Molecular characterisation of Salmonella strains isolated from outbreaks and sporadic cases of diarrhoea occurred in Paraná State, South of Brazil.

A total of 46 strains of Salmonella isolated from patients with sporadic diarrhoea or involved in foodborne outbreaks were analysed by PCR for genus identification and serotyping. Subtyping was performed using pulsed-field gel electrophoresis (PFGE) and multiple amplification of phage locus typing (MAPLT) for seven variable loci. Bacteria were identified as belonging to serotype Enteritidis (33 strains; 71·7%) or Typhimurium (13 strains; 28·3%). A high similarity coefficient (94·6%) was observed in the Salmonella Enteritidis group for which were found three related PFGE profiles and only one MAPLT; strains representing profile PA/P1/MI were prevalent (27; 81·8%). Two Salmonella Typhimurium isolates were untypeable by PFGE. The remaining 11 strains had eight PFGE and three MAPLT profiles. The discriminatory power of MAPLT was lower than that of PFGE. Salmonella Enteritidis of clonal nature is predominant in Paraná State, with the most prevalent profile PA/P1/M1 associated with sporadic diarrhoea and with seven of nine reported outbreaks. In conclusion, PFGE shows higher discriminatory power among Salmonella strains.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories.

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions.

Proteins differentially expressed by Shiga toxin-producing Escherichia coli strain M03 due to the biliar salt sodium deoxycholate.

Shiga toxin-producing Escherichia coli (STEC) can cause conditions ranging from diarrhea to potentially fatal hemolytic uremic syndrome. Enteropathogen adaptation to the intestinal environment is necessary for the development of infection, and response to bile is an essential characteristic. We evaluated the response of STEC strain M03 to the bile salt sodium deoxycholate through proteomic analysis. Cell extracts of strain M03 grown with and without sodium deoxycholate were analyzed by two-dimensional electrophoresis; the differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three proteins were found to be differentially expressed due to sodium deoxycholate. Glycerol dehydrogenase and phosphate acetyltransferase, which are involved in carbon metabolism and have been associated with virulence in some bacteria, were downregulated. The elongation factor Tu (TufA) was upregulated. This protein participates in the translation process and also has chaperone activities. These findings help us understand strategies for bacterial survival under these conditions.

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control.

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.

Virulence characteristics and antimicrobial susceptibility of uropathogenic Escherichia coli strains.

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥10(5). The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II >8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%).

The functional polymorphisms -429T>C and -374T>A of the RAGE gene promoter are not associated with gestational diabetes in Euro-Brazilians.

The receptor for advanced glycation end products (RAGE or AGER) is a multiligand member of the immunoglobulin superfamily. RAGE is expressed in several tissues, including human myometrium, chorionic villi and placenta. Advanced glycation end products are the best studied ligands of RAGE; they have pro-inflammatory actions in human gestational tissues, increasing oxidative stress and the release of cytokines and prostaglandins. We investigated the association of RAGE gene promoter polymorphisms -429T>C (rs1800625) and -374T>A (rs1800624) with gestational diabetes. A sample of 750 unrelated European origin pregnant Brazilian women were classified as nondiabetic (control group, N = 600) or having gestational diabetes (N = 150) according to American Diabetes Association 2009 criteria. Genotyping was performed by PCR-RFLP. The frequencies of the rare alleles -429C (6.3 versus 9.1%) and -374A (26 versus 30%) were not significantly different between the gestational diabetes patients and healthy pregnant women. Also, the -429T>C and -374T>A polymorphisms were not associated with body mass index, lipid profile, fasting glycemia, HbA1C, or insulin requirement. We found that functional promoter polymorphisms of the RAGE gene were not associated with gestational diabetes or its complications in these Euro-Brazilian patients.

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhea in Paraná State, Brazil.

AIMS: To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC. METHODS AND RESULTS: A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx-positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx(1)eae ehxA and stx(1) respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested. CONCLUSIONS: These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence. SIGNIFICANCE AND IMPACT OF STUDY: These results indicate that molecular methods are required to diagnosis of STEC infections.

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil.

AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.