Potential Virulence Factors Research Articles

Deteksi Molekuler Gen Penyandi Protein Virb11 pada Brucella abortus Isolat Lokal Asal Pinrang, NTT dan Strain Vaksin

Brucellosis in cattle is a disease caused by Brucella abortus due to the reduction in livestock population caused by abortion, stillbirth, weak birth, infertility and sterility. Brucella abortus has several potential virulence factors, i.e. virB11 gene that encodes VirB11 protein is an important virulence factor acts as an ATPase for assembling organelles when the bacteria replicate, helping to complete the bacterial cycle and agress to another cells. The aim of this study are to re-identification Brucella abortus and detect virB11 gene as encoding of B. abortus VirB11 protein in local isolates from Pinrang, NTT, strain vaccines S19 and RB51. The isolates Brucella abortus were re-cultured in Brucella agar base and re-identification is followed by microscopic with Gram staining and biochemical tested with urease, citrat, indol and TSIA test. virB11gene was detected with PCR method. The PCR result showed virB11 gene have DNA band 720 bp. virB11 gene are present in local isolates from Pinrang, NTT, strain vaccines S19 and RB51.

Methicillin Resistant Staphylococcus aureus in Health Care Workers of a Tertiary Care Infectious Disease Hospital in Nepal

Objectives: Acquisition of mecA gene in infectious strains of Methicillin resistant Staphylococcus aureus (MRSA) are considered as one of the potential virulence factors that enables the host bacteria to carry out several nosocomial and community-acquired infections. The main aim of this study was to determine the prevalence of MRSA, their antibiogram and mecA gene in the bacterial isolates obtained from the asymptomatic healthcare workers (HCWs) working in Sukraraj Tropical and Infectious Disease Hospital (STIDH), Kathmandu Nepal.
 Methods: This prospective cross-sectional study involved the collection of nasal and hands swab of 125 randomly selected HCWs from December 2019 to February 2020. Conventional microbiological methods were used to isolate and identify S. aureus. Antimicrobial susceptibility testing was done by modified Kirby Bauer disc diffusion method. MRSA was confirmed by using cefoxitin disc. Detection of mecA gene in the chromosome which was extracted by Phenol: Chloroform: isoamyl alcohol DNA extraction method, amplified by using PCR and visualized by running agarose gel electrophoresis.
 Results: The overall and MRSA carriage rate among the HCWs was found to be 28% (35/125) and 10.4% (13/125) respectively. S. aureus carriage rate was highest among sanitation staffs (34.2%) followed by pharmacy staffs (33.3%), laboratory personnel (18.8%), doctors (9.1%) and nurses (7.5%). Similarly, 34.2% (13/38) of the S. aureus isolates were resistant to methicillin, 31.6% (12/38) were inducible-clindamycin resistant and 63.2% (24/38) of them were multi-drug resistant (MDR). All the 13 MRSA isolates harbored the mecA gene.
 Conclusions: Carriage rate of MRSA among HCWs was high and alarming, indicating the prompt need of intervention measures to curb the growth and spread of resistant isolates in the hospital settings. Effective surveillance (of infectious diseases) and establishment of advanced diagnostic facilities can assist in estimating the actual burden of the MRSA which in turn helps to formulate and implement the appropriate policies and infection-control programs to address the increasing antimicrobial resistance in the country.

THE H. PYLORI-RELATED VIRULENCE FACTOR CAGA INFLUENCES THE EXPRESSION OF CHEMOKINES CXCL10, CCL17, CCL20, CCL22, AND THEIR RECEPTORS BY PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PEPTIC ULCER PATIENTS

During the Helicobacter pylori (HP) infection, the infiltration of the leukocytes into stomach mucosa is directed by locally produced chemokines that play a decisive role in infection outcome. The CagA is the most potent virulence factor of HP, so that the infection with CagA + strains is associated with more severe complications than infection with CagA - HP. The aim was to determine the expression of chemokines CXCL10, CCL17, CCL20 and CCL22, and their receptors by CagA + HP- and CagA - HP-derived crude extract (HP-CE)-stimulated peripheral blood mononuclear cells (PBMCs) from peptic ulcer (PU) patients. The serum and the PBMCs were collected from 20 HP-infected PU patients, 20 HP-infected asymptomatic subjects (HIA) and 20 non-infected healthy subjects (NHS). The PBMCs were cultured in absence of stimulator or with 10 µg CagA + HP crude extract (CagA + CE), 10 µg CagA - HP crude extract (CagA - CE). Chemokines and receptors were measured by ELISA and real time-PCR respectively. In PU patients, the production of chemokines CXCL10, CCL17, CCL20 and CCL22, and the expression of chemokine receptors CXCR3, CCR4 and CCR6 by CagA + CE-induced PBMCs were significantly higher than non-stimulated and CagA - CE stimulated cultures. The CXCL10 production by CagA + CE stimulated PBMCs from HIA subjects was significantly higher than the equal cultures from PU and NHS groups. The CCL17 and the CCL20 production by non-stimulated, CagA + CE stimulated, and CagA - CE stimulated PBMCs from PU subjects were significantly higher than the equal cultures from NHS and HIA groups. The CCL22 production by non-stimulated, CagA + CE stimulated and CagA - CE stimulated PBMCs from NHS group were significantly higher than the equal cultures from HIA and PU groups. The CagA + CE stimulated PBMCs from HIA subjects expressed lower amounts of CCR6 in comparison with CagA + CE stimulated PBMCs from NHS and PU groups. The serum levels CXCL10 and CCL20 in PU and HIA groups were significantly higher than NHS subjects. NHS and HIA groups displayed higher serum levels of CCL22 in comparison with PU patients. Results indicated that the CagA status of bacterium influence the expression of chemokines and receptors by HP-CE stimulated PBMCs from PU patients.

Identification of protein targets in red complex organisms binding with resveratrol.

Periodontitis is attributed to the dental biofilm formation. Red complex organisms are a group of organisms linked with periodontal diseases. Therefore, it is of interest to identify potential targets from the red complex organisms to bind with the herbal compound resveratrol (E - 5 - (4 - hydroxy styryl) benzene 1,3 diol). We report a list of potential proteins having optimal drug like binding features with the herbal agent Resveratrol for further consideration. We used the STITCH v.5 pipeline VICMPred and VirulentPred tools to identify such targets as potential virulent factors in the red complex organisms. We considered the strains of Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405 and Tannerella forsythia ATCC 43037 in the red complex pathogens for this analysis. Protein targets in the red complex organisms with optimal binding features with the herbal compound resveratrol were thus identified and reported for further consideration.

Porphyromonas gingivalis-Lipopolysaccharide induces cytokines and enzymes of the mouse cementoblasts

Lipopolysaccharide is a potent virulence factor of Porphyromonas gingivalis and has been implicated predominant pathogen in the development and progression of periodontal diseases. The aim of this study was to determine the effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on cementoblasts.Cementoblast (OCCM-30) were evaluated proliferation using real-time cell analyzer. In addition, total RNA was isolated at 8, 16, 24 and 72 h from 1000 ng/mL Pg-LPS treated OCCM-30 cells and mRNA expressions of pro/anti-inflammatory cytokine mediators, extracellular matrix enzymes and their tissue inhibitors and of oxidative stress enzymes were studied by real-time polymerase chain reaction. Proliferation analysis indicated that Pg-LPS slightly decreased proliferation of OCCM-30. Pg-LPS had a time-dependent impact on the expression of cytokines and enzymes. There was statistically significant up-regulation of IL-1β and IL-10 in response to Pg-LPS at 8, 16, 24, 72 h but IL-6 expression was reduced compared to control at 8 h. While IL-8 and IL-17 expressions were determined higher than control group at 16 and 24 h, their expressions were decreased compared to control groups at 72 h (p < 0.01). While MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2 expressions increased, MMP-9 expression reduced at time-points. Also, a time-dependent up-regulation in mRNA levels for oxidative stress enzymes was detected. These results indicated that up-regulation in the transcripts of inflammation-associated cytokines and degradation enzymes were noted in the cementoblasts exposed to Pg-LPS. Cementoblasts infected with the virulence factors of periodontopathogens might also involve to the induction of inflammation and degradation of the periodontal tissues.

Prediction of Burkholderia pseudomallei DsbA substrates identifies potential virulence factors and vaccine targets.

Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.

Integrated Co-functional Network Analysis on the Resistance and Virulence Features in Acinetobacter baumannii.

Acinetobacter baumannii is one of the most troublesome bacterial pathogens that pose major public health threats due to its rapidly increasing drug resistance property. It is not only derived from clinic setting but also emerges from aquaculture as a fish pathogen, which could pass the resistant genes in the food chain. Understanding the mechanism of antibiotic resistance development and pathogenesis will aid our battle with the infections caused by A. baumannii. In this study, we constructed a co-functional network by integrating multiple sources of data from A. baumannii and then used the k-shell decomposition to analyze the co-functional network. We found that genes involving in basic cellular physiological function, including genes for antibiotic resistance, tended to have high k-shell values and locate in the internal layer of our network. In contrast, the non-essential genes, such as genes associated with virulence, tended to have lower k-shell values and locate in the external layer. This finding allows us to fish out the potential antibiotic resistance factors and virulence factors. In addition, we constructed an online platform ABviresDB (https://acba.shinyapps.io/ABviresDB/) for visualization of the network and features of each gene in A. baumannii. The network analysis in this study will not only aid the study on A. baumannii but also could be referenced for the research of antibiotic resistance and pathogenesis in other bacteria.

Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

Protein targets in the red complex organisms binding with an herbal compound silymarin.

Periodontitis is attributed to the dental biofilm formation caused by various microbial changes that occurs in the biofilm. Red complex organisms are a group of organisms linked with periodontal diseases. Therefore, it is of interest to identify potential targets from the red complex organisms to bind with the herbal compound silymarin. We report a list of potential proteins having optimal drug like binding features with the herbal agent silymarin for further consideration. We used the STITCH v.5 pipeline using VICMPred and VirulentPred tools to identify such targets as potential virulent factors in the red complex organisms. We considered the strains of Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405 and Tannerella forsythia ATCC 43037 in the red complex pathogens for this analysis. Protein targets in the red complex organisms with optimal binding features with the herbal compound silymarin were thus identified and reported for further consideration.

Initial safety evaluation of Enterococcus faecium LBB.E81

Due to their antimicrobial activity Enterococcus faecium strains have a growing record of use as beneficial adjunct cultures in animal nutrition or components of probiotic preparations for humans. However, safety concerns accompany the application of enterococci, as clinical strains associated with infection have been isolated, especially antibiotic resistant cultures. With the increasing availability of whole genome sequencing (WGS), valuable safety data can be extracted in the early phase of development of the probiotic dossier of an E. faecium strain. Here we report the results of initial safety assessment of E. faecium LBB.E81 by selecting accessible commercial antibiotic susceptibility test and WGS. Testing against a panel of antibiotic substances showed that E. faecium LBB.E81 was susceptible to all antimicrobials with the exception of its intrinsic, non-transferable resistance to nitrofurantoin. The identified antibiotic resistance genes of E. faecium LBB.E81 aac(6')-Ii, msrC and efmA belonged to a group of intrinsic non-transferable factors contributing to the lower susceptibility of the E. faecium species to aminoglycoside and macrolide antibiotics but with minimal inhibitory concentrations for E. faecium LBB.E81 below the threshold values determined for resistant strains. The only potential virulence factor genes found in the genome of E. faecium LBB.E81 were a defective acm (collagen-binding adhesin) and efaA fm (antigen A – like protein) without any evidence for its involvement in pathogenicity. Other hospital associated virulence factor genes of E. faecium such as IS16, hyl, asa-type genes, cyl; gelE and fsr, sprE and esp(fm) were found to be absent in E. faecium LBB.E81.

Compare Catalase Activity Between Aspergillus flavus and A. fumigatus, Isolated from Clinical and Environmental Specimens

Background: Catalase enzyme is a potential virulence factor for Aspergillus species. Objectives: This study aimed to evaluate the catalase activity in conidium and mycelium of Aspergillus flavus and A. fumigatus isolated from environmental and clinical samples. Methods: Forty Aspergillus species (20 A. fumigatus and 20 A. flavus) were evaluated. Species were identified using the macroscopic and microscopic criteria of the isolates on culture media and the PCR-RFLP method, using the MwoI enzyme. The activity of the enzyme was evaluated using the Amplex red catalase assay kit. The Shapiro Wilk, Kolmogorov-Smirnov, Mann-Whitney, and Wilcoxon Signed Rank tests were used to analyze the data. Results: The mean conidial and mycelial catalase activities in A. flavus clinical and environmental isolates were 58.10, 57.80 mU/mL, and 1328.30, 531.60 mU/mL, respectively. In A. fumigatus clinical and environmental isolates, the activities were 61.10 and 61.40 mU/mL, and 1248.90 and 722.90 mU/mL, respectively. A significant difference was found between conidial and mycelial catalase activity in Aspergillus species (P = 0.01). The mycelial catalase activity of Aspergillus species isolated from clinical samples was higher than the environmental ones (A. flavus P = 0.01 and A. fumigatus P = 0.04). Conclusions: The mycelial catalase activity was higher than conidia. Clinical isolates of A. flavus had similar mycelium activity to A. fumigatus. By using the information provided in the present study, the severity of aspergillosis can be predicted, which paves the way for identifying new antifungal agents.

Molecular characterisation of csgA gene among ESBL strains of A. baumannii and targeting with essential oil compounds from Azadirachta indica

ObjectivesA. baumannii is considered as a “red alert” nosocomial human pathogen and exhibits an extensive antibiotic resistance spectrum. The biofilm formation mediated by the csgA is a potent virulence factor in A. baumannii and targeting the same would be of a novel strategy to control A. baumannii infections. The aim of the present study is thus to evaluate the anti-biofilm activity of essential bio-compounds from Azadirachta indica against the ESBL producing strains of A. baumannii by in-vitro and in-silico studies. MethodsBiofilm formation by Semi-quantitative adherence assay was performed for the 73 strains of ESBL producing A. baumannii. Genomic DNA was extracted and molecular characterization of csgA gene was done by PCR amplification with further sequencing. In-vitro anti biofilm assay from crude extract of A.indica was performed which was then followed by the in-silico docking involving retrieval of csgA protein and ligand optimisation, molinspiration assessment on drug likeliness, docking simulations and visualisations. ResultsBiofilm assay showed 58.9%, 31.5% and 0.09% as high grade, low grade and non-biofilm formers respectively. 20.54% (15/73) of the screened genomes showed positive amplicons for the csgA gene associated with biofilm formation among the ESBL producing strains of A. baumannii. All the ceftazidime, cefipime and cefotaxime resistant strains showed the presence of csgA gene (100%; 15/15), followed by 46.6% (7/15) resistant isolates for ceftriaxone. In-vitro crystal violet viability assay showed MBEC50 and MBEC90 at a concentration of 20 µl and 40 µl respectively. In-silico assessments on the essential oil compounds from neem showed imidazole to exhibit the highest interaction with least docking energy and high number of hydrogen bonds. ConclusionThe current study emphasises thatimidazole from A.indica to be a promising candidate for targeting the csgA mediated biofilm formation in ESBL strains of A. baumannii. However, further in-vivo studies have to be implemented for the experimental validation of the same.

Anti-Proliferative and Anti-Biofilm Potentials of Bacteriocins Produced by Non-Pathogenic Enterococcus sp.

The incidence of cancer is increasing worldwide; likewise, the emergence of antibiotic-resistant biofilm-forming pathogens has led to a tremendous increase in morbidity and mortality. This study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus sp. with a focus on their anti-biofilm and anticancer activities. Three of 79 Enterococcus isolates (FM43, FM65, FM50) were identified as producers of broad-spectrum bioactive molecules and were molecularly characterized as Enterococcus faecium by 16S rRNA sequencing. Phenotypic and genotypic screening for potential virulence factors revealed no factors known to promote pathogenicity. Treatment with proteinase K resulted in diminished antimicrobial activity; PCR-based screening for bacteriocin genes suggested the presence of both entA and entB genes that encode enterocins A and B, respectively. Maximum antimicrobial activity was detected during the early stationary phase, while activity disappeared after 24h in culture. Bacteriocins from these isolates were stable at high temperatures and over a wide range of pH. Interestingly, crude supernatants of Ent. faecium FM43 and Ent. faecium FM50 resulted in significant destruction (80% and 48%, respectively; P < 0.05) of Streptococcus mutans ATCC 25175-associated preformed biofilms. Moreover, in vitro cytotoxicity assays revealed that extracts from Ent. faecium isolates FM43, FM65, and FM50 inhibited Caco-2 cell proliferation by 76.9%, 70%, and 85.3%, respectively. Taken together, the multifunctional capabilities of the microbial-derived proteins identified in our study suggest potentially important roles as alternative treatments for biofilm-associated infections and cancer.

Comparative proteomic analysis reveals novel potential virulence factors of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic pathogen. An increasing number of reports indicate that it has caused substantial economic losses in the aquaculture industry, in addition to threatening human health. However, little is known about its pathogenesis. Exploration of new virulence factors of A. veronii would be helpful for further understanding its pathogenesis. Hence, we comparatively analyzed the proteomes of virulent, attenuated, and avirulent strains of A. veronii using tandem mass tag (TMT) protein labeling and found numerous proteins either up- or downregulated in the virulent strain. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that these differentially expressed proteins (DEPs) were involved mainly in pathways associated with bacterial chemotaxis and microbial metabolism in diverse environments. Furthermore, the expression levels of lysine decarboxylase, endoribonuclease, maltoporin, pullulanase, and aerolysin were positively correlated with the virulence of the strains, suggesting that their function may be closely related to the virulence of A. veronii. The results of qRT-PCR and multiple reaction monitoring for some DEPs were consistent with the results of TMT protein labeling. These results suggest that these DEPs may be novel potential virulence factors and will help to further understand the pathogenesis of A. veronii.

Whole genome sequencing and de novo assembly of three virulent Indian isolates of Leptospira.

Leptospirosis is a re-emerging bacterial zoonosis caused by pathogenic Leptospira, with a worldwide distribution and becoming a major public health concern. Prophylaxis of this disease is difficult due to several factors such as non-specific variable clinical manifestation, presence of a large number of serovar, species and asymptomatic reservoir hosts, lack of proper diagnostics and vaccines. Despite its global importance and severity of the disease, knowledge about the molecular mechanism of pathogenesis and evolution of pathogenic species of Leptospira remains limited. In this study, we sequenced and analyzed three highly pathogenic species of Indian isolates of Leptospira (interrogans, santarosai, and kirschneri). Additionally, we identified some virulence-related and CRISPR-Cas genes. The virulent analysis showed 232 potential virulence factors encoding proteins in L. interrogans strain Salinem and L. santarosai strain M-4 genome. While the genome of L. kirschneri strain Wumalasena was predicted to encode 198 virulence factor proteins. The variant calling analysis revealed 1151, 19,786, and 22,996 single nucleotide polymorphisms (SNPs) for L. interrogans strain Salinem, L. kirschneri strain Wumalasena and L. santarosai strain M-4, respectively, with a maximum of 5315 missense and 12,221 synonymous mutations for L. santarosai strain M-4. The structural analyses of genomes indicated potential evidence of inversions and structural rearrangment in all three genomes. The availability of these genome sequences and in silico analysis of Leptospira will provide a basis for a deeper understanding of their molecular diversity and pathogenesis mechanism, and further pave a way towards proper management of the disease.

Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes

ABSTRACT Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a host-specific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.

Separation of Glycosylated OmpA-Like Proteins from Porphyromonas gingivalis and Tannerella forsythia.

OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.

Characterization of Salmonella spp. isolated from chickens in Central China

BackgroundSalmonella is an important zoonotic pathogen, and chickens are one of its main hosts. Every year, Salmonella infections pose a serious threat to the poultry industry in developing countries, especially China. In this study, a total of 84 Salmonella isolates recovered from sick and healthy-looking chickens in central China were characterized by serotyping, MLST-based strain typing, presence of potential virulence factors, and antimicrobial resistance profiles.ResultData showed that the main serotypes of Salmonella isolates in central China were Salmonella enterica serovar Gallinarum biovar Pullorum, Salmonella enterica serovar Gallinarum biovar Gallinarum, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium, and among them, S. Pullorum was the dominant type in both sick and healthy-looking chickens, accounting for 43.9 and 46.5%, respectively, while S. Enteritidis was only found in healthy-looking chickens. All isolates exhibited higher resistance rates to ampicillin (97.6%), tetracycline (58.3%) and colistin (51.2%), and among these isolates, 49.5% were resistant to more than three drugs in different combinations. S. Enteritidis was the most severe multidrug-resistant serotype, which showed higher resistance rates to colistin, meropenem and ciprofloxacin. Multilocus sequence typing (MLST) revealed that S. Gallinarum and S. Enteritidis isolates were clustered in clade 1, which belonged to two and one STs, respectively. All S. Typhimurium isolates were clustered in clade 3, and belonged to three STs. However, S. Pullorum were distributed in three clades, which belonged to 7 STs. Twenty-seven virulence-associated genes were detected, and expected cdtB, which was absent in all the isolates, the other 26 genes were conserved in the closely related Salmonella serogroup D (S. Enteritidis, S. Pullorum, and S. Gallinarum).ConclusionSalmonella serogroup D was the major subgroup, and S. Pullorum was the most common type in sick and healthy-looking chickens in central China. Drug resistance assays showed serious multiple antimicrobial resistances, and S. Enteritidis was the most severe drug-resistant serotype. MLST showed that there was correlation between serotypes and genotypes in most Salmonella isolates, except S. Pullorum, which showed complicated genetic diversity firstly. These results provide important epidemiological information for us to control Salmonella in chickens.

Probiotics and food safety: an evidence-based review

Probiotics are commonly defined as live microorganisms (yeast or bacteria), when getting ingested in adequate amounts, they exhibit the beneficial effects on the host. During the past two decades, probiotic microorganisms as health-promoting agents have been increasingly added to various types of food products, especially in fermented food and also drugs. Due to the importance of food safety aspects of the human diet and with regards to some adverse effects of probiotics for human, we decided to carry out a review on probiotics and their adverse effects by research in literature. Previous studies indicated that several aspects, including safety, functional and technological characteristics, have to be considered in the selection of probiotic microorganisms. Safety aspects include origin (gastrointestinal tract of healthy human), nonpathogenicity and antibiotic resistance. Some probiotic microorganisms such as enterococci have been considered as an opportunistic pathogen for humans and cause disease, possess agents for antibiotic resistance and potential virulence factors. The bacteria used as a probiotic in food should be completely safe. Probiotic bacteria should be chosen from the healthy human micro-flora and should not have any antibiotic resistance that would prevent treatment of a rare probiotic infection. This review focused on key issues concerning the safety aspects of probiotics added to particular food products for improvement of general health and also discussed the criteria for probiotic selection in details.

Influence of genetic diversity of seventeen Beauveria bassiana isolates from different hosts on virulence by comparative genomics

BackgroundBeauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome.ResultsThere were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in.ConclusionsBased on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.