Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in the Bacillus cereus group

Bacillus cereus is a food contaminant with widely varying enterotoxic potential of its virulence proteins. In this article, phylogenetic analysis of the whole-genome amino acid sequences of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural prediction of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk clusters. The distances of evolutionary transfer relative to housekeeping genes of incomplete virulence genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L 1 were extracellular according to predictive analysis. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed ‘head’ and ‘tail’ domains. The ‘head’ of NheA and Hbl-B, including two α-helices separated by β-tongue strands, might play a special role in Nhe trimers and Hbl trimers, respectively. The ‘cap’ of CytK, which includes two ‘latches’ with many β-sheets, formed a β-barrel structure with pores, and a ‘rim’ balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with β-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus .

Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon’s index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon’s index of 2.49 and 10 clones with a Shannon’s index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2–3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

The complete genome of Chelonus insularis reveals dynamic arrangement of genome components in parasitoid wasps that produce bracoviruses

Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae ( Microplitis demolitor and Cotesia congregata ) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside of the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis , which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized when compared to M. demolitor and C. congregata . Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. Significance Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This manuscript characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis , which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function.

Revealing the diversity of Fusarium micromycetes in agroecosystems of the North Caucasus plains for replenishing the State Collection of Phytopathogenic Microorganisms of the All-Russian Scientific Research Institute of a Phytopathology

In order to prevent crop yield losses from the most dangerous and economically important pathogenic organisms, it is necessary not only to monitor the virulence gene pool, but also to study the nature of pathogen variability and determine the potential for the emergence of new genes and races. This requires centralized collections of fungal cultures characterized by a set of stable strains to provide for phytopathological, immunological, breeding, genetic, toxicological, parasitological and other studies. The State Collection of Phytopathogenic Microorganisms of the ARSRIP is the State Depository of Phytopathogenic microorganisms that are non-pathogenic to humans or farmed animals. Currently, it has more than 4,500 accessions of plant pathogenic strains of fungi, oomycetes, bacteria, viruses, phytoplasmas, and the collection is updated annually. For this purpose, the study of the inter- and intraspecific genetic diversity of genus Fusarium was carried out in agricultural systems of the Krasnodar Territory. In 2020, the State Collection of Phytopathogenic Microorganisms was supplemented with 13 strains of Fusarium fungi isolated from tissues of winter wheat plants collected in several locations of the Krasnodar region. The complex of Fusarium fungi revealed on winter wheat usually included Fusarium oxysporum, F. culmorum, F. lolii, F. graminearum, F. fujikuroi, F. sporotrichioides, etc. The effect of the preceding crop on the frequency of Fusarium species isolated from winter wheat was observed. After series cloning of collected isolates, 21 strains of different fungal species characterized by stable morphology traits and known pathogenic and phytotoxic properties were selected for collection replenishment. Significant differences in pathogenic activity were revealed between fungi belonging to either the same or different species; the manifestation of this activity varied from the absence of any effect of spore suspensions on seedling development to a complete inhibition of their growth. The phytotoxic activity towards wheat seedlings varied from medium to high. Species possessing a high intensity of phytotoxic activities are the most dangerous for wheat, since they promote accumulation of dangerous phytotoxins in plant tissues.

VIRULENCE GENE PROFILING AND PATHOGENICITY OF Streptococcus agalactiae ISOLATED FROM TILAPIA, Oreochromis niloticus FARMS IN INDONESIA

Streptococcosis caused by Streptococcus agalactiae has become a major disease problem in tilapia culture in Indonesia. This study aimed to detect virulence genes of S. agalactiae during streptococcosis disease outbreaks in several tilapia farms in Indonesia and evaluate the correlation between biotype and virulence genes to bacterial virulence. The presence of virulence genes was determined in 10 strains of S. agalactiae isolated from farm-raised tilapia. Polymerase chain reaction (PCR) protocol was used to determine genes for cylE, hylB, lmb, bib A, PI-2b, fbs A, fbs B, gap, PI-1, and cfb in the template DNA. Pathogenicity test was carried out by intraperitoneal injection of 24 hour-cultured S. agalactiae to tilapia with 108 CFU/fish. Four isolates have seven of virulence genes (cylE, hylB, bibA, PI-2b, fbs A, fbs B, and gap genes), three isolates have six virulence genes (hylB, bib A, fbs A, fbs B, gap, cfb genes), one isolate has four virulence gene (hyl B, bib A, fbs, and cfb genes), and one isolate has one virulence gene (PI-2b gene). None of the isolates has lmb or PI-1 genes. Bacteria with more virulence genes showed higher pathogenicity post injection. Mortality of tilapia injected with b-hemolytic bacteria was 100% within the period of 14-19 hours, while non-hemolytic bacteria was 53.3%-86.6% on 14 days post-injection. Pathological changes associated with the infection by either isolate included melanosis, slow response, anorexia, ocular opacity, gasping, erratic, C-shape, and whirling. It can be concluded that S. agalactiae with more virulence genes show a higher level of pathogenicity. The presence of a virulent gene has the potential to be used as a basis for selecting candidate isolates and designing vaccine compositions as an effort to prevent streptococcosis infection in tilapia in Indonesia.

Investigation of Virulence Genes of Staphylococcus aureus Isolated from Sterile Body Fluid Samples and Their Correlation with Clinical Symptoms and Outcomes

Staphylococcus aureus is the major pathogen causing nosocomial human infections and produces a variety of virulence factors that contribute to its ability to colonize and cause diseases. This study was conducted to investigate the virulence genes in S. aureus isolated from sterile body fluid samples and their correlation with clinical symptoms and outcomes. The VITEK 2® Compact system was used to perform biochemical identification and antimicrobial susceptibility tests on 33 S. aureus isolates. Virulence genes were amplified using multiplex PCR. The virulence gene patterns were analyzed by systematic cluster analysis. The frequency of methicillin-resistant S. aureus was 45.45%, and 17 virulence genes were identified. Genes encoding hemolysins showed high frequencies. The frequencies of hla, hlb, hld, and hlgB were 93.94% and that of the luk-F/S-PV was 21.21%. Except for the frequency of splB (51.52%), the remaining genes encoding invasive proteases showed frequencies greater than 81.82%. Among the patients, 100.00% had undergone invasive medical procedures and 24.00% had been treated with more than three types of antibiotic drugs. Invasive medical procedures are the main causes of infection. Resistance to antibiotic drugs and the status of carrying virulence genes were highly related to clinical symptoms and outcomes.

Whole Genome Sequencing and Beta-Lactam Resistant Genes in Klebsiella pneumoniae Strains Clinically Isolated from CU Shah Hospital, Gujarat, India

A simple explanation for antimicrobial-resistant opportunistic infections in immunocompromised patients is Klebsiella pneumoniae which gradually being associated in insidious infections globally with high mortality rate. Eight hundred fifty-six antibiotic resistant K. pneumoniae isolates were collected over 3 years period (from different wards and different specimens) from the Microbiology department of C.U. Shah hospital, whose AST checked by Kirby Bauer disk diffusion method. To study AMR genes, virulome, interference of virulence gene with resistance gene, phylogenomic; 6 clinical isolates were proceeded for whole genome sequencing and bio informatics analysis. Klebsiella pneumoniae is a multidrug-resistant (MDR) opportunistic and one of delegate of ESKAPE pathogens groups. This pathogen causes nosocomial infections, urinary tract infections, liver abscesses, wound infections, meningitis. These strains obtain a multidrug resistant phenotype by way of horizontal transfer of ARG transported by either transposons or plasmids. This transfer is generally facilitated by Integrons. In this study antibiotic resistance profile and antibiotic resistance genes analysis as well as virulence gene of K. pneumoniae strains were investigated. The study was carried out using 853 clinical isolates collected during 3 years from C.U. Shah hospital of Surendranagar. Antibiotic resistance profile test was carried out by the VITEK 2 against 21 antibiotics. Out of that 6 samples were proceed for DNA extraction, WGS illumina sequencer and analysis of those raw sequences by TORMES pipeline. In this study antibiotic resistance profile included 13 beta lactam antibiotics which classified under 3 class (Penicillin, Cephalosporin, Carbapenem) of beta lactam and in AMR gene study got total 15 different ESBL resistance genes from 6 different klebsiella pneumoniae strain. All these genes detected with more than 90% identity by CARD. (TORMES Pipeline) CTX-M-15, NDM-5, OKP-B-6, PDC-2, OXA-1, OXA-181, OXA-362, OXA-50, OXA-9, SHV-1, SHV-11, SHV-187, TEM-1, TEM-150. In this study, we’ve analyzed the pattern of antibiotic resistance pattern as a phenotypic characteristic and antibiotic resistance genes as genotypic characteristic and co related the results. As multidrug resistance is a worrying matter, constant observation and regular clinical recognition of resistant bacteria are essential to avoid terrible public health incidents. So, our data should be inferred as a warning for need for prevention and control of the MDR K. pneumoniae in hospital settings.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections

Introduction. Streptococcus mutans , a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections. Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR. Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes. Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159. Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains. Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

Effect of Enterocins A and B on the Viability and Virulence Gene Expression of Listeria monocytogenes in Sliced Dry-Cured Ham

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced.