Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method

We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing.This scheme efficiently classifies most of MTC phylogeographical groups: (1) confirming and identifying new sublineage-specific SNPs, (2) unraveling phylogenetical relationships between spoligotyping-defined MTC sublineages, (3) appropriately assigning sublineages to some spoligotypes and reassigning sublineages to other mis-labeled spoligotype signatures. This study opens the way to a more meaningful taxonomic, evolutionary and epidemiological classification. It also allows evaluation of spoligotype-signature significance towards a more comprehensive understanding of the evolutionary mechanisms of the clustered regularly interspaced short palindromic repeat (CRISPR) locus in MTC.     

Vaccine escape of piliated Streptococcus pneumoniae strains

IntroductionType1-pilus proteins were suggested as targets of future protein-based vaccines. Here we studied the effect of pneumococcal-conjugate vaccine (PCV7) implementation on the prevalence of piliated strains in a unique study setting which controls for typical confounders; the Palestinian-Israeli Collaborative Research (PICR).MethodsAnnual cross-sectional surveys of pneumococcal carriage were performed during 2009–2011 among two closely related population that live under different health policies (a) Palestinian-Authority (PA) (n = 1773), where PCV7 was not yet introduced (b) East-Jerusalem (EJ) (n = 983) where PCV7 was rapidly implemented. Clinical data were collected, pneumococci identified and characterized and the presence of Type1-pilus genes was determined by rrgC PCR.ResultsFollowing PCV7 implementation in EJ, overall carriage prevalence did not change (∼30%), but VT7 strains decreased from 61.5% to 33.8%. While prevalence of non-piliated-VT7 isolates decreased from 37% to 10%, p < 0.001, the prevalence of piliated-VT7 strains persisted ∼25%. Additionally, piliated non-VT13 strains emerged (1–15%, p < 0.001). These changes were not observed in PA. These dynamics were independent of the bacteria's resistance pattern.ConclusionsA differential effect of PCV7 was observed with a relative resistance of piliated strains to the vaccine. This suggests that Type1-pilus confers an intrinsic advantage for colonization and may be an attractive vaccine target.     

Sequence diversity and positive selection at the Duffy-binding protein genes of Plasmodium knowlesi and P. cynomolgi: Analysis of the complete coding sequences of Thai isolates

Plasmodium knowlesi and P. cynomolgi are simian malaria parasites capable of causing symptomatic human infections. The interaction between the Duffy binding protein alpha on P. knowlesi merozoite and the Duffy-antigen receptor for chemokine (DARC) on human and macaque erythrocyte membrane is prerequisite for establishment of blood stage infection whereas DARC is not required for erythrocyte invasion by P. cynomolgi. To gain insights into the evolution of the PkDBP gene family comprising PkDBPα, PkDBPβ and PkDBPγ, and a member of the DBP gene family of P. cynomolgi (PcyDBP1), the complete coding sequences of these genes were analyzed from Thai field isolates and compared with the publicly available DBP sequences of P. vivax (PvDBP). The complete coding sequences of PkDBPα (n = 11), PkDBPβ (n = 11), PkDBPγ (n = 10) and PcyDBP1 (n = 11) were obtained from direct sequencing of the PCR products. Nucleotide diversity of DBP is highly variable across malaria species. PcyDBP1 displayed the greatest level of nucleotide diversity while all PkDBP gene members exhibited comparable levels of diversity. Positive selection occurred in domains I, II and IV of PvDBP and in domain V of PcyDBP1. Although deviation from neutrality was not detected in domain II of PkDBPα, a signature of positive selection was identified in the putative DARC binding site in this domain. The DBP gene families seem to have arisen following the model of concerted evolution because paralogs rather than orthologs are clustered in the phylogenetic tree. The presence of identical or closely related repeats exclusive for the PkDBP gene family suggests that duplication of gene members postdated their divergence from the ancestral PcyDBP and PvDBP lineages. Intragenic recombination was detected in all DBP genes of these malaria species. Despite the limited number of isolates, P. knowlesi from Thailand shared phylogenetically related domain II sequences of both PkDBPα and PkDBPγ with those from Peninsular Malaysia, consistent with their geographic proximity.     

Sequence conservation in the Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) from hookworm infected individuals in Thailand

The Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) was one of the promising vaccine candidates against the most prevalent human hookworm species as adverse vaccine reaction has compromised further human vaccine trials. To elucidate the gene structure and the extent of sequence diversity, we determined the complete nucleotide sequence of the Na-asp-2 gene of individual larvae from 32 infected subjects living in 3 different endemic areas of Thailand. Sequence analysis revealed that the gene encoding Na-ASP-2 comprised 8 exons. Of 3 nucleotide substitutions in these exons, only one causes an amino acid change from leucine to methionine. A consensus conserved GT and AG at the 5′ and the 3′ boundaries of each intron was observed akin to those found in other eukaryotic genes. Introns of Na-asp-2 contained 23 nucleotide substitutions and 0–18 indels. The mean number of nucleotide substitutions per site (d) in introns was not significantly different from the mean number of synonymous substitutions per synonymous site (d_S) in exons whereas d in introns was significantly exceeded d_N (the mean number of nonsynonymous substitutions per nonsynonymous site) in exons (p < 0.05), suggesting that introns and synonymous sites in exons may evolve at a similar rate whereas functional constraints at the amino acid could limit amino acid substitutions in Na-ASP-2. A recombination site was identified in an intron near the 3′ portion of the gene. The positions of introns and the intron phases in the Na-asp-2 gene comparing with those in other pathogenesis-related-1 proteins of Loa loa, Onchocerca volvulus, Heterodera glycines, Caenorhabditis elegans and human were relatively conserved, suggesting evolutionary conservation of these genes. Sequence conservation in Na-ASP-2 may not compromise further vaccine design if adverse vaccine effects could be resolved whereas microheterogeneity in introns of this locus may be useful for population genetics analysis of N. americanus.     

Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites

The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 – KU680871). Four genes had a remarkable number of informative sites (P = 0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P = 0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D = − 2.2, P < 0.01), while COII and nagt genes were produced through evolutionary processes for both L. tropica and L. major (Tajima's D = 2.85 & 2.91, P < 0.01). More different clinical lesions with extensive phylogenetic and evolutionary analyses should be employed to avoid confusion in the diagnosis of leishmaniasis and development of vaccines for eradicating Leishmania parasites.     

Major tdh+ Vibrio parahaemolyticus serotype changes temporally in the Bay of Bengal estuary of Bangladesh

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006–2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n = 175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh⁺ V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRS^variant, were present only in the pandemic serotype O3:K6 (n = 13) and its serovariant O4:K68 (n = 2). Temporal distribution of the tdh⁺ serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh⁺ serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh⁺ O3:K6 and its pandemic serovariants from the tdh⁺ non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh⁺ strains, including the observed temporal change of the predominant tdh⁺ serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.     

Genetic characterization of HIV-1 strains in Togo reveals a high genetic complexity and genotypic drug-resistance mutations in ARV naive patients

In this study, the genetic diversity of HIV-1 and the presence of genotypic drug-resistance mutations in ARV naive patients in Lomé, the capital city of Togo, was documented for the first time. Between June 2006 and January 2007, 83 plasma samples were collected in Lomé from HIV-1 positive and antiretroviral (ARV) naive individuals. Pol (protease + RT) and env (V3–V5) regions were amplified and sequenced. Phylogenetic and recombination analyses were done to identify the HIV-1 variants. Pol sequences were then inspected to identify presence of drug-resistance mutations based on the WHO list recommended for epidemiological studies. A total of 75 plasma samples were amplified and sequenced in both genomic regions. The phylogenetic analysis showed that CRF02 (48.7% and 51.2%) and G (12.8% and 16.2%) were predominant, followed by A3 (6.4% and 6.2%) and CRF06 (3.8% and 12.5%) in pol and env, respectively. One strain was identified as CRF05 in pol and env. Two divergent subtype A strains in env were undetermined (U) in pol but clustered with a previously described complex recombinant strain, 99GR303. Overall, at least 23/83 (27.7%) strains were recombinant, 19 had a unique recombinant structure in pol, and 4 had discordant subtype/CRF designations between pol and env. The subtypes/CRFs involved in the recombination events corresponded to those already circulating as non-recombinant strains in the country. A total of 8 patients harbored strains with mutations associated to drug resistance: L90M (n = 1), K103N (n = 1), T69N (n = 1), T215S (n = 1), M41L (n = 4).In this study we showed the complexity of the HIV-1 strains circulating in Togo and documented a relative high proportion of ARV naive patients with drug-resistance mutations. The high number of resistant strains observed in Togo needs further attention and additional studies are needed to confirm this trend especially because the national ART program experienced major problems to provide drugs on a regular base.     

Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8⁺ cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.     

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA,lef and cya genes

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA < ΔhtrAΔcya < ΔhtrAΔlef < ΔhtrAΔlefΔcya) in attenuation – up to 10⁸-fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10⁹ spores) or double doses (>10⁷ spores) of the most attenuated triple mutant strain SterneΔhtrAlef^MUTΔcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10⁵ spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30 weeks, respectively.     

Phylogenetic study of clonal complex (CC)198 capsule null locus (cnl) genomes: A distinctive group within the species Neisseria meningitidis

Capsule null locus (cnl) strains, one type of specific unencapsulated Neisseria spp., only have regions D and E of the capsule gene cluster which encodes the genes for capsule biosynthesis, modification, and transportation. Compared with encapsulated strains, regions A and C of cnl strains have been replaced by 113 or 114 bp conserved non-coding sequences. Cnl strains include multiple clonal complexes (CC). According to previous studies, CC198 is the major clonal lineage in both cnl patients and healthy cnl carriers. We hypothesized that CC198 possesses different genome characteristics compared with other cnl strains. In this study, we obtained the draft genomes of two CC198 strains from healthy carriers. Using 75071 single nucleotide polymorphisms located in 1163 core genes, we constructed the phylogenetic relationships between a batch of representative Neisseria meningitidis genomes. CC198 and CC1136 clustered together, but apart from other N. meningitidis strains including CC53. We also aligned the sequences of genes located in regions D and E of the capsule gene locus from encapsulated and unencapsulated strains. A number of possible recombination events were identified in the galE and tex genes between different serogroups of encapsulated N. meningitidis and CC53 strains, especially in tex. In contrast, there is almost no recombination in N. meningitidis CC198 strains. These results showed that CC198 belongs to a phylogenetically distinct group within the species N. meningitidis, which may be directly derived from the cnl-type ancestor of N. meningitidis. The encapsulated strains may acquire other necessary genes for capsule formation by horizontal transfer.     

Serogroup,virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 74) and cockles (Anadara granosa) (n = 74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p < 0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p < 0.001) and trh (10.8% versus 1.4%) (p < 0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.     

Evaluation of novel fusion proteins derived from extracellular matrix binding domains of LigB as vaccine candidates against leptospirosis in a hamster model

Leptospira binds to host extracellular matrix (ECM) through surface exposed outer membrane proteins called adhesin in order to initiate infection. Of various adhesins present on the surface of the spirochete, Leptospira-immunoglobulin like proteins (Lig proteins) and LipL32 are most abundant, widely distributed among pathogenic serovars and well characterized. Various fragments of Lig proteins (Ligcon4, Ligcon4-7.5, LigBcen2) and C-terminus fragment of LipL32 all of that bind to host ECM were fused, expressed and purified in soluble form as fusion proteins. Four week hamsters were immunized subcutaneously with various fusion proteins emulsified in EMULSIGEN-D adjuvant and subsequently boosted at 3 weeks. The protective efficacy of these novel fusion proteins was evaluated against subsequent challenge with highly virulent L. interrogans serovar Pomona (MLD50-100). Our results indicate that fusion protein based vaccine induced significant protection against acute infection with respect to PBS-adjuvant vaccinated controls as revealed by enhanced survival and reduced pulmonary hemorrhage. Moreover, the protection mediated by these novel proteins was higher than that of conserved region of Lig protein (Ligcon, established protective antigen) and correlated to the level of antibodies. LipL32 failed to impart significant protection, however fusing its immunogenic C-terminus domain to Lig fragments slightly delayed the morbidity of the infected animals. Our results demonstrate that this novel strategy could be promising in developing effective subunit vaccine to combat this zoonotic infection.     

Leptospira wolffii,a potential new pathogenic Leptospira species detected in human,sheep and dog

Leptospirosis is the most common zoonotic disease, which is transmitted to humans through contaminated water or direct exposure to the urine of infected animals. In this study, the presence and prevalence of Leptospira species in the infected samples of human (n = 369) and sheep (n = 75) sera and also dogs’ urine (n = 150), collected from four provinces of Iran, were investigated by using nested-PCR/RFLP assay followed by sequencing analysis. Nested-PCR assay detected that 98/369 (26.5%) human, 13/75 (17.33%) of sheep's sera and 33/150 (22%) dogs’ urine samples were positive for Leptospira DNA. RFLP assay detected that all positive cases had either pathogenic or intermediate Leptospira species. By sequence analysis, Leptospira interrogans was the most prevalent species among the examined samples of human (53/82, 64.6%) and sheep (11/13, 84.6%). However, in dog samples, Leptospira wolffii (27/29, 93.1%) was detected for the first time and was the dominant species. The presence of L. wolffii with 100% identity in clinical human samples and animals suspected with Leptospira may provide evidence for circulation of L. wolffii and its role in transmission cycle within human and animal hosts. In addition, this species can be potentially pathogenic to human and probably animal hosts. A large epidemiology survey would be needed to define the presence and the prevalence of this species in global endemic regions.     

DNA vaccines against leptospirosis: A literature review

Leptospirosis is an infectious disease caused by pathogenic Leptospira species. The vaccines that are currently available for leptospirosis are composed of whole-cell preparations and suffer from limitations such as low efficacy, multiple side-effects, poor immunological memory and lack of cross-protection against different serovars of Leptospira spp. In light of the global prevalence of this disease, the development of a more effective vaccine against leptospirosis is of paramount importance. Genetic immunization is a promising alternative to conventional vaccine development. In the last 25 years, several novel strategies have been developed for increasing the efficacy of DNA vaccines. Examples of such strategies include the introduction of novel plasmid vectors, adjuvants, alternate delivery routes, and prime-boost regimens. Herein we discuss the latest and most promising advances that have been made in developing DNA vaccines against leptospirosis. We also deliberate over the future directions that must be undertaken in order to improve results in this field.     

Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n = 32) and Plasmodium vivax (n = 6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species.     

Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70 kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII + V + VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.     

In-vineyard population structure of ‘Candidatus Phytoplasma solani’ using multilocus sequence typing analysis

‘Candidatus Phytoplasma solani’ is a phytoplasma of the stolbur group (16SrXII subgroup A) that is associated with ‘Bois noir’ and causes heavy damage to the quality and quantity of grapevine yields in several European countries, and particularly in the Mediterranean area. Analysis of ‘Ca. P. solani’ genetic diversity was carried out for strains infecting a cv. ‘Chardonnay’ vineyard, through multilocus sequence typing analysis for the vmp1, stamp and secY genes. Several types per gene were detected: seven out of 20 types for vmp1, six out of 17 for stamp, and four out of 16 for secY. High correlations were seen among the vmp1, stamp and secY typing with the tuf typing. However, no correlations were seen among the tuf and vmp1 types and the Bois noir severity in the surveyed grapevines. Grouping the ‘Ca. P. solani’ sequences on the basis of their origins (i.e., study vineyard, Italian regions, Euro-Mediterranean countries), dN/dS ratio analysis revealed overall positive selection for stamp (3.99, P = 0.019) and vmp1 (2.28, P = 0.001). For secY, the dN/dS ratio was 1.02 (P = 0.841), showing neutral selection across this gene. Using analysis of the nucleotide sequencing by a Bayesian approach, we determined the population structure of ‘Ca. P. solani’, which appears to be structured in 3, 5 and 6 subpopulations, according to the secY, stamp and vmp1 genes, respectively. The high genetic diversity of ‘Ca. P. solani’ from a single vineyard reflects the population structure across wider geographical scales. This information is useful to trace inoculum source and movement of pathogen strains at the local level and over long distances.     

Clonal nature and diversity of resistance,toxins and adhesins genes of meticillin-resistant Staphylococcus aureus collected in a Spanish hospital

In this study we determined the prevalence of genes coding for antimicrobial resistance, toxins, enzymes, immunoevasion and adhesins factors among 189 meticillin-resistant Staphylococcus aureus (MRSA) strains isolated from a third level hospital in Valladolid (Spain) between 2005 and 2008 in order to examine the relationship between these pathogenicity determinants, both individually and in combination, and the genetic background of main MRSA strains that are presents in Spanish hospitals. MRSA isolates were first characterised epidemiologically by a combination of molecular typing strategies like spa, SCCmec and multilocus sequence typing, and then, a cluster analysis based on pathogenicity factors genes was performed according to the hybridisation pattern of 65 virulence, 36 resistance, 15 adhesins, and 11 set/ssl genes on a Diagnostic DNA microarray (Alere StaphyType DNA microarray Jena, Germany). CC5-agr type II [ST125-SCCmecIV/VI (32.2%) or ST125-IV (19.1%), ST228-I (19.1%), ST146-IV (13.7%) and ST5- IV (0.5%)] isolates was widely distributed. CC8-agr type I [ST8-IV (11.5%), USA300 clone (0.5%), and ST239-III (1.1%)]; CC45-agr type II [ST45- IV (1.6%)], and the CC97-agr type I [ST97-IV] were also detected. We identified 42 different resistance genes profiles, 22 set/ssl genes profiles, and 91 different virulence profiles. However although the high genetic diversity of MRSA strains, mainly with respect to virulence factors genes, the results of the simultaneous assessment of resistance and virulence genes and the genetic background illustrated a correspondence relationship (p < 0.001) between the different clones and same resistance and virulence genes or clusters of them. During the study period we observed changes in molecular epidemiology of MRSA isolates and as a consequence we report the changes of the resistance and virulence potential of MRSA strains produced over time in our institution.