Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen.

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Recovery and virulence factors of sublethally injured Staphylococcus aureus after ohmic heating.

Ohmic heating (OH) is an alternative thermal processing technique, which is widely used to pasteurize or sterilize food. However, sublethally injured Staphylococcus aureus induced by OH is a great concern to food safety. The recovery of injured S. aureus by OH and virulence factor changes during recovery were investigated in this study. The liquid media (phosphate-buffered saline, buffered peptone water and nutrient broth (NB)), temperature (4, 25 and 37 °C) and pH (6.0, 7.2 and 8.0) influenced the recovery rate and the injured cells completely repaired in NB at 37 °C, pH 7.2 with the shortest time of 2 h. The biofilm formation ability, mannitol fermentation, hemolysis, and coagulase activities decreased in injured S. aureus and recovered during repair process. Quantitative real-time PCR showed the expression of sek, clfB and lukH involved in virulence factors increased during recovery. The results indicated that the virulence factors of injured S. aureus recovered after repair.

Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria.

BACKGROUND: Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. RESULTS: CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. CONCLUSION: Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods.

An Investigation on the Occurrence and Molecular Characterization of Bacillus cereus in Meat and Meat Products in China.

Bacillus cereus is a common foodborne pathogen that can cause both gastrointestinal and nongastrointestinal diseases. In this study, we collected 603 meat and meat products from 39 major cities in China. The positive contamination rate of B. cereus in the collected samples was 26.37% (159/603), and the contamination level in 5.03% (8/159) positive samples exceeded 1100 most probable number/g. The detection rates of virulence genes were 89.7% for the nheABC gene group, 37.1% for the hblACD gene cluster, 82.3% for cytK-2, and 2.9% for cesB. Notably, all isolates presented with multiple antibiotic resistance, and 99.43% of isolates were resistant to five classes of antibiotics. In addition, the multilocus sequence typing results indicated that all isolates were rich in genetic diversity. Collectively, we conducted a systematic investigation on the prevalence and characterization of B. cereus in meat and meat products in China, providing crucial information for assessing the risk of B. cereus occurrence in meat and meat products.

Prevalence, Characterization, and Control of Campylobacter jejuni Isolated from Raw Milk, Cheese, and Human Stool Samples in Beni-Suef Governorate, Egypt.

Our study aimed to determine the prevalence of Campylobacter jejuni isolated from raw milk, cheese, and human stool samples in Beni-Suef Governorate, Egypt, and to characterize the antibiotic resistance profile and virulence genes of the isolates. An additional objective was to evaluate the effectiveness of cinnamon oil and Lactobacillus acidophilus La5 for controlling C. jejuni in cheese. A total of 200 samples of raw milk and dairy products, including 50 samples of raw milk and 150 samples of three different types of cheese were used. Fifty-three human stool samples were also collected. The samples were tested for the presence of C. jejuni using culture and molecular methods. Campylobacter spp. were isolated from 9.5% (19/200) of the raw milk and cheese samples. The highest prevalence was observed in milk samples (18%), followed by Kareish cheese (14%) and Talaga cheese (6%). In contrast, C. jejuni was not found in any of the Feta cheese samples. Of the human stool samples, 21 (39.6%) were positive for C. jejuni. Of the isolates, 60-90% were highly resistant to the antimicrobial agents tested, that is, nalidixic acid, ciprofloxacin, and tetracycline. Virulent cadF and cdtA genes were detected in all isolates. As milk and dairy products are important sources of contamination, reducing the level of C. jejuni in them will lower the risk to consumers. We showed that L. acidophilus La5 was able to control C. jejuni in Kareish cheese, but cinnamon oil was less effective.

Phenotypic and molecular characterization of virulence factors of extra-intestinal pathogenic Escherichia coli isolated from patients of Peshawar, Pakistan.

Extra-Intestinal Escherichia coli (ExPEC) are important cause of Urinary Tract Infections (UTIs) and systemic infections. The purpose of this study was to investigate numerous ExPEC bacterial isolates for phenotypic virulence characteristics including hemolytic activity and resistance pattern and to observe their association with genetic traits via Polymerase Chain Reaction (PCR). A total of 367 ExPEC isolates were collected from patients admitted in Khyber Teaching Hospital (KTH) Peshawar, Pakistan. Standard techniques were used for identification of isolates, determination of hemolytic potential and antimicrobial susceptibility testing. PCR was used for screening of virulence genes using specific primers. A total of 367 ExPEC isolates were characterized, among which 62.7, 24.3, 7.1 and 6% were isolated from urine, pus, sputum and wound specimens, respectively. Majority of the isolates (82.8%) were hemolysin positive. Multi drug resistance pattern was shown by 41% of the isolates and harbored at least one virulence gene (71.7%), of which sat was the most prevalent (64.3%). The highest resistance was found to cefotaxime (99.2%), ampicillin (97.5%) and aztreonem (89.6%). 15 different virulence genes combinations were observed in the current study. A total of 16 virotypes (15 of positive virulence genes and one of no virulence gene) were observed in the current study. The current investigation showed a high prevalence of sat and hlyA genes among ExPEC isolate, suggesting a role of these genes in the pathogenesis of ExPEC.

An improved quantitative real-time polymerase chain reaction technology for Helicobacter pylori detection in stomach tissue and its application value in clinical precision testing.

BACKGROUND: Helicobacter pylori (H. pylori) infection is a serious human health threat. The empiric H. pylori treatment paradigm guided by traditional testing technologies has led to antibiotic resistance. Here, we improved the qPCR method to provide technical support for precision H. pylori diagnosis and treatment. METHODS: Two pairs of primers and probes targeting the glmM gene were designed to detect H. pylori, and a multiplex qPCR method was established for virulence factor detection. Then, a rapid urease test (RUT), culturing and qPCR were performed on 141 specimens collected from Xinqiao Hospital of China in 2017 to evaluate the qPCR detection capability. Finally, the H. pylori infectious amount and virulence genes were detected by qPCR. RESULTS: 1. The improved qPCR method which used two pairs of primers had a higher detection rate (100%) and better accuracy (p = 0.000), compared with the qPCR using a pair of primers. It also had better consistency with the bacterial culture than with RUT (Kappa =0.440, p < 0.001). 2. The H. pylori infectious amount was significantly positively associated with gastritis in corpus (p = 0.003) and gastric erosion (p = 0.043). The H. pylori infectious amount in gastric precancerous patients was significantly lower than that in H. pylori-positive patients (p < 0.05), and the infectious H. pylori-vacA s1+ amount was significantly greater than that of H. pylori-vacA s1- (p < 0.05). 3. The vacA s1 frequency was significantly higher than that of vacA m1/cagA+/babA2+ in chronic superficial gastritis (p = 0.000), peptic ulcer (p = 0.037) and gastric erosion (p = 0.009). The H. pylori-vacA+/cagA+/babA2+ frequency showed a significant positive correlation (p < 0.05). CONCLUSIONS: The H. pylori infectious amount and presence of H. pylori virulence factors showed complex correlations with gastric disease occurrence and development. The improved qPCR with good detection performance can be used for quantitative H. pylori detection and testing for the virulence genes vacA s1, vacA m1, cagA and babA2 simultaneously. These findings will provide valuable information for disease diagnosis and treatment.

Characterization of Pseudomonas aeruginosa isolated from various environmental niches: New STs and occurrence of antibiotic susceptible "high-risk clones".

The aim of this study was to investigate the antimicrobial phenotypes, major virulence factors, and the molecular typing of 66 P. aeruginosa isolates collected from various sources: human patients and hospital environment, raw milk, poultry meat, chicken/sheep fecal samples, wastewater, thermal water, and seawater. All isolates, except one, were susceptible to all tested antibiotics. exoA, lasB, rhlR, and lasR genes were harbored by 60 isolates. Forty-six, 18, and 2 isolates amplified exoS, exoU, and exoS+exoU genes, respectively. Twenty-one isolates showed high elastase and pigment production. The PFGE typing identified 26 pulsotypes. Some pulsotypes included isolates from different environmental niches and areas. Twelve selected isolates were typed by MLST and eight different STs were found, three of them were new. Our results highlighted the dissemination of some clones amongst different settings and the occurrence of antibiotic susceptible 'high-risk clones' that might be very harmful when acquiring genes encoding antibiotic resistance.

Virulence Factor Identification in the Banana Pathogen Dickeya zeae MS2.

The phytopathogen Dickeya zeae MS2 is a particularly virulent agent of banana soft rot disease. To begin to understand this banana disease and to understand the role of quorum sensing and quorum-sensing-related regulatory elements in D. zeae MS2, we sequenced its genome and queried the sequence for genes encoding LuxR homologs. We identified a canonical LuxR-LuxI homolog pair similar to those in other members of the genus Dickeya The quorum-sensing signal for this pair was N-3-oxo-hexanoyl-homoserine lactone, and the circuit affected motility, cell clumping, and production of the pigment indigoidine, but it did not affect infections of banana seedlings in our experiments. We also identified a luxR homolog linked to a gene annotated as encoding a proline iminopeptidase. Similar linked pairs have been associated with virulence in other plant pathogens. We show that mutants with deletions in the proline iminopeptidase gene are attenuated for virulence. Surprisingly, a mutant with a deletion in the gene encoding the LuxR homolog shows normal virulence.IMPORTANCEDickeya zeae is an emerging banana soft rot pathogen in China. We used genome sequencing and annotation to create an inventory of potential virulence factors and virulence gene regulators encoded in Dickeya zeae MS2, a particularly virulent strain. We created mutations in several genes and tested these mutants in a banana seedling infection model. A strain with a mutated proline iminopeptidase gene, homologs of which are important for disease in the Xanthomonas species phytopathogens, was attenuated for soft rot symptoms in our model. Understanding how the proline iminopeptidase functions as a virulence factor may lead to insights about how to control the disease, and it is of general importance as homologs of the proline iminopeptidase occur in dozens of plant-associated bacteria.

Heme uptake and utilization by hypervirulent Acinetobacter baumannii LAC-4 is dependent on a canonical heme oxygenase (abHemO).

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in critically ill and immune compromised patients. The ability to acquire iron from the hosts iron and heme containing proteins is critical to their survival and virulence. The majority of A. baumannii hypervirulent strains encode a heme uptake system that includes a putative heme oxygenase (hemO). Despite reports indicating A. baumannii can grow on heme direct evidence of extracellular heme uptake and metabolism has not been shown. Through isotopic labeling (13C-heme) we show the hypervirulent A. baumannii LAC-4 metabolizes heme to biliverdin IXα (BVIXα), whereas ATC 17978 that lacks the hemO gene cluster cannot efficiently utilize heme. Expression and purification of the protein encoded by the A. baumannii LAC-4 hemO gene confirmed catalytic conversion of heme to BVIX. We further show inhibition of abHemO with previously characterized P. aeruginosa HemO inhibitors in a fluorescence based assay that couples HemO catalytic activity to the BVIXα binding phytochrome IFP1.4. Furthermore, the hemO gene cluster encodes genes with homology to heme-dependent extra cytoplasmic function (ECF) σ factor systems. The hemophore-dependent ECF system in Pseudomonas aeruginosa has been shown to play a critical role in heme sensing and virulence within the host. The prevalence of a hemO gene cluster in A. baumannii LAC4 and other hypervirulent strains suggests it is required within the host to adapt and utilize heme and is a major contributor to virulence.

Chemistry and Biology of Siderophores from Marine Microbes.

Microbial siderophores are multidentate Fe(III) chelators used by microbes during siderophore-mediated assimilation. They possess high affinity and selectivity for Fe(III). Among them, marine siderophore-mediated microbial iron uptake allows marine microbes to proliferate and survive in the iron-deficient marine environments. Due to their unique iron(III)-chelating properties, delivery system, structural diversity, and therapeutic potential, marine microbial siderophores have great potential for further development of various drug conjugates for antibiotic-resistant bacteria therapy or as a target for inhibiting siderophore virulence factors to develop novel broad-spectrum antibiotics. This review covers siderophores derived from marine microbes.

Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum.

The virulence and broad host range of Fusarium graminearum is associated with its ability to secrete an arsenal of phytotoxic secondary metabolites, including the regulated mycotoxins belonging to the deoxynivalenol family. The TRI genes responsible for the biosynthesis of deoxynivalenol and related compounds are usually expressed during fungal infection. However, the F. graminearum genome harbors an array of unexplored biosynthetic gene clusters that are also co-induced with the TRI genes, including the nonribosomal peptide synthetase 8 ( NRPS8) gene cluster. Here, we identify two bicyclic lipopeptides, gramillin A (1) and B (2), as the biosynthetic end products of NRPS8. Structural elucidation by high-resolution LC-MS and NMR, including 1H-15N-13C HNCO and HNCA on isotopically enriched compounds, revealed that the gramillins possess a fused bicyclic structure with ring closure of the main peptide macrocycle occurring via an anhydride bond. Through targeted gene disruption, we characterized the GRA1 biosynthetic gene and its transcription factor GRA2 in the NRPS8 gene cluster. Further, we show that the gramillins are produced in planta on maize silks, promoting fungal virulence on maize but have no discernible effect on wheat head infection. Leaf infiltration of the gramillins induces cell death in maize, but not in wheat. Our results show that F. graminearum deploys the gramillins as a virulence agent in maize, but not in wheat, thus displaying host-specific adaptation.

Detection of CTX-M-15 harboring Escherichia coli isolated from wild birds in Tunisia.

BACKGROUND: The spreading of antibiotic resistant bacteria is becoming nowadays an alarming threat to human and animal health. There is increasing evidence showing that wild birds could significantly contribute to the transmission and spreading of drug-resistant bacteria. However, data for antimicrobial resistance in wild birds remain scarce, especially throughout Africa. The aims of this investigation were to analyze the prevalence of ESBL-producing E. coli in faecal samples of wild birds in Tunisia and to characterize the recovered isolates. RESULTS: One hundred and eleven samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml). ESBL-producing E. coli isolates were detected in 12 of 111 faecal samples (10.81%) and one isolate per sample was further characterized. ß-lactamase detected genes were as follows: blaCTX-M-15 (8 isolates), blaCTX-M-15 + blaTEM-1b (4 isolates). The ISEcp1 and orf477 sequences were found respectively in the regions upstream and downstream of all blaCTX-M-15 genes. Seven different plasmid profiles were observed among the isolates. IncF (FII, FIA, FIB) and IncW replicons were identified in 11 CTX-M-15 producing isolates, and mostly, other replicons were also identified: IncHI2, IncA/C, IncP, IncI1 and IncX. All ESBL-producing E. coli isolates were integron positive and possessed "empty" integron structures with no inserted region of DNA. The following detected virulence genes were: (number of isolates in parentheses): fimA (ten); papC (seven); aer (five); eae (one); and papGIII, hly, cnf, and bfp (none). Molecular typing using pulsed-field gel electrophoresis and multilocus sequence typing showed a low genetic heterogeneity among the 12 ESBL-producing strains with five unrelated PFGE types and five different sequence types (STs) respectively. CTX-M-15-producing isolates were ascribed to phylogroup A (eleven isolates) and B2 (one isolate). CONCLUSION: To our knowledge, this study provides the first insight into the contribution of wild birds to the dynamics of ESBL-producing E. coli in Tunisia.

Identification of oligopeptide-binding protein (OppA) and its role in the virulence of Streptococcus suis serotype 2.

The oligopeptide permease (Opp) cassette, an oligopeptide transport system belongs to the superfamily of ATP-binding cassette (ABC) transporter, is widely distributed in bacteria, including Streptococcus suis (S. suis). It is encoded by the opp operon containing oppA, oppB, oppC, oppD, and oppF. In addition to the uptake of peptide, the oppA gene also plays an important role in virulence of many pathogens. In this study, an oppA homologue from the highly virulent S. suis serotype 2 (S. suis 2) strain 05ZYH33 was identified. Flow cytometry and Western blot confirmed that OppA is a surface immunogenic protein and is expressed during S. suis 2 infection. To explore the role of oppA in S. suis 2 growth and pathogenicity, an isogenic 05ZYH33 mutant of oppA (△oppA) was obtained by homologous recombination. Although the complementary strain was not obtained due to the △oppA strain is not transformable, the current data revealed that deletion of the oppA gene in S. suis 2 has greatly affected its growth and virulence. Our data revealed that the growth rate is significantly slow for the △oppA. Adherence of the △oppA strain to human epithelial cells is greatly reduced comparing to the wild strain. Mouse infection experiment showed that inactivation of oppA greatly attenuated the high pathogenicity of S. suis 2. The observed results suggest that OppA is a surface-exposed protein and plays important roles in the growth and pathogenicity of S. suis 2.

High frequency of hybrid Escherichia coli strains with combined Intestinal Pathogenic Escherichia coli (IPEC) and Extraintestinal Pathogenic Escherichia coli (ExPEC) virulence factors isolated from human faecal samples.

BACKGROUND: Classification of pathogenic Escherichia coli (E. coli) has traditionally relied on detecting specific virulence associated genes (VAGs) or combinations thereof. For E. coli isolated from faecal samples, the presence of specific genes associated with different intestinal pathogenic pathovars will determine their classification and further course of action. However, the E. coli genome is not a static entity, and hybrid strains are emerging that cross the pathovar definitions. Hybrid strains may show gene contents previously associated with several distinct pathovars making the correct diagnostic classification difficult. We extended the analysis of routinely submitted faecal isolates to include known virulence associated genes that are usually not examined in faecal isolates to detect the frequency of possible hybrid strains. METHODS: From September 2012 to February 2013, 168 faecal isolates of E. coli routinely submitted to the Norwegian Institute of Public Health (NIPH) from clinical microbiological laboratories throughout Norway were analysed for 33 VAGs using multiplex-PCR, including factors associated with extraintestinal pathogenic E. coli (ExPEC) strains. The strains were further typed by Multiple Locus Variable-Number Tandem-Repeat Analysis (MLVA), and the phylogenetic grouping was determined. One isolate from the study was selected for whole genome sequencing (WGS) with a combination of Oxford Nanopore's MinION and Illumina's MiSeq. RESULTS: The analysis showed a surprisingly high number of strains carrying ExPEC associated VAGs and strains carrying a combination of both intestinal pathogenic E. coli (IPEC) and ExPEC VAGs. In particular, 93.5% (101/108) of isolates classified as belonging to an IPEC pathovar additionally carried ExPEC VAGs. WGS analysis of a selected hybrid strain revealed that it could, with present classification criteria, be classified as belonging to all of the Enteropathogenic Escherichia coli (EPEC), Uropathogenic Escherichia coli (UPEC), Neonatal meningitis Escherichia coli (NMEC) and Avian pathogenic Escherichia coli (APEC) pathovars. CONCLUSION: Hybrid ExPEC/IPEC E. coli strains were found at a very high frequency in faecal samples and were in fact the predominant species present. A sequenced hybrid isolate was confirmed to be a cross-pathovar strain possessing recognised hallmarks of several pathovars, and a genome heavily influenced by horizontal gene transfer.

A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells.

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

Metabolite Transporter PEG344 Is Required for Full Virulence of Hypervirulent Klebsiella pneumoniae Strain hvKP1 after Pulmonary but Not Subcutaneous Challenge.

Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathotype that is capable of causing tissue-invasive and organ- and life-threatening infections in healthy individuals from the community. Knowledge on the virulence factors specific to hvKP is limited. In this report, we describe a new factor (PEG344) that increases the virulence of hvKP strain hvKP1. peg-344 is present on the hvKP1 virulence plasmid, is broadly prevalent among hvKP strains, and has increased RNA abundance when grown in human ascites. An isogenic derivative of hvKP1 (hvKP1Δpeg-344) was constructed and compared with its wild-type parent strain in in vitro, ex vivo, and infection model studies. Both survival and competition experiments with outbred CD1 mice demonstrated that PEG344 was required for full virulence after pulmonary challenge but, interestingly, not after subcutaneous challenge. In silico analysis suggested that PEG344 serves as an inner membrane transporter. Compared to hvKP1, a small but significant decrease in the growth/survival of hvKP1Δpeg-344 was observed in human ascites, but resistance to the bactericidal activity of complement was similar. These data suggested that PEG344 may transport an unidentified growth factor present in ascites. The data presented are important since they expand our limited knowledge base on virulence factors unique to hvKP, which is needed to lay the groundwork for translational approaches to prevent or treat these devastating infections.

PfmA, a novel quorum-quenching N-acylhomoserine lactone acylase from Pseudoalteromonas flavipulchra.

Many bacteria, such as Proteobacteria, Cyanobacteria and Bacteroidetes, use N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules for communication. Enzymatic degradation of AHLs, such as AHL acylase and AHL lactonase, can degrade AHLs (quorum quenching, QQ) to attenuate or disarm the virulence of pathogens. QQ is confirmed to be common in marine bacterial communities. Many genes encoding AHL acylases are found in marine bacteria and metagenomic collections, but only a few of these have been characterized in detail. We have reported that the marine bacterium Pseudoalteromonas flavipulchra JG1 can degrade AHLs. In the present study, a novel AHL acylase PfmA, which can degrade AHLs with acyl chains longer than 10 carbons, was identified from strain JG1. Ultra-performance liquid chromatography (UPLC) and electrospray ionization mass spectrometry (ESI-MS) analysis demonstrated that PfmA functions as an AHL acylase, which hydrolysed the amide bond of AHL. The purified PfmA of P. flavipulchra JG1 showed optimum activity at 30 °C and pH 7.0. PfmA belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily and showed homology to a member of penicillin amidases, but PfmA can degrade ampicillin but not penicillin G. The residue Ser256 in PfmA is the active site according to site-directed mutagenesis. Furthermore, PfmA reduced AHL accumulation and the production of virulence factors in Vibrio anguillarum VIB72 and Pseudomonas aeruginosa PAO1, and attenuated the virulence of P. aeruginosa to increase Artemia survival, which suggested that PfmA can be considered as a therapeutic agent to control AHL-mediated pathogenicity.

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence.

UNLABELLED: Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-ß promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE: Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-ß and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

Intestinal Colonization Dynamics of Vibrio cholerae.

To cause the diarrheal disease cholera, Vibrio cholerae must effectively colonize the small intestine. In order to do so, the bacterium needs to successfully travel through the stomach and withstand the presence of agents such as bile and antimicrobial peptides in the intestinal lumen and mucus. The bacterial cells penetrate the viscous mucus layer covering the epithelium and attach and proliferate on its surface. In this review, we discuss recent developments and known aspects of the early stages of V. cholerae intestinal colonization and highlight areas that remain to be fully understood. We propose mechanisms and postulate a model that covers some of the steps that are required in order for the bacterium to efficiently colonize the human host. A deeper understanding of the colonization dynamics of V. cholerae and other intestinal pathogens will provide us with a variety of novel targets and strategies to avoid the diseases caused by these organisms.