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

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

Phenotypic CS5 levels in LB alone and LB supplemented with bile or individual bile salts.

<p>CS5 expression was quantified by inhibition ELISA after overnight culture to stationary phase of strains E1777, E1779, E1785, E2265, and E3003. CS5 surface expression tifwas induced by NaGCH, but not by the corresponding unconjugated bile salt NaCH or its tauro-conjugated counterpart TCA (representative data).</p

Dose-dependent induction of phenotypic CS5 expression by NaGCH.

<p>Expression of CS5 was determined by inhibition ELISA in strain E1777 after overnight culture to stationary phase in LB medium supplemented with NaGCH or crude bile. Bars indicate means and standard errors of the means of two measurements in one experiment.</p

Levels of <i>csfD</i> and <i>cssB</i> transcription compared to in LB alone after one hour of culture.

<p>Level of <i>csfD</i> (A) and <i>cssB</i> (B) transcription in LB supplemented with crude bile or individual bile salts standardized to the level of transcription in LB medium alone, after one hour of culture. Transcription was measured by reverse transcriptase real time PCR. Bars show means and standard errors of the means of three separate experiments (strains E1785, E2265, and E3003, respectively). *, P = <0.05</p