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.

The rise and rise of enteropathogenic Escherichia coli.

First described in 1885, Escherichia coli gradually achieved recognition as a cause of diarrhoea. Strains of E. coli which belonged to a limited number of O-serogroups and had been associated with outbreaks of diarrhoea in hospitalised children were designated 'enteropathogenic' E. coli (EPEC) to distinguish them from E. coli strains that cause other types of infection. The discovery that some strains of E. coli can produce toxins or invade epithelial cells in a similar fashion to established pathogens, such as Vibrio cholerae and Shigella species, shed new light on E. coli virulence. As EPEC do not exhibit either of these properties, however, their pathogenicity was brought into question. Further studies revealed that EPEC constitute a distinctive group of pathogenic bacteria that display characteristic adherence to cultured epithelial cells and produce distinctive histopathological changes, termed 'attaching-effacing lesions', in the intestinal epithelium. The ability to evoke these lesions, which are essential for virulence, is associated with a series of linked genes, known as the locus for enterocyte effacement, on the bacterial chromosome. In addition, the pathogenicity of some EPEC strains is associated with the presence of a plasmid-encoded, bundle-forming pilus. Naturally occurring strains of EPEC which lack this pilus are known as atypical EPEC and are an emerging cause of diarrhoea throughout the world.

A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli.

A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.

Brachyspira aalborgi infection in four Australian children.

AIM: The clinical presentation of four children and adolescents (two males and two females with a mean age of 12.4 years; range 9-16 years) with colorectal spirochetosis is discussed. RESULTS: Symptoms included persistent diarrhea (n = 2), rectal bleeding (n = 1) and abdominal pain (n = 2). In all patients, colorectal spirochetosis was an unanticipated finding on colonic histology, and the presence of spirochetes was confirmed by the use of electron microscopy. Spirochetes were identified as Brachyspira aalborgi by using PCR amplification of the bacterial 16S rRNA and nicotinamide adenine dinucleotide oxidase sequences in all four patients. No other enteric pathogens were found. CONCLUSIONS: Although all patients appeared to respond to antibiotic treatment, the clinical significance of B. aalborgi as a human pathogen requires further investigation.

Nationwide study of haemolytic uraemic syndrome: clinical, microbiological, and epidemiological features.

AIMS: To establish the incidence and aetiology of haemolytic uraemic syndrome (HUS) in Australia and compare clinical and microbial characteristics of sporadic and outbreak cases. METHODS: National active surveillance through the Australian Paediatric Surveillance Unit with monthly case notification from paediatricians, July 1994 to June 1998. Children under 15 years presenting with microangiopathic haemolytic anaemia, thrombocytopenia, and acute renal impairment were identified. RESULTS: Ninety eight cases were identified (incidence 0.64 per 10(5) children <15 years/annum and 1.35 per 10(5) children <5 years/annum). Eighty four were associated with diarrhoea (64 sporadic, 20 constituting an outbreak) and 14 were atypical. Shiga toxin producing Escherichia coli (STEC) O111:H- was the most common isolate in sporadic HUS and caused the outbreak. However O111:H- isolates from outbreak and sporadic cases differed in phage type and subtyping by DNA electrophoresis. STEC isolates from sporadic cases included O26:H-, O113:H21, O130:H11, OR:H9, O157:H-, ONT:H7, and ONT:H-. STEC O157:H7 was not isolated from any case. Only O111:H- isolates produced both Shiga toxins 1 and 2 and possessed genes encoding E coli attaching and effacing gene (intimin) and enterohemolysin. Outbreak cases had worse gastrointestinal and renal disease at presentation and more extrarenal complications. CONCLUSIONS: Linking national surveillance with a specialised laboratory service allowed estimation of HUS incidence and provided information on its aetiology. In contrast to North America, Japan, and the British Isles, STEC O157:H7 is rare in Australia; however, non-O157:H7 STEC cause severe disease including outbreaks. Disease severity in outbreak cases may relate to yet unidentified virulence factors of the O111:H- strain isolated.

EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri.

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic Escherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with Shigella flexneri protein VirA, and the cloned espG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPEC espG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned orf3 gene could also rescue invasion in a Shigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

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.

Immune response to infection with Mycobacterium ulcerans.

Mycobacterium ulcerans is a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses to M. ulcerans and M. bovis BCG in patients with M. ulcerans disease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-gamma). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-gamma production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or current M. ulcerans disease showed significantly reduced proliferation and production of IFN-gamma in response to stimulation with live M. ulcerans or M. bovis than PBMC from healthy, tuberculin test-positive subjects (P < 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P > 0.2). Serum from 9 of 11 patients with M. ulcerans disease, but no control subject, contained antibodies to M. ulcerans. The results indicate that patients with M. ulcerans infection mount an immune response to M. ulcerans as evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features of M. ulcerans-induced disease.

Contribution of plasmid-encoded fimbriae and intimin to capacity of rabbit-specific enteropathogenic Escherichia coli to attach to and colonize rabbit intestine.

Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal eae gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H-), in which the ralE and eae genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a delta eae Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its eae-positive parent and far more extensively than an eae(+) delta ral strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a delta eae delta ralE double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin.

Effect of short-term high dose of steroids on the healing of microvascular anastomoses in a rabbit model: pilot study.

Short-term high doses of steroids have been used in head and neck surgery to reduce swelling in free tissue transfers. This study was set up to assess the effect of this treatment on the healing of microvascular anastomoses in a rabbit model. Anastomoses were made in the common carotid artery and anterior facial vein in 40 animals, half of which were given high doses of methylprednisolone (2.5 mg/kg/day) intravenously for 48 h peri-operatively. The patency of the vessels was assessed and they were harvested for histological examination at 1, 3, 7, 14, and 90 days postoperatively. Steroids tended to reduce the swelling in the vessel walls during the early stages of healing (24 h and 3 days), and also to reduce the inflammatory cell count in the lumen of the vessels at 3 days. It seems likely from these initial observations that steroids are more likely to improve than jeopardize the healing of microvascular anastomoses.

Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) are food-borne intestinal pathogens with a low infectious dose. Adhesion of some EHEC strains to epithelial cells is attributed, in part, to intimin, but other factors may be required for the intestinal colonizing ability of these bacteria. In order to identify additional adherence factors of EHEC, we generated transposon mutants of a clinical EHEC isolate of serotype O111:H-, which displayed high levels of adherence to cultured Chinese hamster ovary (CHO) cells. One mutant was markedly deficient in CHO cell adherence, human red blood cell agglutination and autoaggregation. Sequence analysis of the gene disrupted in this mutant revealed a 9669 bp novel chromosomal open reading frame (ORF), which was designated efa1, for EHEC factor for adherence. efa1 displayed 28% amino acid identity with the predicted product of a recently described ORF from the haemolysin-encoding plasmid of EHEC O157:H7. The amino termini of the putative products of these two genes exhibit up to 38% amino acid similarity to Clostridium difficile toxins A and B. efa1 occurred within a novel genetic locus, at least 15 kb in length, which featured a low G+C content, several insertion sequence homologues and a homologue of the Shigella flexneri enterotoxin ShET2. DNA probes prepared from different regions of efa1 hybridized with all of 116 strains of attaching-effacing E. coli (AEEC) of a variety of serotypes, including enteropathogenic E. coli (EPEC) and EHEC, but with none of 91 non-AEEC strains. Nevertheless, efa1 was not required for the attachment-effacement phenotype, and the efa1 locus was not physically linked to the locus for enterocyte effacement (LEE) pathogenicity island, which is responsible for this phenotype in EPEC. These findings suggest that efa1 encodes a novel virulence-associated determinant of AEEC, which contributes to the adhesive capacity of these bacteria.

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.

Characterization of the interaction between Yersinia enterocolitica biotype 1A and phagocytes and epithelial cells in vitro.

Yersinia enterocolitica strains of biotype 1A are increasingly being recognized as etiological agents of gastroenteritis. However, the mechanisms by which these bacteria cause disease differ from those of highly invasive, virulence plasmid-bearing Y. enterocolitica strains and are poorly understood. We have investigated several biotype 1A strains of diverse origin for their ability to resist killing by professional phagocytes. All strains were rapidly killed by polymorphonuclear leukocytes but persisted within macrophages (activated with gamma interferon) to a significantly greater extent (survival = 40.5% +/- 17.4%) than did Escherichia coli HB101 (9.3% +/- 0.7%; P = 0.0001). Strains isolated from symptomatic patients were significantly more resistant to killing by macrophages (survival = 48.9% +/- 19.5%) than were strains obtained from food or the environment (survival = 32.1% +/- 10.3%; P = 0.04). Some strains which had been ingested by macrophages or HEp-2 epithelial cells showed a tendency to reemerge into the tissue culture medium over a period lasting several hours. This phenomenon, which we termed "escape," was observed in 14 of 15 strains of clinical origin but in only 3 of 12 nonclinical isolates (P = 0.001). The capacity of bacteria to escape from cells was not directly related to their invasive ability. To determine if escape was due to host cell lysis, we used a variety of techniques, including lactate dehydrogenase release, trypan blue exclusion, and examination of infected cells by light and electron microscopy, to measure cell viability and lysis. These studies established that biotype 1A Y. enterocolitica strains were able to escape from macrophages or epithelial cells without causing detectable cytolysis, suggesting that escape was achieved by a process resembling exocytosis. The observations that biotype 1A Y. enterocolitica strains of clinical origin are significantly more resistant to killing by macrophages and significantly more likely to escape from host cells than are strains of nonclinical origin suggest that these properties may account for the virulence of these bacteria.

Identification and characterization of IS2404 and IS2606: two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR.

Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606. IS2404 was identified by complete sequencing of a previously described repetitive DNA segment from M. ulcerans. This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida (AsIs1), Escherichia coli (H repeat element), Vibrio cholerae (VcIS1), and Porphyromonas gingivalis (PGIS2). The second IS, IS2606, was discovered by sequence analysis of a HaeIII fragment of M. ulcerans genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS1554 from M. tuberculosis. Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti (ISRm3), Burkholderia cepacia (IS1356), Corynebacterium diphtheriae, and Yersinia pestis. PCR screening of DNA from 45 other species of mycobacteria with primers for IS2404 confirm that this element is found only in M. ulcerans. However, by PCR, IS2606 was also found in Mycobacterium lentiflavum, another slow-growing member of the genus Mycobacterium that is apparently genetically distinct from M. ulcerans. Testing the sensitivity of PCR based on IS2404 and IS2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations of M. ulcerans among large numbers of other environmental mycobacteria.

Production of enterotoxin by Yersinia bercovieri, a recently identified Yersinia enterocolitica-like species.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

Ability of clinical isolates of group A streptococci to adhere to and invade HEp-2 epithelial cells.

Individual strains of group A streptococci (GAS) differ in virulence, but the reasons for these differences are incompletely understood. To determine if the ability of GAS to cause invasive disease corresponded with their capacity to adhere to or invade epithelial cells, 63 clinical isolates of GAS (40 from patients with systemic infection and 23 from superficial disease) were examined in quantitative assays of bacterial adhesion to and invasion of HEp-2 cells, a continuous line of human pharyngeal epithelial cells. The results showed that individual isolates of GAS varied considerably in their ability to adhere to and penetrate HEp-2 cells. However, on the whole, strains from patients with invasive disease adhered to cells in numbers c.1.5 greater than those from superficial infection. Paradoxically, strains from patients with invasive disease invaded HEp-2 cells to a significantly lesser extent than those from superficial sites, with a two-fold difference in invasion index (defined as the percentage of cell-associated bacteria located intracellularly). To determine if these differences were caused by differences in the production of hyaluronic acid capsule or M protein by the two groups of bacteria, the adherence and invasive capacities of bacteria carrying defined mutations in the genes for these factors were examined. Although M6-protein-deficient [corrected] bacteria were less adherent to HEp-2 cells than the wild-type, neither the hyaluronic acid capsule nor the M protein had a significant influence on the ability of GAS to adhere to or invade HEp-2 cells. The results of this study demonstrate that there are biological differences between GAS isolates associated with invasive and superficial diseases and that these differences can be demonstrated by an assay of bacterial adherence to and invasion of HEp-2 epithelial cells.