Non-virulent Strains Research Articles

Virulence Factors and Antibiotic Resistance of Klebsiella pneumoniae Strains Isolated From Neonates With Sepsis.

Introduction: Klebsiella pneumoniae is one of the most important infectious agents in neonates. There are “classic” and hypervirulent strains of K. pneumoniae. The “classic” non-virulent strain of K. pneumoniae, producing extended-spectrum beta-lactamases (ESBLs), is associated with nosocomial infections. Hypervirulent K. pneumoniae strains are associated with invasive infections in previously healthy adult people, and most of them exhibit antimicrobial susceptibility. The role of virulent strains of K. pneumoniae (including hv-KP) in neonatal infections is unknown. The aim of the study was the assessment of the impact of virulence factors and antibiotic resistance of K. pneumoniae strains on clinical features and outcomes of neonatal infection.Materials and Methods: Two groups of infants were enrolled. The first group consisted of 10 neonates with sepsis caused by K. pneumoniae. The second group consisted of 10 neonates with urinary tract infection (UTI) caused by K. pneumoniae. We investigated the susceptibility of K. pneumoniae isolates to antibiotics, the ability of the microorganism to produce ESBL, and virulence factors, including the rmpA gene, aerobactin, and colibactin genes. In neonates with sepsis, we investigated K. pneumoniae isolates, which was taken from the blood, in neonates with UTI—from the urine.Results: In neonates with sepsis testing of K. pneumoniae isolates for ESBL production was positive in 60% of cases, in neonates with UTI—in 40% of cases. All blood and urine ESBL producing K. pneumoniae isolates were resistant to ampicillins, including protected ones, and third-generation cephalosporins. At the same time, these isolates were sensitive to meropenem, amikacin, and ciprofloxacin. The rmpA gene was detected in four blood, and three urine K. pneumoniae isolates. In neonates with sepsis rmpA gene in two cases was detected in ESBL-producing K. pneumoniae isolates. They were infants with meningitis, and both cases were fatal. In the group of infants with UTI, the rmpA gene was detected only in K. pneumoniae isolates not producing ESBL. Aerobactin and colibactin genes were detected in two neonates with sepsis and in three neonates with UTI. In all cases, aerobactin and colibactin genes were detected only in rmpA-positive K. pneumoniae isolates. Out of three fatal outcomes, two cases were caused by hv-KP producing ESBL.Conclusion: The prevalence of virulent strains of K. pneumoniae among neonates with sepsis and other neonatal infection is higher than we think. The most severe forms of neonatal sepsis with an unfavorable outcome in our study were due to virulent strains of K. pneumoniae.

CHARACTERISTICS OF THE FAR EASTERN POPULATION OF TICK-BORNE ENCEPHALITIS VIRUS AND SOLVING ISSUES OF SPECIFIC PREVENTION

The subdivision of the viral population into subtypes and clusters based on the molecular genetic characteristics of the tick-borne encephalitis virus (TBEV) strains predetermines not only the differences in the biological properties of these strains, but also their different responses to specific antibodies in persons vaccinated against TBE.The aimof the present study is to show the differences in biological properties on the model of two strains of Far Eastern TBEV subtype belonging to different clusters and to substantiate the need for a personalized approach to the vaccine prophylaxis of tick-borne encephalitis.Results.Two strains of TBEV were used in the studies. On the basis of full genome sequencing the Dal’negorsk strain (FJ402886, GenBank) is referred to the typical representative of Sofjin-like, and Primorye-437 (JQ825162, GenBank) – to Oshima-like TBEV strains of the Far Eastern subtype.The experiment shows the levels of specific antibodies capable of neutralizing virulence strains of tick- borne encephalitis virus. Low antibody titers (1:100 and 1:400) can neutralize only a low dose of a non-virulent strain of tick-borne encephalitis virus. Reliable protection against the disease of people infected with virulent strains of the TBEV can provide only a high level of specific antibodies.Conclusion.If the level of specific antibodies of IgG is 1:400 or lower, the vaccination course should be continued, at a titer of antibodies above 1:400, revaccination can be postponed subject to annual monitoring of specific immunity parameters in the pre-epidemic TBE season.

Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR.

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 104 and 105 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.

Fog, phenolic acids and UV-A light irradiation: A new antimicrobial treatment for decontamination of fresh produce

This study evaluates synergistic interactions of food grade phenolic acids (gallic and ferulic acid) and UV-A light to achieve decontamination of fresh produce using a fog to improve dispersion of the phenolic acids on produce surface. Nonvirulent strains of Escherichia coli O157:H7 and Listeria innocua were used as model bacteria and spinach was selected as a model fresh produce. Synergistic combination of a fog deposited phenolic acid and a UV-A light treatment achieved reduction in bacterial plate count up to 2 log CFU/cm2 independently of the initial load of the bacteria (104 or 106 CFU/cm2). Following the treatment, fog deposited gallic and ferulic acid could be easily removed from the surface of produce by immersion in water and the treatment did not significantly alter the total endogenous phenolic content of spinach. The treatment also did not affect the texture, but impacted the color of the spinach leaves on a Hunter's Lab scale although the visual color changes were small. Overall, this technology may aid in developing alternative approaches for decontamination processes using food grade compounds.

Following Acute Encephalitis, Semliki Forest Virus is Undetectable in the Brain by Infectivity Assays but Functional Virus RNA Capable of Generating Infectious Virus Persists for Life.

Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the brain, by standard infectivity assays, for around ten days. As we have shown previously, in severe combined immunodeficient (SCID) mice, infectious virus is detectable for months in the brain. Here we show that in MHC-II-/- mice, with no functional CD4 T-cells, infectious virus is also detectable in the brain for long periods. In contrast, in the brains of CD8-/- mice, virus RNA persists but infectious virus is not detectable. In SCID mice infected with SFV, repeated intraperitoneal administration of anti-SFV immune serum rapidly reduced the titer of infectious virus in the brain to undetectable, however virus RNA persisted. Repeated intraperitoneal passive transfer of immune serum resulted in maintenance of brain virus RNA, with no detectable infectious virus, for several weeks. When passive antibody transfer was stopped, antibody levels declined and infectious virus was again detectable in the brain. In aged immunocompetent mice, previously infected with SFV, immunosuppression of antibody responses many months after initial infection also resulted in renewed ability to detect infectious virus in the brain. In summary, antiviral antibodies control and determine whether infectious virus is detectable in the brain but immune responses cannot clear this infection from the brain. Functional virus RNA capable of generating infectious virus persists and if antibody levels decline, infectious virus is again detectable.

Type I and III interferon signaling contributes to severe pathogenesis of human metapneumovirus clinical isolates

Abstract Human metapneumovirus (HMPV) is a leading cause of respiratory tract infection in infants and children. The disease outcome of HMPV infection ranges from mild infection to severe bronchiolitis and fatal pneumonia. HMPV was discovered in 2001 and its disease mechanisms are not fully understood. To gain insight into HMPV pathogenesis, we tested nine HMPV clinical isolates in an established C57BL/6 mouse model. HMPV clinical isolates induced variable disease severity ranging from mild to fatal disease. Mice infected by non-virulent strain TN/94-49 did not show significant weight loss, but mice infected with virulent isolate C2-202 showed dramatic weight loss and 40% mortality within 5 days post-infection. These findings were confirmed in other inbred mouse strains. C2-202 infected mice also suffered from impaired pulmonary function post-recovery. Lung virus replication did not correlate with disease severity, suggesting immune-mediated pathogenesis. Further investigation revealed increased type I and III interferons, pro-inflammatory cytokine production, and neutrophil infiltration in C2-202 infected mice. HMPV C2-202 infection also inhibited dendritic cell recruitment and down-regulated MHC-II surface expression on macrophages. Stat1/Stat2 double knockout mice lacking interferon signaling exhibited reduced weight loss, lessened disease severity, and increased dendritic cell recruitment after C2-202 infection. Our results indicate that severe disease caused by HMPV clinical isolates was due to exuberant interferon response leading to immuno-pathological tissue damage. These data suggest interferon signaling plays an important role in HMPV pathogenesis, and thus serves as a potential therapeutic target.

Deep Sequencing-Based Transcriptome Profiling Reveals Avian Interferon-Stimulated Genes and Provides Comprehensive Insight into Newcastle Disease Virus-Induced Host Responses.

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide, with variations in NDV pathogenicity due to the differences in virulence between strains. However, there is limited knowledge regarding the avian innate immune response to NDV infection. In this study, transcriptional profiles were obtained from chick embryo fibroblasts (CEFs) that were infected with the highly virulent NDV Herts/33 strain or the nonvirulent LaSota strain using RNA-seq. This yielded 8433 transcripts that were associated with NDV infection. This list of candidate genes was then further examined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. It showed a high enrichment in the areas of cellular components and metabolic processes, with the cellular components possibly being associated with NDV pathogenicity. Among these 8433 transcripts, 3616 transcripts associated with interferon-stimulated genes (ISGs) were obtained; these transcripts are involved in metabolic processes, including protein phosphorylation and protein modification. These results provide further insight into the identification of genes that are involved in NDV infection. The global survey of changes in gene expression performed herein provides new insights into the complicated molecular mechanisms underlying virus and host interactions and will enable the use of new strategies to protect chickens against this virus.

New Vaccine Formulations Containing a Modified Version of the Amastigote 2 Antigen and the Non-Virulent Trypanosoma cruzi CL-14 Strain Are Highly Antigenic and Protective against Leishmania infantum Challenge.

Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.

Contamination sources, biofilm‐forming ability and biocide resistance of Shiga toxin‐producing Escherichia coli O157:H7 and non‐O157 isolated from tilapia‐processing facilities

AbstractContamination sources, biofilm‐forming ability and biocide resistance of Shiga toxin‐producing Escherichia coli O157:H7 and non‐O157 from two tilapia‐processing plants were assessed. The application of unsuitable handling and sanitizing procedures caused a wide contamination by thermotolerant coliforms in Factories A (51.2% of points) and B (66.7%), exceeding the legal limits for frozen tilapia fillets. E. coli was also detected in both factories, but at low concentrations. RAPD‐PCR using primers DAF4, GEN15001, and M13 characterized seven E. coli strains. Biofilm‐forming ability of stx2d‐carriers and E. coli O157:H7 strains (E3, E4, E5, E6, E7) was higher than nonvirulent strains (E2, E8). Biofilm formation was correlated with the type of origin surface (abiotic or biotic) and with the presence of fliC H7. Biofilms resisted doses of peracetic acid and sodium hypochlorite higher than those recommended by manufacturers. E. coli was more susceptible to peracetic acid than sodium hypochlorite, but their similar mechanisms of action could increase the risk of crossresistance.Practical applicationsCase‐by‐case approaches are recommended to determine the sources and degree of contamination present in food‐processing facilities, which would allow applying precise responses that avoid, or at least reduce, the persistence of bacterial pathogens and the risk of antimicrobial resistance. Data obtained in this study showed that current strategies to control thermotolerant coliforms and E. coli in tilapia‐processing facilities are ineffective and the application of sublethal doses have allowed the emergence of antimicrobial resistant strains. Therefore, novel working‐safe, environmentally friendly and cost‐effective biocides, and the development of innovative sanitizing procedures are required to control biofilms formed by bacterial pathogens in food‐processing environments.

Increased exposure to extended-spectrum β-lactamase-producing multidrug-resistant Enterobacteriaceae through the consumption of chicken and sushi products

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromID ESBL agar and further phenotypic (antimicrobial study on MicroScan© NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant β-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MLST E. coli isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.

Kinetics of parasite distribution after reinfection with genetically distinct strains of Toxoplasma gondii

Recent data shows that prior infection by Toxoplasma gondii does not protect the host from subsequent reinfection even after the development of immunological memory. Although animal models for T. gondii reinfection were proposed after cases of natural human reinfection were described, little is known about the events that occur immediately after challenge. To further understand these events, BALB/c mice were chronically infected with D8 non-virulent strain (genotype ToxoDB#8 BrIII) and challenged with two different virulent strains: EGS (genotype ToxoDB #229) or CH3 strain (genotype ToxoDB #19). Primary infection protected animals from lethal challenge and morbidity was reduced. Reinfection was confirmed by PCR-RFLP, showing differences in the way the parasites spread in challenged animals. Parasites reached the lungs during early infection and a parasitism delay in the intestine was observed in D8+CH3 group. Parasites from challenge strains were not detected in the brain of D8+CH3 and in the intestine and brain of D8+EGS group. Previous infection with D8 strain of T. gondii protected against lethal challenges, but it did not prevent parasite spread to some organs.

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Case of Meningitis in a Neonate Caused by an Extended-Spectrum-Beta-Lactamase-Producing Strain of Hypervirulent Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the most important infectious agents among neonates. This pathogen has a potential to develop an increased antimicrobial resistance and virulence. The classic non-virulent strain of K. pneumoniae, producing an extended-spectrum beta-lactamases (ESBL), is associated with nosocomial infection mainly in preterm neonates. Hypervirulent K. pneumoniae strains are associated with invasive infection among previously healthy ambulatory patients, and most of them exhibit antimicrobial susceptibility. During the last few years, several cases of diseases caused by hypervirulent K. pneumoniae producing ESBL have been registered in different geographical regions of the world. However, reports of such cases in neonates are rare. Here, we reported that this pathogen can cause pyogenic meningitis in full-term neonate with poor prognosis. A previously healthy, full-term, 12-day-old neonate was admitted to the infectious diseases hospital with suspected meningitis. The clinical symptoms included loss of appetite, irritability, fever, seizures, and a bulging anterior fontanelle. The analysis of the cerebrospinal fluid confirmed the diagnosis of meningitis. Blood and cerebrospinal fluid cultures were positive for K. pneumoniae, producing ESBL. K. pneumoniae isolates were resistant to aminopenicillins, 3rd generation cephalosporins but were sensitive to imipenem and meropenem. The “string test” was positive. The study of the virulence factors of K. pneumoniae by PCR revealed the presence of the rmpA gene. A combination of K. pneumoniae virulence and drug resistance complicated by cerebral oedema led to the death of the neonate. We concluded that both the risk of developing severe forms of infection and the outcome of the disease due to K. pneumonia are associated with the phenotypic features of the pathogen such as its antibiotic susceptibility and virulence factors. Emergence of the ESBL-producing strain of hypervirulent K. pneumoniae could represent a new serious threat to public health, suggesting an urgent need to enhance clinical awareness and epidemiological surveillance.

Association between virulence profile, biofilm formation and phylogenetic groups of Escherichia coli causing urinary tract infection and the commensal gut microbiota: A comparative analysis

Variety of virulence factors are involved in the pathogenicity of Escherichia coli, the common cause of the urinary tract infections (UTIs). The aim of this study was to determine some virulence factors involved in the pathogenicity and the phylogenetic grouping of E. coli from UTIs compared with the E. coli isolates from gut microbiota (fecal flora). The isolates were tested for biofilm formation, haemagglutination, cell surface hydrophobicity (CSH), hemolysin production, phylogenetic grouping and the distribution of 6 known virulence genes. Isolates from UTIs showed a significantly higher prevalence of haemagglutination and hemolysin production compared with fecal flora (P ≤ 0.05), while biofilm formation and cell surface hydrophobicity (CSH) were not significantly different among the groups. Prevalence of virulence genes fimH, kpsMT ll, iutA, sat, hlyA, and cnf1 among all isolates were: 94.5%, 66.95%, 67.8%, 39%, 23.07% and 21.08%, respectively. The genes for hlyA, cnf1, kpsMT ll were found to be higher in UTI isolates compared to fecal flora (P ≤ 0.05). The frequency of the isolates in the phylogenetic groups B2, D, A and B1 were 36.7%, 31.3%, 16.2% and 15.6%, respectively. All the virulence genes except fimH were found to be significantly higher in the isolates of groups B2 and D. The results suggests that certain factors are necessary for the host colonization and infection and they are common in both virulent and non-virulent strains, and that the strains in the groups A and B1 having the lower virulence factors must acquire these factors when the condition is in favor of their dissemination to the urinary tract. In contrast the isolates in the groups B2 and D appeared to be potentially virulent.

Genomic comparison of virulent and non-virulent serotype V ST1 Streptococcus agalactiae in fish

Streptococcus agalactiae or Group B Streptococcus (GBS) is the major pathogen causing pneumonia and meningitis in human, mastitis in dairy cows, and streptococcal disease in tilapia. Previous studies have shown that fish GBS strains are correlated with human GBS strains in evolution and might have cross-host infection ability. Although the invasive disease caused by ST1 GBS in non-pregnant adults and cows is increasing worldwide, infection of fish by ST1 GBS has not been reported. The aim of this study was to determine whether ST1 GBS was virulent in fish and to investigate the genomic characteristics of ST1 GBS strains with different pathogenicity in tilapia. The human-derived serotype V ST1 GBS strains NNA048 and NNA038 were used to intraperitoneally challenge Nile tilapia (Oreochromis niloticus) with doses of 1.0×109CFU/fish, 1.0×107CFU/fish, and 1.0×105CFU/fish, respectively. The cumulative mortality rates of NNA048 infection at three different doses were 100.00%, 83.33%, and 40.00%. In contrast, there were no any sick or dead fish in NNA038 infection group. Histopathological results indicated that challenge of tilapia with NNA048 caused different degree of degeneration and necrosis in brain, liver, spleen, head kidney, and gut, and a large number of blue-stained Streptococcus granules were observed in the tissues. In contrast, there were no any lesions in the tissues of tilapia that were challenged with NNA038. Genome comparison showed that the major genome differences between NNA048 and NNA038 were attributed to the different phage sequences, and there was a 49.8kb length, intact phage sequence encoding 68 proteins in NNA048 genome. SNV and Indels analysis between NNA038 and NNA048 genomes indicated that there were a total of 96 SNVs, 5 deletions and 1 insert. Taken together, serotype V ST1 GBS was comprised of virulent and nonvirulent strains to tilapia, and gene rearrangement might be the main reason of causing different levels of virulence between strains.

Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors.

Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H.pylori, as vector for H.pylori antigens for possible use as a vaccine strain against H.pylori. This was done by recombinant expression of the H.pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V.cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V.cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V.cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H.pylori bacteria. Our results demonstrate that a non-virulent V.cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V.cholerae strains expressing HpaA, and possibly also other H.pylori antigens, may have the potential as oral inactivated vaccine candidates against H.pylori.

Response of life-history traits to artificial and natural selection for virulence and nonvirulence in a Drosophila parastitoid, Asobara tabida.

Co-evolution of host-parasitoid interactions is determined by the costs of host resistance, which received empirical evidence, and the costs of parasitoid virulence, which have been mostly hypothesized. Asobara tabida is a parasitoid, which mainly parasitizes Drosophila melanogaster and D. subobscura, the first species being able to resist to the parasitoid development while the second species is not. To parasitize resistant hosts, including D. melanogaster, A. tabida develops sticky eggs, which prevent encapsulation, but this virulence mechanism may be costly. Interindividual and interpopulation variation in the proportion of sticky eggs respectively allowed us to (i) artificially select and compare life-history traits of a virulent and a nonvirulent laboratory strain, and (ii) compare a virulent and a nonvirulent field strain, to investigate the hypothetical costs of virulence. We observed strong differences between the 2 laboratory strains. The nonvirulent strain invested fewer resources in reproduction and walked less than the virulent one but lived longer. Concerning the field strains, we observed that the nonvirulent strain had larger wings while the virulent one walked more and faster. All together, our results suggest that virulence may not always be costly, but rather that different life histories associated with different levels of virulence may coexist at both intra- and interpopulation levels.

Characterization of putative virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura

Two Serratia marcescens strains, SEN and ICC-4, isolated from diseased insect cadavers were observed to differ considerably in their virulence towards Spodoptera litura. The present study was aimed to characterize the possible virulence factors present in the virulent Serratia marcescens strain SEN. Both the S. marcescens strains were evaluated for the presence of various lytic enzymes such as chitinase, lipase, protease and phospholipase. The virulent S. marcescens strain SEN was observed to possess considerably higher activity of chitinase and protease enzymes; activity of phospholipase enzyme was also higher. Although, all the three toxin genes shlA, phlA and swr could be detected in both the S. marcescens strains, there was a higher expression of these genes in the virulent strain SEN. S. marcescens strain ICC-4 showed greater reduction in overall growth yield in the post-exponential phase in the presence of midgut juice and hemolymph of S. litura larvae, as compared to S. marcescens strain SEN. Proliferation of the S. marcescens strain SEN was also considerably higher in foregut, midgut and hemolymph of S. litura larvae, as compared to strain ICC-4. Peritrophic membrane treated with broth culture of the S. marcescens strain SEN showed higher damage as compared to strain ICC-4. The peritrophic membrane of larvae fed on diet treated with the virulent strain showed considerable damage while the peritrophic membrane of larvae fed on diet treated with the non-virulent strain showed no damage. This is the first report documenting the fate of ingested S. marcescens in S. litura gut and the relative expression of toxin genes from two S. marcescens strains differing in their virulence towards S. litura.

Identification of a surface epitope specific of virulent strains of Haemophilus parasuis

Haemophilus parasuis is a bacterium from the Pasteurellaceae family that comprises strains of different degree of virulence. Non-virulent strains are considered components of the upper respiratory tract microbiota, while virulent strains can invade systemic organs and cause fibrinous polyserositis (Glässer’s disease). Genomic comparison of virulent and non-virulent strains led to the identification of a family of genes differentially associated to virulence, the virulence-associated trimeric autotransporters (vtaA). Monoclonal antibody 69C6 reacted with the surface of virulent strains and has allowed now the identification of an epitope in the C terminus of the passenger domain of the VtaAs from virulent strains. Protein modelling indicated that the epitope is probably exposed, although sera from pigs vaccinated with the passenger domain of VtaA9 and from convalescent animals did not react with the 69C6 epitope. Induction of antibodies against the 69C6 epitope by vaccination would allow a response targeting specifically virulent strains of H. parasuis.