primer 6) or composite of RAPD (Lis11)/ribotyping-PCR is expected the most promising approach for typing field isolated Listeria spp. strains.

Genetic characterization of pseudomonas syringae pv. syringae strains from stone fruits in california

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.

PCR-based molecular discrimination of Verticillium chlamydosporium isolates

PCR-based assays were performed to resolve the genetic variation between 28 different isolates of Verticillium chlamydosporium using primers designed to amplify ribosomal internal transcribed spacers (ITS) and intergenic spacers (IGS). Different isolates of V. chlamydosporium were also differentiated using primers matching enterobacterial repetitive intergenic consensus (ERIC) sequences and repetitive extragenic palindromic (REP) elements. Restriction fingerprinting of PCR-amplified ITS products failed to yield intraspecific polymorphism, and different levels of discrimination between V. chlamydosporium isolates were not achieved. However restriction patterns of ITS products digested with Hae III and Hin f I were useful in differentiating between some of the closely related isolates of V. chlamydosporium , plant pathogenic Verticillium species and some common soil fungi. PCR amplification of IGS was found to be the most sensitive method which enabled the detection of 22 variants within the sample of 28 isolates of V. chlamydosporium and six different plant pathogenic Verticillium species. By using ERIC and REP-PCR fingerprinting, isolates were categorized in 20 and 13 genotypes, respectively. In general, PCR-based procedures can differentiate between closely related isolates of V. chlamydosporium within IGS genotypes. This also could be achieved by ERIC and REP-PCR, and may be considered a rapid tool for the genetic characterization and detection of different isolates of V. chlamydosporium .