Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. isolated from chili pepper fruit exhibiting symptoms of anthracnose in Thailand

Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.     

Construction of a recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strain expressing a fusion protein of Omp22 and HpaA from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> for oral vaccine development

Objectives

To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.

Results

The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).

Conclusions

This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

     

Methods of <Emphasis Type="BoldItalic">Candida dubliniensis</Emphasis> identification and its occurrence in human clinical material

Candida dubliniensis was reported as a new species in 1995. This species is often misidentified as Candida albicans. The aims of this work were to determine the occurrence of C. dubliniensis in various clinical materials, to evaluate several ways to identify it and to examine the genetic variability of isolates. Among 7706 isolates originally identified as C. albicans, 237 were identified as C. dubliniensis (3.1%). Most of the C. dubliniensis isolates were obtained from the upper and lower respiratory tract (61.4 and 22.9%). Five phenotypic methods including latex agglutination were used (cultivation on CHROMagar Candida, on Staib agar, at 42 °C and in medium with 6.5% NaCl), but only cultivation on the medium with an increased concentration of NaCl and latex agglutination gave reliable results. Species-specific polymerase chain reaction was used as the confirmation method. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided less reliable results. In fact, 78.9% of C. dubliniensis isolates had scores above 1.7. However, the rest of them (21.1%) were also identified as C. dubliniensis even when the scores were lower than 1.7. Divergences among C. dubliniensis strains were evaluated by means of pulsed-field gel electrophoresis. Eighty-six selected C. dubliniensis isolates showed a 69.6% level of similarity. The results of this study expand the knowledge of the incidence, means of identification and genotypic divergence of C. dubliniensis isolates.     

Development of a Multiplex-PCR probe system for the proper identification of <Emphasis Type="Italic">Klebsiella variicola</Emphasis>

Background

Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.

Result

This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.

Conclusions

This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

     

Taxonomy and phylogeny of <Emphasis Type="Italic">Cryptocoryneum</Emphasis> (Pleosporales,Dothideomycetes)

Species of Cryptocoryneum were taxonomically reassessed on the basis of morphological observations and the results of molecular phylogenetic analysis. Eighteen isolates of Cryptocoryneum species, namely two strains from Africa, three from Europe, and 13 from Japan, were phylogenetically analysed using sequences of nuclear rDNA internal transcribed spacers (ITS) and the partial sequence of the translation elongation factor 1α gene (TEF1). The phylogenetic analysis indicated that Cryptocoryneum species formed a monophyletic clade and were closely related to Lophiotrema (Lophiotremataceae) and Aquasubmersa (incertae sedis) in the Pleosporales (Dothideomycetes). We examined holotype specimens of C. fasciculatum, C. hysterioides, and Torula uniformis and concluded that these species are conspecific, with C. hysterioides having priority. Although C. hysterioides has long been regarded as a synonym of C. condensatum, we consider C. hysterioides to be a distinct species within the genus. We found several cryptic species that were morphologically similar to C. condensatum sensu lato, but that could be separated on the basis of conidial size and the number of conidial arms and conidial septa, characters that seem to be informative for species delimitation within Cryptocoryneum. A total of seven new species, namely C. akitaense, C. brevicondensatum, C. congregatum, C. japonicum, C. longicondensatum, C. paracondensatum, and C. pseudorilstonei, are described and illustrated. A key to species accepted in Cryptocoryneum is provided.     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

Clinical management of fungal diseases is compromised by the emergence of antifungal drug resistance in fungi, which leads to elimination of available drug classes as treatment options. An understanding of antifungal resistance at molecular level is, therefore, essential for the development of strategies to combat the resistance. This study presents the assessment of molecular mechanisms associated with fluconazole resistance in clinical Candida glabrata isolates originated from Iran. Taking seven distinct fluconazole-resistant C. glabrata isolates, real-time PCRs were performed to evaluate the alternations in the regulation of the genes involved in drug efflux including CgCDR1, CgCDR2, CgSNQ2, and CgERG11. Gain-of-function (GOF) mutations in CgPDR1 alleles were determined by DNA sequencing. Cross-resistance to fluconazole, itraconazole, and voriconazole was observed in 2.5 % of the isolates. In the present study, six amino acid substitutions were identified in CgPdr1, among which W297R, T588A, and F575L were previously reported, whereas D243N, H576Y, and P915R are novel. CgCDR1 overexpression was observed in 57.1 % of resistant isolates. However, CgCDR2 was not co-expressed with CgCDR1. CgSNQ2 was upregulated in 71.4 % of the cases. CgERG11 overexpression does not seem to be associated with azole resistance, except for isolates that exhibited azole cross-resistance. The pattern of efflux pump gene upregulation was associated with GOF mutations observed in CgPDR1. These results showed that drug efflux mediated by adenosine-5-triphosphate (ATP)-binding cassette transporters, especially CgSNQ2 and CgCDR1, is the predominant mechanism of fluconazole resistance in Iranian isolates of C. glabrata. Since some novel GOF mutations were found here, this study also calls for research aimed at investigating other new GOF mutations to reveal the comprehensive understanding about efflux-mediated resistance to azole antifungal agents.     

Multigene phylogeny reveals a new species and novel records and hosts in the genus <Emphasis Type="Italic">Ramularia</Emphasis> from Iran

Ramularia is a species-rich genus in the order Capnodiales (Dothideomycetes, Ascomycota) that includes numerous phytopathogenic taxa, several of which are economically important plant pathogens. In this study, six isolates of Ramularia were recovered from leaf spot symptoms on six herbaceous and woody plants from Guilan, East and West Azarbaijan provinces in the north and northwest of Iran. The isolates were studied by a polyphasic approach involving morphological and cultural data, and multi-gene phylogeny (ITS, TEF1-α, ACT, HIS, RPB2 and GAPDH). The isolates were grouped in three species clades of the R. eucalypti species complex. Of these, R. mali is recorded for the first time in Asia and R. glennii represents a new record for the mycobiota of Iran. Ramularia taleshina on Alnus subcordata is described as a new species. Ramularia taleshina is phylogenetically related to R. mali, but they can be differentiated by morphological and cultural characters as well as molecular data. Acalypha australis, Ficus carica and Platanus sp. are reported as new hosts of R. glennii, and Prunus cerasus and Vitis vinifera as new hosts of R. mali.     

Variation of cassiicolin genes among Chinese isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Corynespora cassiicola is a species of fungus that is a plant pathogen of many agricultural crop plants, including severe target spot disease on cucumber. Cassiicolin is an important effector of pathogenicity of this fungus. In this study, we collected 141 Corynespora isolates from eighteen hosts, and the casscolin gene was detected in 82 C. cassiicola strains. The deduced protein sequences revealed that 72 isolates contained the Cas2 gene, two strains from Gynura bicolor harboured the Cas2.2 gene, and 59 isolates without a cassiicolin gene were classified as Cas0. Phylogenetic analyses was performed for the 141 isolates using four loci (ITS, ga4, caa5, and act1) and revealed two genetic clusters. Cluster A is composed of four subclades: subcluster A1 includes all Cas2 isolates plus 18 Cas0 strains, subcluster A2 includes the eight Cas5 isolates and one Cas0 isolate, and subclusters A3 and A4 contain Cas0 strains. Cluster B consists of 21 Cas0 isolates. Twenty-two C. cassiicola strains from different toxin classes showed varying degrees of virulence against cucumber. Cas0 or Cas2 strains induced diverse responses on cucumber, from no symptoms to symptoms of moderate or severe infection, but all Cas5 isolates exhibited avirulence on cucumber.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Molecular Genotyping of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> Species Complex

Background

The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures.

Methods

The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites.

Results

Upon the analysis of 182 strains of C. parapsilosis complex species, 10–17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21–32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value).The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization.

Conclusions

These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

     

Multi-approach analysis of the diversity in <Emphasis Type="Italic">Colletotrichum cliviae</Emphasis> sensu lato

Colletotrichum cliviae is a fungal species reported both as pathogen and endophyte with broad geographical distribution. Some purported isolates of this species have been assigned to different taxa, including Colletotrichum aracearum, Colletotrichum orchidearum and Colletotrichum. sichuanensis, for which a preliminary analysis of extensive multilocus (ACT, GAPDH, ITS, TUB2) data in this study revealed high sequence similarity with C. cliviae. We further reassessed the species delineation by using the coalescent method of the generalized mixed Yule-coalescent (GMYC) and Poisson Tree Processes (PTP). Single and multilocus gene trees strongly supported a C. cliviae s. lat. clade including the four species. This clade unfolded eight subclades grouped into three distinct lineages, but no monophyly of any of the four species. GMYC and PTP analyses confidently supported the evolutionary independence of these lineages. C. sichuanensis and C. cliviae, except one isolate, formed the largest lineage. The second lineage was made up of isolates named C. aracearum and some of C. orchidearum sharing the haplotype and the third lineage accommodated two isolates named C. cliviae and C. orchidearum. This finding suggests the synonymization of C. sichuanensis with C. cliviae whereas the taxonomic status of C. aracearum and C. orchidearum still needs clarification. This study lays great stress upon the use of comprehensive data for sequence-based characterisation of species in the C. cliviae s. lat. It also presents the first report of C. cliviae in tropical Africa and on citrus host.     

Conspecificity of <Emphasis Type="Italic">Endothia eugeniae</Emphasis> and <Emphasis Type="Italic">Cryphonectria cubensis</Emphasis>: a re-evaluation based on morphology and DNA sequence data

Cryphonectria cubensis and Endothia eugeniae are fungal pathogens of Eucalyptus and clove that were reduced to synonymy on the basis of results of cross-inoculation studies, isozyme analysis, cultural studies, and morphology. A previous phylogenetic study on Cryphonectria, based on sequence variation in the ITS region of the ribosomal RNA operon, also supported the conspecificity of C. cubensis and E. eugeniae, but was based on only one E. eugeniae isolate. New collections from clove in Brazil and Indonesia have become available, providing the opportunity to reconsider the conspecificity of C. cubensis and E. eugeniae. The occurrence of C. cubensis on clove was confirmed based on morphological comparisons and phylogenetic analyses of ribosomal DNA and β-tubulin gene sequence data. In addition to C. cubensis, other fungi morphologically similar to Cryphonectria species on the basis of their orange stromata were present on some clove specimens, but no isolates were available for these fungi. Furthermore, some isolates, for which no herbarium material exists, grouped separately from the C. cubensis clade and closer to the Cryphonectria clade. The presence of more than one closely related fungus on clove raises questions relating to the legitimacy of the synonymy of E. eugeniae and C. cubensis. Based on the presence of C. cubensis on the type specimen of E. eugeniae, we recognize the synonymy of the two fungi but provide evidence that other fungi, more closely related to Cryphonectria spp. than to C. cubensis, are present on clove.     

<Emphasis Type="Italic">Allophoma hayatii</Emphasis> sp. nov., an undescribed pathogenic fungus causing dieback of <Emphasis Type="Italic">Lantana camara</Emphasis> in Iran

Dieback and canker diseases are a major problem in ornamental shrubs and trees of Ahvaz, southwestern Iran. Symptomatic stems and branches were collected from two urban parks in the downtown regions of Ahvaz to identify the dieback-causing agents of Lantana camara. Accordingly, two isolates of a new species, Allophoma hayatii sp. nov., were obtained, which are described and illustrated. This species is identified based on morphological characters and analyses of nucleotide sequences of four regions, including internal transcribed spacer 1 and 2 and 5.8S nrDNA (ITS), partial large subunit 28S nrDNA (LSU-D1/D2), a partial sequence of the β-tubulin (tub2) and part of the RNA polymerase II (rpb2). The isolates of A. hayatii generated a well-supported clade in the trees constructed from multi-locus phylogenetic analysis, distinct from other previously known species of Allophoma. Pathogenicity of both isolates was verified by the inoculation of stem fragments of L. camara. These findings confirm A. hayatii as the causal agents of dieback and canker of L. camara in Ahvaz.     

<Emphasis Type="Italic">A2144G</Emphasis> Is the Main Mutation in the <Emphasis Type="Italic">23S</Emphasis> rRNA Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Associated with Clarithromycin Resistance

To detect point mutations A2115C, A2143G/C, and A2144G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.     

Identification of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. associated with fruit rot of <Emphasis Type="Italic">Capsicum annuum</Emphasis> in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.     

<Emphasis Type="Italic">Calcarisporium xylariicola</Emphasis> sp. nov. and introduction of <Emphasis Type="Italic">Calcarisporiaceae</Emphasis> fam. nov. in Hypocreales

During a survey of fungicolous fungi, a novel taxon from the surface of stroma of an unidentified Xylaria species was collected. Phylogenetic analyses showed that this taxon clustered with Calcarisporium sp. and C. arbuscula isolates, but was resolved as a distinct species. A detailed morphological examination coupled with phylogenetic analysis indicated that the taxon represented a new species. Calcarisporium xylariicola sp. nov. is thus introduced. The new taxon is characterized by short conidiophores with swollen bases and less length/width ratio of conidia that distinguish it from other Calcarisporium species. Calcarisporium is presently placed in Hypocreales genera, incertae sedis genus. Species in the genus are largely fungicolous, or occasionally caulicolous or foliicolous, and have hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiation. A phylogenetic analysis of combined SSU, ITS, LSU, TEF and RPB2 sequence data from Calcarisporium species and other taxa in Hypocreales indicate that Calcarisporium is a distinct lineage from other families. Therefore, a new family, Calcarisporiaceae, in Hypocreales is introduced.     

Comparative clustering and genotyping of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from broiler and turkey feces by using RAPD-PCR and ERIC-PCR analysis

To compare the genetic profiles of Campylobacter jejuni (C. jejuni) isolates of broiler and turkey reservoirs sampled in Semnan City, Iran, 60 C. jejuni isolates (30 from broilers and 30 from turkeys) were genotyped by RAPD-PCR- and ERIC-PCR-based methods. RAPD-PCR identified 6 genotypes and ERIC-PCR identified 21 genotypes among the 60 C. jejuni isolates. Both techniques were able to discriminate the C. jejuni isolates. Results demonstrated that one single genotype was identical to broiler and one single genotype was identical to turkey isolates at 83% similarity level in RAPD UPGMA clustering. Also, one single profile was identical to turkey isolates at 73% similarity level in ERIC-PCR clustering. The existence of high genetic similarity in some C. jejuni isolates from both hosts suggests the presence of some overlap between isolates from different sources and boosts the power of RAPD-PCR- and ERIC-PCR-based methods in discriminating C. jejuni isolates from various sources.     

Porcine and bovine <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 078 isolates demonstrate similar growth and toxigenic properties

Clostridioides (C.) difficile are found in cows, pigs and poultry suggesting that this pathogen is present and more importantly animals could act as a reservoir, via food or environment, of human C. difficile infection. Molecular methods together with phenotypical characterisation bring integrated and important tools to describe diversity and nature of bacteria including C. difficile. Moreover, similar or identical C. difficile types are found in different farm animals. This study aimed to phenotypically characterise C. difficile isolates belonging to ribotype 078 and to identify differences such as growth and toxicity between porcine and bovine isolates. C. difficile isolates were assessed for the growth behaviour (turbidimetry), metabolic potential (Biolog AN) and toxin production (ELISA method) in vitro. The concentration of released either toxin A (TcdA) or toxin B (TcdB) varied greatly between the isolates tested; however, it did not differ between the porcine and bovine ribotypes. Also, the TcdA/TcdB ratio of the isolates did not show a difference either. The most common metabolised substrates were pyruvic acid followed by α-ketobutyric acid. The results show that both porcine and bovine C. difficile RT 078 share similar phenotypical characteristics including growth and production of toxins. The findings may help understand the virulence of C. difficile RT 078 in porcine and bovine species.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.