Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains

Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E. coli involved in diarrheal diseases. A total of 1.5% of them belonged to the enterotoxigenic E. coli pathotype, but none belonged to the enteroinvasive E. coli, enterohemorrhagic E. coli, or enteropathogenic E. coli pathotypes. Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe. Most of the strains involved in diarrhea belonged to the diffusely adhering E. coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells. Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells. The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E. coli strains should be considered pathogens. Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe. Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe. In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane. This cytotoxic effect was correlated with the synthesis of a hemolysin. The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.     

Enteroaggregative Escherichia coli strains as a cause of traveler's diarrhea: a case-control study

To elucidate the importance of enteroaggregative Escherichia coli (EAggEC) strains as a cause of traveler's diarrhea in Spanish travelers, a prospective case-control 1:1 study was done in a university hospital clinic for travelers. EAggEC strains were isolated from 23 of 165 case-patients and from 4 of 165 controls (P = .0003). In 16 patients, this was the only isolate recovered. Six of the EAggEC-positive isolates from the case-patients and 2 from the controls were positive for the enteroaggregative stable toxin type 1 gene. Other enteropathogens were also isolated. Shigella and enterotoxigenic E. coli strains showed significant differences between cases and controls (P = .0023 and P < .0001, respectively). Geographic distribution of the EAggEC strains was homogeneous, and the clinical symptom, secretory diarrhea, did not differ statistically with that for the enterotoxigenic E. coli strains. EAggEC strains are a cause of secretory diarrhea in Spaniards traveling to developing countries.     

Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro, Brazil

Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.     

A case-control study of diarrhoea in children caused by Escherichia coli producing heat-stable enterotoxin (EAST-1)

Escherichia coli strains associated with diarrhoeal disease have been classified into several types according to the pathogenic mechanism. Among these, enteroaggregative E. coli strains (EAggEC) have been associated with persistent childhood diarrhoea. Some strains of EAggEC produce a heat-stable toxin (EAST-1) that differs from others described previously. The main goal of this case-control study was to determine the prevalence of EAggEC and EAST-1-producing E. coli strains as a cause of diarrhoea in children in Spain and to study their in-vitro susceptibility to 21 antimicrobial agents. In the case group (115 children) 22 (19%) isolates and four (3.5%) isolates were EAST-1-producing E. coli and EAggEC, respectively, whereas in the control group (79 children) four (5%) isolates produced EAST-1 (p = 0.005) and three (3.8%) isolates were EAggEC. The present study suggests that EAST-1-producing E. coli strains are associated with diarrhoeal diseases in Spanish children, whereas EAggEC strains are not. Moreover, EAST-1-producing E. coli strains showed a high susceptibility to all the antimicrobial agents tested except for ampicillin.     

Clinico-pathological investigation for infantile brain stem glioma: report of two cases

A total of 132 isolates of the Havana serovar of Salmonela enterica were studied for their adhesion to and invasion of Hela cells in the presence of D-mannose. A total of 124 (93.9%) isolates were adherent, and 51% of these were invasive. Of these 124 strains 82 (66%) adhered to HeLa cells with a pattern typical for enteroaggregative (EAgg) Escherichia coli whereas the remaining 34% adhered in a non-aggregative (non-EAgg) pattern. Of the 82 strains showing the EAgg pattern 60% were invasive whereas only 33% of the 49 non-EAgg isolates invaded HeLa cells (P < 0.01). The action of mannose-sensitive type 1 pili was inhibited by the addition of D-mannose. Therefore we conclude that type 1 pili are not important in adhesion and invasion of Havana.     

Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29:H21 strain

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmids containing genes for diffuse adherence have not been described previously.     

Hemagglutination ability and adherence to the Buffalo green monkey kidney cell line of uropathogenic Escherichia coli

The hemagglutination ability and adherence capacity to the Buffalo green monkey (BGM) kidney cell line of 160 wild-type strains of Escherichia coli isolated from bacteriuric patients were investigated. It was found that P-fimbriated E. coli strains adhered significantly better to BGM cells than did strains in which P-fimbriae were not detected, which is in accordance with the capacity of P-fimbriated strains to cause unobstructive pyelonephritis and with receptor distribution for P-fimbriae in the urinary tract. The strains which exhibited other adhesions, alone or simultaneously, showed reduced adherence to BGM cells, while non-agglutinating strains, mostly isolated from urine of patients with asymptomatic bacteriuria, did not adhere at all or adhered poorly to the utilized cell line. The BGM cells served as a good experimental model for investigation of uropathogenic E. coli adherence; because these cells originate from the upper urinary tract, they are viable and not coated with Tamm-Horsfall protein.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

Adhesion of enteroaggregative Escherichia coli to formalin-fixed intestinal and ureteric epithelia from children

The adhesion characteristics of enteroaggregative Escherichia coli (EAggEC) to the mucosal surfaces of formalin-fixed paediatric intestinal and ureteral tissue were studied. The technique offers a means of overcoming the problem of limited tissue access in childhood and a way of examining the initial steps of bacterial adhesion. Five EAggEC strains isolated from children with diarrhoea in the UK and a well characterised, prototype EAggEC strain (221) were examined. Five of the six EAggEC strains showed preferential adhesion to jejunal mucosa with limited adhesion to ileum and colon. Five of the six also adhered to ureteric tissue. EAggEC can adhere to proximal, as well as distal, regions of the gastrointestinal tract in children, a previously unrecognised characteristic.     

Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes

A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E. coli (EPEC). Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA). The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains. Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period. The second pattern was represented by strains which hybridized with the eaeA gene only. Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype). The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe. Most (80%) of these strains exhibited the LA-like phenotype. Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not. The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs). ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H-. The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype. The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype. Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.     

Production and role of interleukin-10 in concanavalin A-induced hepatitis in mice

Coliform colonies from children whose stools were submitted for microbiologic analysis were studied prospectively to determine the frequency of shedding of enteropathogenic Escherichia coli (EPEC). In total, 2225 isolates from 445 patients were probed with eaeA (encoding intimin) and the EAF (EPEC adherence factor) probe, and adherence and actin-aggregating phenotypes were determined. Twenty-five patients (5.6%) shed non-O157:H7 eaeA+ EAF- E. coli. Of these 25 patients, isolates from 5 produced Shiga toxins and from 3 possessed bfpA (encoding the bundle-forming pilus) sequences. Non-O157:H7 eaeA+ E. coli from 21 (84%) of 25 patients adhered locally to and aggregated actin in HeLa cells. Four patients shed nonadherent EAF+ eaeA- E. coli. Non-O157:H7 eaeA+ and EAF- isolates belonged to diverse electrophoretic types and classical and nonclassical enteropathogenic serotypes. EPEC are relatively common in stools submitted for analysis in this North American pediatric hospital. Their etiologic role in childhood diarrhea warrants elucidation.     

Relative importance of enterotoxigenic and invasive enteropathogenic bacteria in infantile diarrhoea

Swedish children and adults (648 patients) with acute diarrhoea were investigated for enterotoxigenic strains in stool cultures. A total number 74 strains were isolated from 28 patients and assayed in the rabbit intestinal loop test and the adrenal cell test. Only three of the enterotoxigenic E. coli (ETEC) isolates belonged to classical enteropathogenic serotypes of E. coli (EPEC). Two enterotoxigenic strains of Proteus morganii, two of Enterobacter hafniae and one of Citrobacter freundii were isolated. None of 67 EPEC strains were found to produce a heat-labile enterotoxin (LT) in either of the two test systems. A number of Yersinia enterocolitica and Pseudomonas aeruginosa strains from stool cultures often produced toxic effects in the cell test but no enterotoxin activity was detected for any of the strains investigated either in the adrenal cell test for heat-labile enterotoxin (LT) or the suckling mouse test for heat-stable enterotoxin (ST). All EPEC isolates were also tested for ST and for invasive properties in the Sereny test; each isolate was negative in both test systems. It is concluded that production of LT and ST enterotoxin were common in stool isolates from Ethiopian children but a rare phenomenon among Swedish children with acute infantile diarrhoea. Isolation of aerobic stool bacteria with invasive properties seems to be uncommon both in Ethiopian and Swedish children. However, since both LT and ST as well as invasive properties seem to be very unstable genetic properties in many of these stool isolates improved sensitive methods for the last two properties will probably change this picture in the future.     

Adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from children with and without diarrhea in S?o Paulo city, Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in S?o Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.     

A clinical and epidemiological study of gastro-enteritis

Over a 13-year period, 2,903 Asian, Coloured and White children with gastro-enteritis were admitted to the Wilkins Infectious Diseases Hospital, Salisbury. During a pilot study of 250 patients conducted over the first 2 years, a definite seasonal variation in the occurrence of gastro-enteritis, with a peak incidence in winter, was noticed, and this led to a more detailed study of 2,653 patients over the next 11 years. Bacterial pathogens were recovered from 27% of patients, strains of Shigella accounting for 12% Salmonella for 7% and Escherichia coli for 8%. A severe outbreak of E. coli infection occurred between October 1971 and December 1972, and 4 of a total of 9 deaths occurred during this period, all in children suffering from E. coli 0111/B4 infections. The bacterial recovery rate was highest in the summer (40%) and lowest in the winter (12%). Winter diarrhoea broke out over 3-month periods during each year and accounted for 37% of the cases, but none of the children who contracted it died. The clinical picture of winter diarrhoea differed from the others and the most serious illness was that caused by enteropathogenic strains of E. coli. The over-all mortality in the series was 0,27%.     

HEp-2 cell-adherent Escherichia coli in patients with human immunodeficiency virus-associated diarrhea

Diarrhea occurs commonly in African human immunodeficiency virus (HIV) infections. A case-control (HIV-positive vs. -negative) study of adults with diarrhea was done in Lusaka, Zambia, to determine the prevalence of intestinal infection by HEp-2 cell-adherent Escherichia coli. Adherent E. coli were more common in HIV-positive patients with acute diarrhea than among HIV-negative controls (60% vs. 33%) and were found significantly more often in HIV-positive patients with chronic diarrhea than among HIV-negative controls with chronic diarrhea (79% vs. 17%, P < .002). Adherent strains were found significantly more often among HIV-positive patients (69%) than in 22 asymptomatic subjects (36%, P < .02). The HEp-2 cell adherence of the E. coli strains did not show a common pattern. Adherent bacteria were also observed in colonic biopsies from 32% of Zambians with chronic diarrhea who underwent endoscopy. Adherent E. coli may be an important cause of HIV-associated diarrhea in Zambia.     

Prevalence of the "high-pathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans

The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this "high pathogenicity island" (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregative Escherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella enterica strains investigated. Polypeptides encoded by the fyuA, irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess the hms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the juA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.     

An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.     

Diarrheagenic Escherichia coli

Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler's diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (entero-pathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.     

Activation of host cell protein kinase C by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.     

Influence of age and low afterload on the stress-velocity relation of the left ventricle

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using EcoRI and HindIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, EcoRI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When HindIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when EcoRI and HindIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake.