BackgroundSeveral species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services.MethodsMultiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp.ResultsBy molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites.ConclusionsThis study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

Sequencing of DNA from 15 expanded-spectrum cephalosporin (e.g., ceftriaxone)-resistant Salmonella isolates obtained in the United States revealed that resistance to ceftriaxone in all isolates was mediated by cmy-2. Hybridization patterns revealed three plasmid structures containing cmy-2 in these 15 isolates. These data suggest that the spread of cmy-2 among Salmonella strains is occurring through mobilization of the cmy-2 gene into different plasmid backbones and consequent horizontal transfer by conjugation.Salmonellosis is primarily a food-borne disease that affects an estimated 1.4 million people each year in the United States (14). Expanded-spectrum cephalosporins (e.g., ceftriaxone and cefotaxime) are the antimicrobial agents of choice in the treatment of pediatric patients with invasive Salmonella infections (9). Until recently, resistance to expanded-spectrum cephalosporins was rarely reported among Salmonella strains (8). Review of 1996 data from the National Antimicrobial Resistance Monitoring System (NARMS) in the United States identified only 1 (0.1%) ceftriaxone-resistant Salmonella isolate among 1,272 human Salmonella isolates. However, by 1999 almost 2% of Salmonella isolates were ceftriaxone resistant, as determined by review of 1999 NARMS data (6). Comparisons of these ceftriaxone-resistant isolates found divergent strains, indicating multiple probable sources. The isolates either were different serotypes or, among patients infected with Salmonella enterica serotype Typhimurium, were distinguishable by their pulsedfield gel electrophoresis patterns, thus demonstrating that these ceftriaxone-resistant human isolates did not represent the epidemic spread of a clonal strain (6). This study was undertaken to confirm the identity of the ␤-lactamase conferring resistance to expanded-spectrum cephalosporins and characterize the associated plasmids from the apparently sporadic human Salmonella isolates collected through NARMS from 1996 to 1998. MATERIALS AND METHODSThe 15 bacterial strains used in the study are listed in Table 1. Thirteen of the isolates were obtained by the Centers for Disease Control and Prevention through NARMS. These 13 isolates represented 87% of the total expandedspectrum cephalosporin-resistant Salmonella isolates (n ϭ 15) obtained by the Centers for Disease Control and Prevention from 1996 to 1998 (6). Isolate SS034 was isolated in Nebraska, whereas isolate 922 was isolated in Ohio. Susceptibility testing of the Salmonella isolates and the Escherichia coli transconjugants and transformants was performed by the disk diffusion methodology according to NCCLS standards (16). The MIC for the pACYC184 construct containing cmy-2 was tested by the E-test (AB Biodisk, Solna, Sweden) methodology. The MICs of ceftiofur (kindly provided by Pharmacia/Upjohn) were determined by broth microdilution (15, 17). Plasmid DNA was extracted either by the method of Kado and Liu (10) or with the Concert Purification Midi kit (Life Technologies, Milan, Italy) and digested with PstI (Roche, Indianapoli...

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extendedspectrum beta-lactamase gene, bla SHV-5 , was identified 3.5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla SHV-5 -In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expandedspectrum cephalosporins and aminoglycosides.Although antibiotic resistance is becoming a major threat to human health worldwide, information concerning the dissemination and geographical distribution of antibiotic-resistant bacterial pathogens remains scattered (8,14,26). During the last decade many hospital outbreaks caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have been reported, and most of the ESBL-producing strains were found to carry the bla TEM-1 , bla TEM-2 , and bla SHV-1 gene derivatives (5,6,9,11,19,27). These genes are prevalently located on large conjugative plasmids of the incompatibility (Inc) groups IncC, IncFI, IncH12, and IncL/M (3,5,7,13,18,20,23,28).More recent reports have highlighted the emergence of ESBL-producing strains endowed with an extremely wide spectrum of antibiotic resistance, including resistance to sulfonamides, trimethoprim, streptomycin, kanamycin, gentamicin, and amikacin (17,21,23). However, the molecular mechanisms involved in the acquisition and/or transmission of ESBL-encoding genes are poorly understood.In previous work we identified an IncL/M plasmid, referred to as pSEM, in eight epidemiologically unrelated, multipledrug-resistant strains of Salmonella enterica serotype Typhimurium. This plasmid contains a sul1-type class 1 integron (In-t3) carrying the aacA4, aacC1, and aadA1 gene cassettes and conferring resistance to sulfonamides, kanamycin, gentamicin, and amikacin (29). The streptomycin and spectinomycin-resistance determinant, aadA1, is poorly expressed in In-t3 because it is located in a distal position relative to the main integron promoter P ant (29).The Salmonella strains harboring pSEM were analyzed in more detail with regard to their antibiotic resistance profile. Microdilution susceptibility tests demonstrated that all eight strains were resistant to ceftazidime (Ն16 g/ml), cefotaxime (Ն32 g/ml), ceftriaxone (Ն64 g/ml), cefamandole (Ն32 g/ ml), cefoxitin (Ն8 g/ml), and aztreonam (Ն16 g/ml) but sensitive to imipenem (Ͻ4 g/ml).To clone the gene responsible for resistance to expandedspectrum cephalosporins, an EcoRI genomic library was constructed in the pUC18 vector using total DNA extracted from the prototypic Salmonella 202 strain. The library was introduced by transformation in Escherichia coli DH5␣ competent cells, and selection was performed on Luria-Bertani agar plates containing ceftaz...

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ TM reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidiumspecific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells. Molecular & Cellular Proteomics 6:346 -355, 2007.