2a Strain Research Articles

Comparative proteomics of Shigella flexneri 2a strain 301 using a rabbit ileal loop model reveals key proteins for bacterial adaptation in host niches.

Many studies focusing on changes in the host following Shigella spp invasion have been reported in recent years. However, the key factors required for the adaptation of these pathogens to host niches have usually been neglected. In this study, a comparative proteomic analysis was performed to examine changes in the protein expression profile of Shigella flexneri within the host using a rabbit ileal loop model to reveal proteins that are associated with pathogenic adaptation. The protein expression profiles of bacteria isolated from the ileum and colon were very similar, although they differed slightly from that of bacteria isolated from the cecum. When compared with the sample in vitro, the expressions of seven proteins were found to be upshifted in vivo (OmpA, YgiW, MglB, YfiD, MetK, TktA, and AhpF), while two proteins were down-regulated (ElaB and GlnH). The abundance of nine proteins changed in vivo, suggesting that these proteins may contribute to adaptation to the intestinal lumen.

Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity.

The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.

Genotype Distribution and Molecular Epidemiology of Hepatitis C Virus in Hubei, Central China.

BackgroundLittle is known about the molecular epidemiology of hepatitis C virus (HCV) infection in Central China.Methodology/Principal FindingsA total of 570 patients from Hubei Province in central China were enrolled. These patients were tested positive for HCV antibody prior to blood transfusion. Among them, 177 were characterized by partial NS5B and/or Core-E1 sequences and classified into five subtypes: 1b, 83.0% (147/177); 2a, 13.0% (23/177); 3b, 2.3% (4/177); 6a, 1.1% (2/177); 3a, 0.6% (1/177). Analysis of genotype-associated risk factors revealed that paid blood donation and transfusion before 1997 were strongly associated with subtypes 1b and 2a, while some subtype 2a cases were also found in individuals with high risk sexual behaviors; subtypes 3b, 6a, and 3a were detected only in intravenous drug users. Phylogeographic analyses based on the coalescent datasets demonstrated that 1b, 2a, 3b, and 6a were locally epidemic in Hubei Province. Among them, subtype 1b Hubei strains may have served as the origins of this subtype in China, and 2a and 3b Hubei strains may have descended from the northwest and southwest of China, respectively, while 6a Hubei strains may have been imported from the central south and southwest.Conclusion/SignificanceThe results suggest that the migration patterns of HCV in Hubei are complex and variable among different subtypes. Implementation of mandatory HCV screening before donation has significantly decreased the incidence of transfusion-associated HCV infection since 1997. More attention should be paid to intravenous drug use and unsafe sexual contact, which may have become new risk factors for HCV infection in Hubei Province.

Phenotypic and genotypic characterization of Brucella strains isolated from autochthonous livestock reveals the dominance of B. abortus biovar 3a in Nigeria

Brucellosis is a worldwide widespread zoonosis caused by bacteria of the genus Brucella. Control of this disease in a given area requires an understanding of the Brucella species circulating in livestock and humans. However, because of the difficulties intrinsic to Brucella isolation and typing, such data are scarce for resource-poor areas. The paucity of bacteriological data and the consequent imperfect epidemiological picture are particularly critical for Sahelian and Sub-Sahara African countries. Here, we report on the characterization of 34 isolates collected between 1976 and 2012 from cattle, sheep and horses in Nigeria. All isolates were identified as Brucella abortus by Bruce-ladder PCR and assigned to biovar 3 by conventional typing. Further analysis by enhanced AMOS-ERY PCR showed that all of them belonged to the 3a sub-biovar, and MLVA analysis grouped them in a cluster clearly distinct from that formed by European B. abortus biovar 3b strains. Nevertheless, MLVA detected heterogeneity within the Nigerian biovar 3a strains. The close genetic profiles of the isolates from cattle, sheep and horses, suggest that, at least in some parts of Nigeria, biovar 3a circulates among animal species that are not the preferential hosts of B. abortus. Consistent with previous genetic analyses of 7 strains from Ivory Cost, Gambia and Togo, the analysis of these 34 Nigerian strains supports the hypothesis that the B. abortus biovar 3a lineage is dominant in West African countries.

Detection of the NS3 Q80K polymorphism by Sanger and deep sequencing in hepatitis C virus genotype 1a strains in the UK

The Q80K polymorphism in the hepatitis C virus (HCV) NS3 enzyme reduces susceptibility to simeprevir and other novel protease inhibitors. The aims of this study were to determine the prevalence of Q80K in treatment-naïve HCV-1a carriers in the North West region (NW) and South East region (SE) of England, investigate the occurrence of Q80K as a minority variant, and characterize viral phylogeny. Plasma samples from subjects who were naïve to anti-HCV therapy were subjected to conventional (Sanger) and deep (Illumina-Miseq, 1% interpretative cut-off) sequencing of NS3. Q80K occurred in 44 of 238 subjects (18.5%, 95% CI 13.6–23.4%), including 19 of 70 (27.1%) in the NW and 25 of 168 (14.9%) in the SE (p 0.0425), with no difference in HCV RNA load or human immunodeficiency virus (HIV) status. Q80K frequencies in reads of samples subjected to Illumina sequencing were >40% in all cases. Among subjects with Q80K, five of 44 (11.4%) showed one additional major resistance-associated mutation in NS3, detected at frequencies of >10% (V36L and V55A) or <10% (V36M). Phylogenetic analyses identified the two recognized HCV-1a lineages with (clade I) and without (clade II) Q80K. Overall, 148 of 238 (62.2%) sequences occurred within regional or inter-regional clusters, each comprising 3–20 sequences. There was no unique clustering of English sequences relative to strains from continental Europe and North America. In conclusion, Q80K was found at a high prevalence among treatment-naïve HCV-1a carriers in England, and was reliably detected by conventional sequencing, with no increased detection by deep sequencing. English sequences were highly interspersed with sequences from elsewhere in Europe (clade II) and North America (clade I), and their phylogeny was consistent with multiple introductions from different areas.

Analysis of iron acquisition and storage-related genes in clinical and non-clinical strains of Yersinia enterocolitica biovar 1A.

Possession of mechanisms for iron acquisition and its storage enhances the ability of the bacteria to survive in the iron-limiting environment of the host. In this study, 81 strains of Yersinia enterocolitica biovar 1A isolated from various clinical (n=51) and non-clinical (n=30) sources were investigated for the presence of the genes related to iron acquisition and storage. Important genes which were present in more than 85% of the strains included hasA, foxA, bfr, bfd, ftnA, and hmsT as well as the fhuCDB, fepBDGCfesfepA, feoAB, yfuABCD, hemPRSTUV, and hmsHFRS gene clusters. Majority of these genes is being reported for the first time in biovar 1A strains and showed significant homology with genes present in the known pathogenic biovars of Y.enterocolitica. However, no significant difference was observed in the distribution of iron acquisition and storage-related genes among clinical and non-clinical biovar 1A strains. Thus, it may be suggested that the presence of iron acquisition and storage-related genes per se might not be responsible for the supposedly better ability of clinical biovar 1A strains to cause infections in humans. However, in the backdrop of this data, the need to undertake functional studies are highly recommended.

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.

Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008.It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein.Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains.The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations.Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

Prevalence of hepatitis C virus (HCV) variants resistant to NS5A inhibitors in naïve patients infected with HCV genotype 1 in Tunisia.

BackgroundHepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibitors have been recently developed to inhibit NS5A activities and have been approved for the treatment of HCV infection. However the drawback of these direct acting antivirals (DAAs) is the emergence of resistance mutations. The prevalence of such mutations conferring resistance to HCV-NS5A inhibitors before treatment has not been investigated so far in the Tunisian population. The aim of this study was to detect HCV variants resistant to HCV-NS5A inhibitors in hepatitis C patients infected with HCV genotype 1 before any treatment with NS5A inhibitors.MethodsAmplification and direct sequencing of the HCV NS5A region was carried out on 112 samples from 149 untreated patients.ResultsIn genotype 1a strains, amino acid substitutions conferring resistance to NS5A inhibitors (M28V) were detected in 1/7 (14.2 %) HCV NS5A sequences analyzed. In genotype 1b, resistance mutations in the NS5A region (R30Q; L31M; P58S and Y93H) were observed in 17/105 (16.2 %) HCV NS5A sequences analyzed. R30Q and Y93H (n = 6; 5.7 %) predominated over P58S (n = 4; 3.8 %) and L31M (n = 3; 2.8 %).ConclusionsMutations conferring resistance to HCV NS5A inhibitors are frequent in treatment-naïve Tunisian patients infected with HCV genotype 1b. Their influence in the context of DAA therapies has not been fully investigated and should be taken into consideration.

Draft Genome Sequence of 24570, the Type Strain of Shigella flexneri.

Shigella flexneri is a diarrheal pathogen that causes a large disease burden worldwide. We sequenced the genome of the publicly available type strain (S. flexneri 2a strain 24570) of this bacterial species to increase its utility as a reference. We present genome assembly results and comparisons with other reference strains.

Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/Ribavirin Therapy in Chronic Hepatitis C.

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.

Mouse polyomavirus tropism alters magnitude and functionality of virus-specific CD8 T cells (VIR6P.1173)

Abstract Mouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. Human JC polyomavirus variants associated with progressive multifocal leukoencephalopathy have discrete amino acid mutations in VP1, the major capsid protein, that affects tropism for glial cells. A glutamic acid (E) to glycine (G) difference at position 92 of VP1, shifts the profile to tumors induced by MPyV from epithelial to mesenchymal cell origin. Here, we asked if this shift in tropism affects the MPyV-specific CD8 T cell response, which controls MPyV infection/tumorigenesis. We found that the A2 (92E-VP1) and RA (92G-VP1) MPyV strains differ sharply in MPyV-specific CD8 T cell responses. A2 virus generates a robust, high magnitude virus-specific CD8 T cell response, whereas RA virus elicits a markedly smaller anti-MPyV CD8 T cell response. Infection by an A2 92G-VP1 mutant virus recapitulates the antiviral CD8 T cell response seen with RA virus. Notably, the antiviral CD8 T cell recall response during persistent infection by this A2 mutant significantly exceeded that of A2 virus. Using immunization with peptide-loaded dendritic cells in the setting of bystander inflammation by A2 or the mutant virus, we found that A2 virus stimulates a higher magnitude MPyV-specific CD8 T cell response; this difference is negated in DC-deficient Flt3-/- mice. Together, these data indicate that viral tropism for DCs is a major determinant of the quantity and quality of the anti-MPyV CD8 T cell response.

The effect of quorum sensing system for growth competitiveness on Shigella flexneri.

Quorum sensing (QS) regulates the onset of bacterial social responses related to cell density. Comparison between the gene sequences of all components of QS system of Escherichia coli and Shigella strains, shows that the QS system is generally lost or mutated in Shigella. Since AI-2 is produced and processed by the lsr operon, we analyzed the potential function of the lsr operon. We first detected AI-2 in Shigella flexneri 2a strain 301 through the reporter bacteria Vibrio harveyi BB170, indicating that S. flexneri can produce AI-2. Then, the lsr operon of E. coli MG1655 was cloned into S. flexneri using the Golden Gate method. Colony counting experiments showed that the QS system recovery strain had growth advantage over the wild-type strain when they were mixed and cultured. The preliminary comparative proteomics analysis showed that the lsr operon could be expressed and the abundance of stress response proteins also changed when the QS system was introduced into S. flexneri.

Proteomic analysis of Yersinia enterocolitica biovar 1A under iron-rich and iron-poor conditions indicate existence of efficiently regulated mechanisms of iron homeostasis

The pathogenicity of Yersinia enterocolitica biovar 1A strains is controversial as these lack most of the known virulence factors. Acquisition of iron and presence of well-regulated iron homeostasis in bacteria represents an important virulence trait. Differential abundance of proteins was examined under iron-rich and iron-poor conditions in a clinical Y. enterocolitica biovar 1A strain IP27407. Whole cell protein profiles were analysed by 2D gel electrophoresis (2D-GE). Following statistical and MALDI-TOF MS analyses, 28 differentially abundant proteins were identified. Significant iron-responsive changes were observed in the proteins involved in iron acquisition or storage namely, hemin receptor (HemR), periplasmic Fe2+ transport protein (Tpd), periplasmic chelated iron-binding protein (YfeA) and bacterioferritin (Bfr). Quantitative real-time PCR (qRT-PCR) of eight mRNA transcripts revalidated the differential protein abundance. In silico analysis of iron homeostasis mediated by the bacterioferritin and bacterioferritin-associated ferredoxin (Bfr–Bfd) complex suggested two pathways for the release of reserve iron which might be operating under conditions of different iron availability. The study, for the first time, showed the existence of highly competent iron homeostasis mechanisms in Y. enterocolitica biovar 1A and identified the key proteins involved thereof. Such mechanisms might have implications for the pathogenicity of Y. enterocolitica biovar 1A strains. Biological significanceAlthough, a few studies have identified the differentially abundant bacterial proteins in response to iron starvation, little information is available in this regard for Y. enterocolitica (especially, the biovar 1A strains). In the present study, differential abundance of several proteins was identified under iron-rich and iron-poor conditions by 2D-GE and MALDI-TOF/MS analysis. These included proteins which may not only be directly implicated in iron acquisition or storage but also play crucial role in cellular metabolism. Given the absence of most known virulence factors in Y. enterocolitica biovar 1A strains, demonstration of well-regulated mechanisms for efficient iron homeostasis constitutes an important observation. The proteins, as identified in the present study, provide useful insights to further unravel the potential pathogenicity of the biovar 1A strains.

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage, SfMu, of Shigella flexneri.

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri.

Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle.

Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing. The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.

Kinetic modeling of thermal inactivation of antimicrobial peptides produced by Lactobacillus sakei subsp. sakei 2a

The combination of bacteriocins with heat may facilitate the application of mild thermal treatments in food processing. In this context, the knowledge on thermal inactivation kinetics of antimicrobial peptides is important to allow their adequate use as biopreservatives in the food industry. Lactobacillus sakei subsp. sakei 2a (Lb. sakei 2a), a strain isolated from Brazilian sausage, produces bacteriocins active against Listeria monocytogenes. In this work, the kinetics of thermal inactivation of antimicrobial peptides produced by Lb. sakei 2a was evaluated. The bacteriocin was heated in sealed tubes in the range of 90–120°C for over 180min and residual antimicrobial activity was determined using L. monocytogenes Scott A as an indicator microorganism. Applicability of various inactivation models available in the literature was critically evaluated. The Weibull model provided the best description for inactivation kinetics over the selected temperature range. Inactivation reliable time (tR) ranged from 291 to 65min, with a z value of 46°C and activation energy of 220kJmol−1. The antimicrobial activity of the bacteriocin decreased when exposed to a temperature of 120°C for 180min but was not fully suppressed, which shows high thermal stability of bacteriocins produced by the Lb. sakei 2a strain. The results indicate that this bacteriocin can be used under pasteurization conditions, maintaining part of its antimicrobial activity after the thermal processing.

Complete Genome Sequence of the Human Herpesvirus 6A Strain AJ from Africa Resembles Strain GS from North America.

The genome sequence of human herpesvirus 6A (HHV-6A) strain AJ was determined in a comparison of target enrichment and long-range PCR using next-generation sequencing methodologies. The analyses show 85 predicted open reading frames (ORFs), conservation with sequenced HHV-6A reference strain U1102, and closest identity to the recently determined GS strain, despite different geographic origins (United States and Gambia).

Bioprospecting of pseudomonas aeruginosa for their potential to produce siderophore, process optimization and evaluation of its bioactivity.

Siderophores produced by Pseudomonas spp . play a vital role in controlling several plant diseases. In this study, two siderophore producing bacterial strains were isolated from waste water sample and labeled them as RM1 and RM2 . Among these two isolates, RM1 strain was found to produce more siderophore and therefore selected for further study. 16s rRNA analysis and biochemical characterization as described in the Bergey’s manual of determinative bacteriology, identified the RM1 strain as Pseudomonas aeruginosa 6A (bc4). Siderophore production was determined by more sensitive and reliable Chrome azurol- S (CAS) method. Chemical and spectrophotometric assays showed that P. aeruginosa 6A strain produced 99% siderophore units. Two siderophores, hydroxamate (Pyoverdine) and phenol catecholate (pyochelin) were mainly found to be produced by this strain. Different parameters such as temperature, pH, carbon and nitrogen sources were optimized for maximum production of siderophores. Maximum production (95.65%) was observed on succinate medium, pH 7.0 and at 40⁰c temperature. Several amino acids were used as carbon and nitrogen sources. Among them serine resulted in a maximum production of siderophore (99.80% unit). The Pseudomonas spp . were also tested as seed inoculants and found to be very effective in seed germination and plant growth promotion of Glycine max JS-335 plant under pot culture conditions. It also exhibits antifungal activity against Aspergilus spp . In summary, the present study has identified a novel siderophore producing bacterial strain, which can also be used as an antifungal agent.

The first pathogenic Yersinia enterocolitica bioserotype 4/O:3 strain isolated from a hunted wild boar (Sus scrofa) in Poland.

The objective of this study was to identify the bioserotypes and virulence markers of Yersinia enterocolitica strains isolated from wild boars in Poland. Bacteriological examination of 302 rectal swabs from 151 wild boars resulted in the isolation of 40 Y. enterocolitica strains. The majority of the examined strains (n = 30), belonged to bioserotype 1A/NI. The presence of individual Y. enterocolitica strains belonging to bioserotypes 1B/NI (3), 1A/O:8 (2), 1A/O:27 (2), 2/NI (1), 2/O:9 (1) and 4/O:3 (1) was also demonstrated. Amplicons corresponding to ail and ystA genes were observed only in one Y. enterocolitica strain--bioserotype 4/O:3. The ail and ystB gene amplicons were noted in 11 Y. enterocolitica biotype 1A strains, although single amplicons of ystB gene were found in 28 of the tested samples. In four out of eight cases when two Y. enterocolitica strains were isolated from the same animal, the strains differed in biotype, serotype or virulence markers. The European population of wild boars continues to grow and spread to new areas, therefore, wild boars harbouring potentially pathogenic Y. enterocolitica 4/O:3 strains pose a challenge to public health.