Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. CONCLUSION/SIGNIFICANCE: The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors

Pseudomonas boanensis sp. nov., a bacterium isolated from river water used for household purposes in Boane District, Mozambique

A Gram-negative rod with a single polar flagellum was isolated from a freshwater reservoir used for household purposes in Boane District, near Maputo, Mozambique, and designated as strain DB1T. Growth was observed at 30-42 °C (optimum, 30-37 °C) and with 0.5-1.5 % NaCl. Whole-genome-, rpoD- and 16S rRNA-based phylogenies revealed this isolate to be distant from other Pseudomonas species with Pseudomonas resinovorans, Pseudomonas furukawaii and Pseudomonas lalkuanensis being the closest relatives. Phenotypic analyses of strain DB1T showed marked differences with respect to type strains P. resinovorans CCUG 2473T, P. lalkuanensis CCUG 73691T, P. furukawaii CCUG 75672T and Pseudomonas otiditis CCUG 55592T. Taken together, our results indicate that strain DB1T is a representative of a novel species within the genus Pseudomonas for which the name Pseudomonas boanensis is proposed. The type strain is DB1T (=CCUG 62977T=CECT 30359T).SIDA 2012 and FORMAS-Sida 2010.https://www.microbiologyresearch.org/content/journal/ijsemVeterinary Tropical Disease

Expression of Colonization Factor CS5 of Enterotoxigenic Escherichia coli (ETEC) Is Enhanced In Vivo and by the Bile Component Na Glycocholate Hydrate

Enterotoxigenic Escherichia coli (ETEC) is an important cause of acute watery diarrhoea in developing countries. Colonization factors (CFs) on the bacterial surface mediate adhesion to the small intestinal epithelium. Two of the most common CFs worldwide are coli surface antigens 5 and 6 (CS5, CS6). In this study we investigated the expression of CS5 and CS6 in vivo, and the effects of bile and sodium bicarbonate, present in the human gut, on the expression of CS5. Five CS5+CS6 ETEC isolates from adult Bangladeshi patients with acute diarrhoea were studied. The level of transcription from the CS5 operon was approximately 100-fold higher than from the CS6 operon in ETEC bacteria recovered directly from diarrhoeal stool without sub-culturing (in vivo). The glyco-conjugated primary bile salt sodium glycocholate hydrate (NaGCH) induced phenotypic expression of CS5 in a dose-dependent manner and caused a 100-fold up-regulation of CS5 mRNA levels; this is the first description of NaGCH as an enteropathogenic virulence inducer. The relative transcription levels from the CS5 and CS6 operons in the presence of bile or NaGCH in vitro were similar to those in vivo. Another bile salt, sodium deoxycholate (NaDC), previously reported to induce enteropathogenic virulence, also induced expression of CS5, whereas sodium bicarbonate did not

Studies on the Expression and Regulation of Enterotoxins and Colonization Factors in Enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute watery diarrhoea in developing countries, particularly among local children less than five years and is also the most common cause of diarrhoea in travellers to ETEC endemic areas. The infection is transmitted by ingestion of contaminated food and water and the disease is established in the small intestine. Colonization factors (CFs) on the bacterial surface mediate adhesion to the intestinal epithelium and diarrhoea is manifested by the actions of a heat-stable (ST) and / or a heat-labile (LT) enterotoxin. Two of the most common CFs in strains isolated world-wide are coli surface antigens 5 (CS5) and 6 (CS6). In this thesis the expression and regulation of these important virulence factors as well as the genetic variability among ETEC strains have been studied. Using ETEC strains isolated directly from diarrhoeal stool specimens of Bangladeshi patients without sub-culturing the gene expression of the two enterotoxins as well as the two CFs were studied in vivo. By also quantifying the transcription levels of the respective genes after in vitro culture we found that there was no significant up- or down-regulation of transcription of the genes encoding ST (estA) or LT (eltB) in vivo as compared to in vitro; however, the CS5 operon was up-regulated 100-fold and CS6 operon 10-fold in vivo. By culturing clinical strains under various conditions in vitro, ST, LT, CS5 and CS6 were shown to be differentially regulated by certain environmental factors, i.e. the presence of bile salts, lack of oxygen and different carbon sources (glycerol, glucose and amino acids). Thus, secretion of ST was down-regulated by glucose as carbon source under certain conditions but up-regulated by casamino acids, LT was only secreted in complex media in the absence of bile salts and presence of oxygen, phenotypic expression of CS5 on the bacterial surface was induced by bile salts and down-regulated by lack of oxygen, and expression of CS6 was up-regulated by lack of oxygen. An important finding was that the regulation of expression of these virulence factors does not seem to occur at the transcriptional level of the virulence operons. A majority of wild-type LT-only ETEC strains that were genotypically positive for CS6, but that did not express CS6 on the bacterial surface, were shown to contain truncating mutations within the functional chaperone subunit. This mutation was predicted to severely affect the capacity of the chaperone to bind to the structural subunits, thus indicating a requirement for a functional chaperone for surface expression of CS6. In addition, a single-point mutation was identified in the non-coding region up-stream of the chaperone-encoding gene in these strains; this mutation was found in strains isolated in diverse geographical areas and belonging to different clonal groups. By investigating the genetic relationship between ST-only CS6 positive strains isolated from children in a region highly endemic for ETEC, i.e. Guatemala, and adult travellers to the same region we found that these two groups may be infected by strains of the same genetic background and that ST-only CS6 positive strains belonging to several clonal complexes circulate in this area. We suggest that an ST-only CS6 positive ETEC strain belonging to the most common clonal complex, which was present during several years and found in strains isolated both from children and adults, may be considered as a candidate vaccine strain

Role of different genes in the CS6 operon for surface expression of Enterotoxigenic Escherichia coli colonization factor CS6

Coli surface antigen 6, CS6, is one of the most prevalent colonization factors (CFs) associated with Enterotoxigenic Escherichia coli (ETEC) bacteria, the most common cause of diarrhea among infants and children in developing countries. The CS6 operon encodes two structural subunit proteins, CssA and CssB, a chaperon, CssC, and an usher, CssD. Since little is known about the relationship of the genes and their role in expression and surface assembly of CS6, the operon was cloned into a laboratory E. coli strain, and mutants were constructed by creating deletions in each of the genes. Examination of protein expression by different methods, using monoclonal antibodies with specificities for the individual CS6 structural proteins, suggested that the usher (CssD) was not involved in assembly or surface expression of CS6 whereas deletion of the chaperon (CssC) significantly reduced levels of CssA, but not of CssB. Binding studies with CaCo-2 cells demonstrated that there is a combined effect of CssC and CssD on receptor binding, presumably associated with their activities in assembly and transport of the structural subunits. These findings may have important implications for the construction of strains expressing high levels of CS6 on the surface for eventual use in an ETEC vaccine

In vivo expression of the heat stable (estA) and heat labile (eltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro

Enterotoxigenic Escherichia coli multilocus sequence types in Guatemala and Mexico

The genetic backgrounds of 24 enterotoxigenic Escherichia coli (ETEC) strains from Mexico and Guatemala expressing heat-stable toxin (ST) and coli surface antigen 6 (CS6) were analyzed. US travelers to these countries and resident children in Guatemala were infected by ETEC strains of sequence type 398, expressing STp and carrying genetically identical CS6 sequences

IgA and IgG serum antibody responses to CS5 and CS6 in individual patients.

<p>The responses to CS5 (broken lines) and CS6 (solid lines) were measured by ELISA in sera from patients infected with strains E1777, E1785 and E1779, on different days after hospitalization. Serum from the patient infected with strain E2265 was not available.</p