Factors associated with typical enteropathogenic Escherichia coli infection among children <5 years old with moderate-to-severe diarrhoea in rural western Kenya, 2008-2012.

Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 µM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Otitis media among high-risk populations: can probiotics inhibit Streptococcus pneumoniae colonisation and the risk of disease?

Otitis media is the second most common infection in children and the leading cause for seeking medical advice. Indigenous populations such as the Inuits, indigenous Australians and American Indians have a very high prevalence of otitis media and are considered to be high-risk populations. Streptococcus pneumoniae, one of the three main bacterial causes of otitis media, colonises the nasopharynx prior to disease development. In high-risk populations, early acquisition of high bacterial loads increases the prevalence of otitis media. In these settings, current treatment strategies are insufficient. Vaccination is effective against invasive pneumococcal infection but has a limited impact on otitis media. Decreasing the bacterial loads of otitis media pathogens and/or colonising the nasopharynx with beneficial bacteria may reduce the prevalence of otitis media. Probiotics are live microorganisms that offer health benefits by modulating the microbial community and enhancing host immunity. The available data suggest that probiotics may be beneficial in otitis media. This review discusses the potential use of probiotics to reduce pathogen colonisation and decrease the prevalence of otitis media, providing justification for further investigation.

Lactobacillus GG treatment during pregnancy for the prevention of eczema: a randomized controlled trial.

BACKGROUND: Probiotic supplementation in early life may be effective for preventing eczema. Previous studies have suggested that prenatal administration may be particularly important for beneficial effects. OBJECTIVE: We examined whether prenatal treatment with the probiotic Lactobacillus rhamnosus GG (LGG) can influence the risk of eczema during infancy. METHODS: We recruited 250 pregnant women carrying infants at high risk of allergic disease to a randomized controlled trial of probiotic supplementation (LGG 1.8 × 10(10) cfu/day) from 36 weeks gestation until delivery. Infants were assessed during their first year for eczema or allergic sensitization. Immunological investigations were performed in a subgroup. Umbilical cord blood was examined for dendritic cell and regulatory T cell numbers and production of TGFß, IL-10, IL-12, IL-13, IFN-γ and TNFα. Maternal breast milk was examined for total IgA, soluble CD14 and TGFß. RESULTS: Prenatal probiotic treatment was not associated with reduced risk of eczema (34% probiotic, 39% placebo; RR 0.88; 95% CI 0.63, 1.22) or IgE-associated eczema (18% probiotic, 19% placebo; RR 0.94; 95% CI 0.53, 1.68). Prenatal probiotic treatment was not associated with any change in cord blood immune markers, but was associated with decreased breast milk soluble CD14 and IgA levels. CONCLUSIONS: Prenatal treatment with Lactobacillus rhamnosus GG was not sufficient for preventing eczema. If probiotics are effective for preventing eczema, then a postnatal component to treatment or possibly an alternative probiotic strain is necessary.

Effects of Lactobacillus GG treatment during pregnancy on the development of fetal antigen-specific immune responses.

BACKGROUND: Several clinical trials suggest that probiotics may have a role in the prevention of eczema. The optimal timing and mechanisms underlying this intervention are not clear. In particular it is not known whether such treatment works during pregnancy or whether postnatal exposure is important. OBJECTIVE: We investigated whether the probiotic Lactobacillus rhamnosus strain GG (LGG) influences fetal immune responses when administered to pregnant women, as a possible mechanism for its protective effects against the development of eczema. METHODS: Peripheral blood mononuclear cell from 11 adults treated with LGG, and cord blood mononuclear cells (CBMCs) from 73 women participating in a randomized controlled trial of LGG treatment were cultured with heat-killed LGG, ovalbumin (OVA) or without stimulus. Cells were analysed by flow cytometry and real-time PCR for markers of dendritic cell (DC) phenotype, T cell proliferation and regulation. Cytokine secretion was analysed in culture supernatants by multiplex cytokine assay. RESULTS: LGG treatment of adults led to systemic immune responses suggestive of antigen-specific tolerance including reduced CD4(+) T cell proliferation to heat-killed LGG (30% reduction; P=0.03). LGG treatment of pregnant women did not influence CD4(+) T cell proliferation, forkhead box P3 expression, DC phenotype or cytokine secretion in CBMCs cultured with heat-killed LGG or OVA. CONCLUSION: LGG treatment of pregnant women fails to influence fetal antigen-specific immune responses. This suggests that modulation of fetal immune responses may not be a major mechanism by which probiotics such as LGG prevent eczema.

Role of YopH in the suppression of tyrosine phosphorylation and respiratory burst activity in murine macrophages infected with Yersinia enterocolitica.

Tyrosine phosphorylation is an important component of the signaling pathways responsible for the activation of the macrophage respiratory burst. Because the virulence plasmid of Yersinia enterocolitica encodes a phosphotyrosine phosphatase, YopH, it is possible that the pathogenic strategy of Y. enterocolitica involves the disruption of tyrosine phosphorylation in the macrophage leading to inhibition of respiratory burst activity. We have investigated the effects of Yersinia infection on tyrosine phosphorylation and respiratory burst activity in murine bone marrow-derived macrophages. Infection of macrophages with virulent [Ye(pYV+)] but not avirulent [Ye(pYV-)] strains of Y. enterocolitica was found to suppress both tyrosine phosphorylation and respiratory burst activity in response to zymosan. Mutational inactivation of YopH reversed the suppressive effect of Ye(pYV+) on zymosan-induced tyrosine phosphorylation, indicating that YopH is responsible for the dephosphorylation of macrophage phosphotyrosine-containing proteins observed in macrophages infected with the virulent strain of Y. enterocolitica. In contrast, mutational loss of YopH failed to reverse the inhibitory effect of Ye(pYV+) on the zymosan-triggered respiratory burst. We conclude that the inhibition of the macrophage respiratory burst by Y. enterocolitica involves a plasmid-encoded virulence protein(s) other than, or in addition to, YopH.

Analysis of the urease gene complex of members of the genus Yersinia.

The urease gene complex of Yersinia enterocolitica is relatively conserved within the species, although this conservation may not extend to other members of the genus. Spontaneous urease-negative isolates of Y. enterocolitica appear to have arisen as a result of large deletions within this complex, while Y. pestis shows no significant deletions within the complex, despite being urease negative.

Characterisation of the urease-encoding gene complex of Yersinia enterocolitica.

A cosmid gene library of chromosomal DNA from Yersinia enterocolitica A2635 (serogroup O:8) was constructed in Escherichia coli. Subcloning of a urease-positive (Ure+) clone revealed a region of 6.6 kb that was sufficient for expression of Ure activity in E. coli. Sequencing of this fragment disclosed seven ORFs transcribed in the same direction. On the basis of homology to known Ure, these were designated ureA, ureB, ureC, ureE, ureF, ureG and ureD, which are predicted to encode polypeptides of 11.1, 17.9, 61.0, 29.5, 25.0, 24.1 and 36.4 kDa, respectively. The polypeptides encoded by the ure gene complex of Y. enterocolitica are significantly divergent from those encoded by the ure operons of other Enterobacteriaceae, which appear to be closely related to each other. This suggests that the ure genes were acquired by Y. enterocolitica from an unrelated organism or alternatively, that they diverged from those of other Enterobacteriaceae some considerable time ago.

Effect of Shiga toxin and Shiga-like toxins on eukaryotic cells.

Shigella dysenteriae and Shiga-toxin-producing Escherichia coli (STEC) elaborate the AB holotoxins, Shiga or Shiga-like toxins (Stx). Stx play a major role in the pathogenesis of haemorrhagic colitis and haemolytic uremic syndrome. This review provides an overview of the mechanisms of action of Stx and a model of the pathogenesis of Stx-induced disease.

The fate of ingested lactobacilli in the proximal small intestine.

A freeze-dried commercial preparation of Lactobacillus acidophilus and Lactobacillus bulgaricus (Lactinex) dissolved in skim milk was ingested by four nonfasting and seven fasting informed community volunteers in the Isolation Ward of the Center for Vaccine Development. Samples of jejunal fluid withdrawn from the volunteers at varying intervals were cultured for lactobacilli on a selective medium. Quantitative counts varied considerably amongst the individuals studied and also in the same person examined on two consecutive occasions. In general, however, it was shown that lactobacilli entered the small intestine and persisted in elevated numbers for about 3 h in fasting subjects and for up to 6 h in nonfasting individuals. By 4 h, counts in fasting volunteers had returned to base-line levels. Although both Lactobacillus species in Lactinex entered the intestine in approximately equal numbers, L. acidophilus was recovered from more samples and in slightly greater number than L. bulgaricus.

Elimination of extracellular bacteria by antibiotics in quantitative assays of bacterial ingestion and killing by phagocytes.

In vitro assays to quantify the bactericidal capacity of phagocytes require the removal of extracellular bacteria from the reaction mixture before intracellular bacteria are released from the phagocytic cells and counted. This may be achieved by using an antibiotic, such as gentamicin, which has a rapid bactericidal action coupled with limited ability to cross cytoplasmic membranes. In this study, we investigated the susceptibility of Escherichia coli and Yersinia enterocolitica to killing by mouse peritoneal macrophages. Extracellular bacteria in the reaction mixture were eliminated with gentamicin, whereafter the antibiotic was enzymatically inactivated using gentamicin acetyl transferase (GAT). The method has few washing stages, thus reducing the likelihood of inadvertent removal of macrophages or bacteria from the reaction mixture. The assay disclosed clear differences in the ability of E. coli and Y. enterocolitica to survive phagocytosis by macrophages.

Effect of bacterial invasion of macrophages on the outcome of assays to assess bacterium-macrophage interactions.

In vitro assays to quantify killing of bacteria by macrophages provide useful insights into host-pathogen relations. In the present study, we used strains of Yersinia enterocolitica and Escherichia coli which varied in their ability to invade mammalian cells to evaluate these assays. The results showed that 30 min and 24 h after incubation with murine bone marrow-derived macrophages, strains of Y. enterocolitica and E. coli which expressed invasin (an outer membrane protein which allows bacteria to penetrate mammalian cells) achieved significantly greater numbers in macrophages than otherwise isogenic bacteria which lacked this protein (P < 0.01). When the 24-h data were corrected for the number of bacteria ingested by macrophages initially, the differences between invasin-positive and -negative bacteria were no longer evident (P> 0.2). This study has shown (1) that invasin-mediated penetration of macrophages by bacteria is not associated with enhanced intracellular survival, and (2) that invasion of macrophages by bacteria may influence the interpretation of assays for bactericidal capacity unless allowance is made for the number of bacteria ingested during the early phase of the assay.

Campylobacter upsaliensis gastroenteritis in childhood.

BACKGROUND: Campylobacter upsaliensis can cause gastroenteritis and bacteremia. Data on its epidemiology and role in pediatric gastroenteritis are limited. OBJECTIVE: To describe the incidence and clinical features of enteric C. upsaliensis infection in children and to compare these with similar data for Campylobacter jejuni. DESIGN AND METHODS: Medical records of all patients with enteric C. upsaliensis infection between 1992 and 1999 at the Royal Children's Hospital, Melbourne, were reviewed. A case-control study (age-matched 1:2) was performed to compare the severity of clinical disease and associated risk factors for infection with C. upsaliensis and C. jejuni. RESULTS: Of 18,516 specimens 666 (3.6%) were positive for C. jejuni and 19 (0.1%) were positive for C. upsaliensis. Records were available for 18 patients with C. upsaliensis gastroenteritis (mean age, 1.6 years; median age, 1.3 years; range, 3 months to 7 years; 14 male). Eleven patients (61%) presented with acute and 7 (39%) with chronic or intermittent diarrhea. The case-control study showed that fever (P = 0.03), acute diarrhea (P = 0.05) and rectal bleeding (P = 0.0006) were significantly less common in C. upsaliensis than in C. jejuni infection. CONCLUSION: C. upsaliensis is a rare cause of gastroenteritis in young children and, compared with C. jejuni infection, is associated with significantly lower rates of fever, acute diarrhea and rectal bleeding.

Thrombocytopenia in trypanosomiasis.

In all of four patients with African trypanosomiasis, thrombocytopenia was present on admission to hospital or developed during the course of the illness. One patient with severe thrombocytopenia died following gastrointestinal haemorrhage shortly after admission to hospital. Kinetic studies in the other three patients showed marked pooling of platelets in the spleen in all, but severe shortening of platelet life-span in only one. Evidence of disseminated intravascular coagulation (DIC) was found in 3 patients, 2 of whom received heparin therapy. These findings provide evidence that thrombocytopenia is a feature of African trypanosomiasis and is due mainly to hypertrophy of the reticuloendothelial system which accompanies the infection. In some patients immune damage to platelets or platelet consumption as part of DIC may be an additional factor contributing to the thrombocytopenia.

Treatment of acute nonspecific gastroenteritis of infants and young children with erythromycin.

A double-blind placebo-controlled trial of erythromycin ethylsuccinate was conducted in 65 infants and young children hospitalized with acute nonspecific gastroenteritis. Etiologic agents included rotaviruses (29%), Campylobacter jejuni (17%), "classical" enteropathogenic Escherichia coli (12%), enterotoxigenic E. coli (11%), Salmonella (9%), Shigella (2%), and Giardia lamblia (2%). No pathogens were obtained from 25 (38%) children. Treatment with erythromycin had no effect on the course of the illness in terms of the time required for hydration, stool frequency and temperature to return to normal, or for vomiting to be abolished. Children treated with erythromycin, however, experienced a marginally, but significantly (P less than 0.05), shorter period of abnormal stool consistency compared with control subjects. This effect was most pronounced in children from whom no enteropathogens were isolated.

In vitro association between the virulence proteins, YopD and YopE, of Yersinia enterocolitica.

The virulence plasmid (pYV) of Y. enterocolitica encodes a set of anti-host proteins, known as Yops, which contribute to survival of the bacteria in the host. Several Yops directly influence the interaction of pathogenic bacteria with host cells which in the case of at least four Yops, YopE, YopH, YopM and YpkA (YopO), involves translocation of the Yop across the eukaryotic cell membrane into the cytoplasm of target cells. Translocation requires the presence of two other pYV-encoded proteins, YopB and YopD, but it is not clear how these proteins mediate translocation of the effector Yops. We have used an affinity blot technique (overlay) to examine potential binding between YopD and other Yops. The results indicated that under the in vitro conditions used in this study, YopD bound preferentially to YopE and YopB. To investigate the interaction of YopD and YopE in more detail we produced different regions of the YopD protein in Escherichia coli by creating fusions of YopD with glutathione S-transferase. These studies showed that YopE bound the central hydrophobic region of YopD. This is the first demonstration of protein interactions between an effector Yop and a putative Yop translocator.

A novel mechanism of urease regulation in Yersinia enterocolitica.

Yersinia enterocolitica produces the enzyme urease which hydrolyses urea, resulting in the production of carbonic acid and ammonia and a net increase in pH. In the presence of urea, urease enhances survival of Y. enterocolitica in the stomach and presumably in other acidic environments the bacteria encounter during the course of infection. In this study we show that Y. enterocolitica urease is a cytosolic enzyme which has a low Km value (0.15 +/- 0.01 mM urea), suggesting that it functions at close to maximum velocity even at the low concentrations of urea available to Y. enterocolitica in gastric fluid and other tissues. Y. enterocolitica urease was active over a wide pH range, but unlike most other bacterial ureases, displayed an optimal activity at pH 3.5-4.5, suggesting a physiological role in protecting the bacteria from acid. Higher levels of urease activity were attained at 28 degrees C than at 37 degrees C, and investigation of the regulation of urease production revealed that the enzyme was not induced by urea, or by nitrogen limitation. Instead maximal activity was attained during the stationary phase of growth which coincides with the period of maximum acid tolerance of the bacteria. This type of regulation has not been described for any other ureolytic bacteria and seems to be unique to Y. enterocolitica.

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature.

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y. enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y. enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y. frederiksenii hybridization group 2 (unnamed genomospecies 2). This findings was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified.

Examination of Serpulina pilosicoli for attachment and invasion determinants of Enterobacteria.

The spirochaete, Serpulina pilosicoli, is the agent of intestinal spirochaetosis, a diarrhoeal disease of humans and other species. By mechanisms as yet unknown, large numbers of these spirochaetes intimately attach to the colonic mucosa by one cell end. In some infected individuals, the spirochaetes may invade the lamina propria and adjacent tissues, and they may cause spirochaetaemia. To examine S. pilosicoli for pathogenic determinants homologous with Enterobacteria, DNA was extracted from six strains of S. pilosicoli and hybridised at low stringency with DNA probes derived from the inv, ail and yadA genes of Yersinia enterocolitica, the eae gene from enteropathogenic Escherichia coli and a probe derived from the virulence plasmid of Shigella flexneri. No hybridisation of the enterobacterial probes to S. pilosicoli DNA was detected, indicating that these gene sequences, which are known to be involved in the attachment and invasion processes of the other intestinal pathogens, were not present in the spirochaetes.

Serological response of sheep to plasmid-encoded proteins of Yersinia species following natural infection with Y. enterocolitica and Y. pseudotuberculosis.

A prospective study of the serological response to natural infection with Yersinia enterocolitica and Y. pseudotuberculosis was performed in an experimental flock of sheep. A preliminary investigation with immunoblotting techniques showed that lambs infected with virulent Yersinia spp. produced antibodies to several yersinia outer-membrane proteins (yops) encoded by a virulence plasmid (pYV) of Y. enterocolitica or Y. pseudotuberculosis. Thereafter, an enzyme immunoassay (EIA) was developed to measure antibodies to yops. Criteria for interpreting the EIA were established by examining sera from a negative control population of lambs which had not been infected with Yersinia spp. since birth. Test samples comprised 25 pairs of pre- and post-infection sera from animals with bacteriologically proven infections with Yersinia spp. The results showed that infection of lambs with pYV-bearing strains of Y. enterocolitica or Y. pseudotuberculosis invariably evoked a significant antibody response to yops, even though all the infections were subclinical. No animal infected with so-called "environmental", pYV-negative Yersinia spp. seroconverted to yops. EIA with yops as antigen provided a sensitive and specific means to diagnose subclinical infection of lambs with virulent Yersinia spp.