RFLP clusters of Mycobacterium tuberculosis strains from the Indian Ocean Region: local and South Asian characteristics

This is the first study describing the genetic polymorphism of Mycobacterium tuberculosis strains in the Indian Ocean Region. Using IS6110 RFLP analysis, 475 M. tuberculosis isolates from Madagascar, Comoros, Mauritius, Mozambique and La Reunion were compared. Of the 332 IS6110 profiles found, 43 were shared by clusters containing 2-65 strains. Six clusters were common to at least two countries. Of 52 families of strains with similar IS6110 profiles, 10 were common to at least two countries. Interestingly, another characteristic was the frequency (16.8%) of IS6110 single-copy strains. These strains could be distinguished using the DR marker. This preliminary evaluation suggests genetic similarity between the strains of the Indian Ocean Region. However, additional markers would be useful for epidemiological studies and to assess the ancient transmission of strains between countries of this region.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9%) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6%) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

Evaluation of the synergistic potential of vancomycin combined with other antimicrobial agents against methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp strains

Methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus spp (CNS) are the most common pathogens that cause serious long term infections in patients. Despite the existence of new antimicrobial agents, such as linezolid, vancomycin (VAN) remains the standard therapy for the treatment of infections caused by these multidrug-resistant strains. However, the use of VAN has been associated with a high frequency of therapeutic failures in some clinical scenarios, mainly with decreasing concentration of VAN. This work aims to evaluate the synergic potential of VAN plus sulfamethoxazole/trimethoprim (SXT), VAN plus rifampin (RIF) and VAN plus imipenem (IPM) in sub-minimum inhibitory concentrations against 22 clinical strains of MRSA and CNS. The checkerboard method showed synergism of VAN/RIF and VAN/SXT against two and three of the 22 strains, respectively. The combination of VAN with IPM showed synergistic effects against 21 out of 22 strains by the E-test method. Four strains were analyzed by the time-kill curve method and synergistic activity was observed with VAN/SXT, VAN/RIF and especially VAN/IPM in sub-inhibitory concentrations. It would be interesting to determine if synergy occurs in vivo. Evidence of in vivo synergy could lead to a reduction of the standard VAN dosage or treatment time.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Virulence variation among epidemic and non-epidemic strains of Saint Louis encephalitis virus circulating in Argentina

Saint Louis encephalitis virus caused an outbreak of febrile illness and encephalitis cases in Córdoba, Argentina, in 2005. During this outbreak, the strain CbaAr-4005 was isolated from Culex quinquefasciatus mosquitoes. We hypothesised that this epidemic variant would be more virulent in a mouse model than two other non-epidemic strains (78V-6507 and CorAn-9275) isolated under different epidemiological conditions. To test this hypothesis, we performed a biological characterisation in a murine model, including mortality, morbidity and infection percentages and lethal infection indices using the three strains. Mice were separated into age groups (7, 10 and 21-day-old mice) and analysed after infection. The strain CbaAr-4005 was the most infective and lethal of the three variants, whereas the other two strains exhibited a decreasing mortality percentage with increasing animal age. The strain CbaAr-4005 produced the highest morbidity percentages and no significant differences among age groups were observed. The epidemic strain caused signs of illness in all inoculated animals and showed narrower ranges from the onset of symptoms than the other strains. CbaAr-4005 was the most virulent for Swiss albino mice. Our results highlight the importance of performing biological characterisations of arbovirus strains likely to be responsible for emerging or reemerging human diseases.

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.

CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Draft genome of the Leptospira interrogans strains, Acegua, RCA, Prea, and Capivara, obtained from wildlife maintenance hosts and infected domestic animals

In the present paper, we announce new draft genomes of four Leptospira interrogans strains named Acegua, RCA, Prea, and Capivara. These strains were isolated in the state of Rio Grande do Sul, Brazil, from cattle, dog, Brazilian guinea pig, and capybara, respectively.