Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.

Secretion of five extracellular enzymes by strains of chromoblastomycosis agents

The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.

The monitoring of hematopoietic stem cell transplant donors and recipients from endemic areas for malaria

Malaria is an unusual complication after hematopoietic stem cell transplantation in non-endemic countries. However, transplant candidates, recipients and donors living in endemic regions frequently report previous episodes of malaria. This fact could represent an important risk for immunosuppressed recipients that could develop severe malaria cases. We report a case of hematopoietic stem cell transplant (HSCT) in which the donor had a history of previous malaria, and close monitoring was performed before and after procedure by parasitological and molecular tests. The donor presented Plasmodium vivax in thick blood smears one month after transplant and was treated according to Brazilian Health Ministry guidelines. The polymerase chain reaction (PCR) was able to detect malaria infection in the donor one week earlier than thick blood film. Even without positive results, the recipient was pre-emptively treated with chloroquine in order to prevent the disease. We highlight the importance of monitoring recipients and donors in transplant procedures with the aim of reducing the risk of malaria transmission.

Cytokines and T-Lymphocute count in patients in the acute and chronic phases of Bartonella bacilliformis infection in an endemic area in peru: a pilot study

Human Bartonellosis has an acute phase characterized by fever and hemolytic anemia, and a chronic phase with bacillary angiomatosis-like lesions. This cross-sectional pilot study evaluated the immunology patterns using pre- and post-treatment samples in patients with Human Bartonellosis. Patients between five and 60 years of age, from endemic areas in Peru, in the acute or chronic phases were included. In patients in the acute phase of Bartonellosis a state of immune peripheral tolerance should be established for persistence of the infection. Our findings were that elevation of the anti-inflammatory cytokine IL-10 and numeric abnormalities of CD4+ and CD8+ T-Lymphocyte counts correlated significantly with an unfavorable immune state. During the chronic phase, the elevated levels of IFN-γ and IL-4 observed in our series correlated with previous findings of endothelial invasion of B. henselae in animal models.

Rickettsia parkeri: a Rickettsial pathogen transmitted by ticks in endemic areas for spotted fever rickettsiosis in southern Uruguay

At first Rickettsia conorii was implicated as the causative agent of spotted fever in Uruguay diagnosed by serological assays. Later Rickettsia parkeri was detected in human-biting Amblyomma triste ticks using molecular tests. The natural vector of R. conorii, Rhipicephalus sanguineus, has not been studied for the presence of rickettsial organisms in Uruguay. To address this question, 180 R. sanguineus from dogs and 245 A. triste from vegetation (flagging) collected in three endemic localities were screened for spotted fever group (SFG) rickettsiosis in southern Uruguay. Tick extracted DNA pools were subjected to PCR using primers which amplify a fragment of the rickettsial gltA gene. Positive tick DNA pools with these primers were subjected to a second PCR round with primers targeting a fragment of the ompA gene, which is only present in SFG rickettsiae. No rickettsial DNA was detected in R. sanguineus. However, DNA pools of A. triste were found to be positive for a rickettsial organism in two of the three localities, with prevalences of 11.8% to 37.5% positive pools. DNA sequences generated from these PCR-positive ticks corresponded to R. parkeri. These findings, joint with the aggressiveness shown by A. triste towards humans, support previous data on the involvement of A. triste as vector of human infections caused by R. parkeri in Uruguay.

USE OF THE POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF ASYMPTOMATIC Leishmania INFECTION IN A VISCERAL LEISHMANIASIS-ENDEMIC AREA

The diagnosis of asymptomatic infection with Leishmania (Leishmania) chagasi has become more important over recent years. Expansion of visceral leishmaniasis might be associated with other routes of transmission such as transfusion, congenital or even vector transmission, and subjects with asymptomatic infection are potential reservoirs. Moreover, the identification of infection may contribute to the management of patients with immunosuppressive conditions (HIV, transplants, use of immunomodulators) and to the assessment of the effectiveness of control measures. In this study, 149 subjects living in a visceral leishmaniasis endemic area were evaluated clinically and submitted to genus-specific polymerase chain reaction (PCR), serological testing, and the Montenegro skin test. Forty-nine (32.9%) of the subjects had a positive PCR result and none of them developed the disease within a follow-up period of three years. No association was observed between the results of PCR, serological and skin tests. A positive PCR result in subjects from the endemic area did not indicate a risk of progression to visceral leishmaniasis and was not associated with a positive result in the serological tests.

HISTOPLASMIN SURVEY IN HIV-POSITIVE PATIENTS: RESULTS FROM AN ENDEMIC AREA IN NORTHEASTERN BRAZIL

SUMMARY Background. Disseminated histoplasmosis is common in AIDS patients with advanced immunosuppression in Ceará, Northeastern Brazil. The goal of this study was to determine the prevalence of Histoplasma infection in patients with HIV/AIDS living in Fortaleza, the capital of Ceará. Methods. Intradermal tests with histoplasmin (mycelial phase) were performed in 161 HIV patients with CD4 ≥ 350 cells/mm 3 . Evidence of recent illness was evaluated with immunodiffusion (ID) tests in 76 of these individuals. Results. A total of 11.8% of patients reacted to histoplasmin and 2.63% had ID test positive to Histoplasma. The presence of mango trees (Mangifera indica) in the patient neighborhood (OR = 2.870; 95% CI = 1.081-7.617; p = 0.040) and past activity involving soil (OR = 2.834; 95% CI = 1.045-7.687; p = 0.045) or visits to a farm (OR = 3.869; 95% CI = 1.189-12.591; p = 0.033) were significantly associated with Histoplasma infection. Conclusions. Patients with HIV living in Fortaleza have an expressive prevalence of infection with Histoplasma.

FIRST REPORT OF METALLO-β-LACTAMASES PRODUCING Enterobacter spp. STRAINS FROM VENEZUELA

Clinical strains of Enterobacter were isolated from Cumana's Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes(7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacaeshowed blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum β-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

SEROLOGICAL AND MOLECULAR SURVEY OF Leptospira spp. AMONG CART HORSES FROM AN ENDEMIC AREA OF HUMAN LEPTOSPIROSIS IN CURITIBA, SOUTHERN BRAZIL

Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

Introduction:American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo.Methods:Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus.Results:Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample.Conclusion:These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci.