Eva Sjögren

Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels.

A natural lactic fermentation of mixtures of water and whole flour of either maize or high-tannin sorghum was obtained either before or after cooking to a weaning gruel: The preparations had a final pH of about 3.8 (range 3.67 to 4.00) and a ratio of lactic acid to acetic acid of 9∶1 (w/w). The growth of added (about 107 c.f.u./g gruel) Gram-negative intestinal pathogenic bacteria, enterotoxigenicEscherichia coli, Campylobacter jejuni, Shigella flexneri andSalmonella typhimurium, was strongly inhibited in the sour gruels, and the effect could primarily be explained by the low pH caused by the formation of lactic and acetic acids during the fermentation process. Of the added Gram-positive bacteria,Bacillus cereus andStaphylococcus aureus showed similar inhibited growth up to 7h after inoculation in the sour gruels. The strain ofStaphylococcus, however, showed only a continued reduction in growth in the fermented gruel samples, which had a viable lactic bacteria culture indicating the presence of a bacteriocin. This implies that a low pH (< 4.0) alone is not sufficient to sustain the inhibition of the growth ofStaphylococcus aureus. The survival studies were carried out at optimal temperatures for each respective enteropathogen.

Campylobacter upsaliensis, C. sputorum sputorum and C. concisus as Common Causes of Diarrhoea in Swedish Children

It has recently been reported that Campylobacter sp. other than Campylobacter jejuni/coli are likely to be the cause of gastroenteritis in humans. Examples are C. upsaliensis, C. jejuni doylei, C. concisus, C. lari and C. mucosalis (1–4).

Inhibition of different strains of enteropathogens in a lactic-fermenting cereal gruel

Twenty-eight strains of enteropathogens, including Campylobacter, Salmonella, Shigella, enterotoxigenic Escherichica coli (ETEC), Staphylococcus and Bacillus were added to cereal gruels prepared from low-tannin sorghum and inoculated with a lactic acid starter culture. Campylobacter strains were not detectable after 6 h, and Salmonella, Shigella and Staphylococcus strains not after 12 h. No viable cells of Bacillus strains were detected after 16 h of fermentation and the ETEC strains were completely inhibited after 24 h. No strain variability was observed within the different genera. In control gruels (no starter culture added), all the enteropathogens increased in number during incubation at 32°C except for the Campylobacter strains which decreased after 12 h of incubation.

pH and acidity in lactic-fermenting cereal gruels: effects on viability of enteropathogenic microorganisms

Survival of Bacillus cereus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Salmonella typhimurium and Shigella flexneri during lactic acid fermentation of cereal gruels prepared from low-tannin (white) and high-tannin (red) sorghum varieties was studied. A previously fermented gruel (starter culture, SC) recycled daily or stored for 7, 14 or 28 days, germinated cereal flour (power flour, PF), or a combination of PF and SC (PF+SC) were used as starters. At 24 h, the pH of all gruels with added starter was ≤4; the pH in control gruels without starter was ≥5.2. pH decrease was significantly faster in gruels made with PF+SC than with either PF or SC alone (P<0.05). A daily recycled SC resulted in a significantly faster decrease in pH (P<0.05) than SC stored for more than 7 days. Acid production was correlated with pH decrease (r=−0.94; P<0.01). In control gruels, the enteropathogens remained at the inoculation level or increased in number. Their growth was inhibited within 24 to 48 h in the fermented gruels, in the order: Bacillus > Campylobacter > Escherichia coli > Salmonella > Shigella. The inhibition rate was significantly faster in fermenting gruel with PF+SC (P<0.05) than in gruel with PF or SC alone and correlated with pH development (r=0.71; P<0.01). Both white and red sorghum gruels gave similar results. Using PF+SC as a starter resulted in a faster decrease in pH as well as a more rapid inhibition of enteropathogenic microorganisms. The effect is optimal if the SC is transferred daily.

Rapid Emergence of Quinolone Resistance in Campylobacter jejuni in Patients Treated with Norfloxacin

15/60 subjects from one center, who all took part in a multicenter double-blind, placebo-controlled study to evaluate the effect of norfloxacin on acute enteritis, had norfloxacin sensitive strains of Campylobacter jejuni in pre-study stool specimens. Eight of the 15 subjects received active drug. In 3 of these 8, high-level quinolone resistant Campylobacter strains of the same serotype as in pre-treatment samples were isolated 4-90 days after the initiation of treatment.

Outbreak of Campylobacter infection in a subartic community.

Abstract A presumably waterborne outbreak of Campylobacter jejuni/coli infection in a subarctic community is described. Drinking water supplied to residents was delivered unchlorinated during a 4-week period. No Campylobacter sp. was recovered from the water supply. Three hundred thirty individuals (15% of the 2200 exposed) became ill. Diarrhoea, abdominal pain, fever, nausea and joint pain occurred in 81%, 30%, 29%, 43% and 21%, respectively. Nine percent reported swelling of joints, and two cases of reactive arthritis occurred. A Campylobacter sp. was isolated from 9 of 33 individuals who became ill and from 1 of 33 healthy controls. All culture-positive individuals, 46% of culture-negative ill persons and 27% of healthy controls were seropositive. All strains recovered had an identical DNA profile.

Campylobacter jejuni/coli and enterotoxigenic Escherichia coli (ETEC) in faeces from children and adults in Tanzania.

The occurrence of Campylobacter and enterotoxigenic E. coli (ETEC) was studied in faecal samples from Tanzanian children (< 5 years of age), adolescents and adults (only Campylobacter) with and without diarrhoea. The Campylobacter strains isolated were tested for subspecies, enterotoxigenicity and serotype. Out of 394 children with diarrhoea 18% were infected with Campylobacter and 20% with ETEC. In 278 samples tested for Campylobacter and 136 tested for ETEC from asymptomatic children the corresponding numbers were 12 and 5%, respectively. In children < 18 months with diarrhoea Campylobacter was noted in 22% and ETEC in 18%, whereas the figures were 11 and 4% respectively in asymptomatic children. In the age group 18 months to 5 years Campylobacter was demonstrated in 2% of the children with diarrhoea and 27% had ETEC, while the figures were 15 and 8% for asymptomatic children. Among adults the prevalence of Campylobacter-positive samples was 1% both for symptomatic and asymptomatic individuals. There were no seasonal differences in the prevalences of both Campylobacter and ETEC either in the symptomatic or the asymptomatic group. Campylobacter jejuni was the dominating Campylobacter species among both symptomatic and asymptomatic individuals. C. jejuni strains from patients with diarrhoea were significantly more often enterotoxigenic than were C. coli strains. The serotype pattern regarding Campylobacter was in general similar for symptomatic and asymptomatic individuals. We conclude that Campylobacter and ETEC are common causes of bacterial diarrhoea in Tanzanian children, and that Campylobacter infections are more important in children younger than 18 months, than in older ones.

Enteropathogenic bacteria in faecal swabs of young children fed on lactic acid-fermented cereal gruels.

The influence of consumption of a lactic acid-fermented cereal gruel togwa with pH < or = 4 on the presence of faecal enteric bacteria such as campylobacter, enterohaemorrhagic Escherichia coli (EHEC:O157), enterotoxigenic Escherichia coli (ETEC), salmonella and shigella was evaluated. Under 5 years old healthy children listed in an ascending order of age were alternatively assigned and given either a lactic-acid fermented cereal gruel togwa (test diet) or an unfermented cereal gruel uji (control diet) once a day for 13 consecutive days. The presence of the enteropathogens was examined in rectal swabs collected from the children at baseline (before feeding session started), on days 7 and 13, and additionally 14 days (follow-up day) after the feeding session had stopped. The swabs were cultured on to different optimal media for respective enteropathogen and confirmed by standard microbiological and serological methods. Campylobacter spp. dominated among the enteropathogens (62% out of total) followed by Salmonella spp., ETEC and Shigella spp. Children with isolated enteropathogens in the togwa group was significantly reduced (P < 0.001) from 27.6% at baseline to 7.8, 8.2 and 12.7% on days 7, 13 and follow-up day, respectively. The effect was more pronounced in those children taking togwa > 6 times during the study period. In the control group, there was a slight decrease from 16.7% at baseline to 11.4% on day 7 and 8.1% on day 13. On the follow-up day, enteropathogens were found in 22.6% of the children, which was significantly higher than in those children taking togwa > 6 times. We conclude, that regular consumption of togwa with pH < or = 4, once a day, three times a week may help to control intestinal colonization with potential diarrhoea-causing pathogens in young children.

Adherence, enterotoxigenicity, invasiveness and serogroups in Campylobacter jejuni and Campylobacter coli strains from adult humans with acute enterocolitis

Two hundred Campylobacter jejuni and Campylobacter coli strains from the same number of adult Swedish patients with acute enterocolitis were tested regarding adherence to and invasiveness in HEp‐2 cells and for enterotoxigenicity by the CHO‐cell assay. The serogroup characteristics, heat‐stable and heat‐labile, for each strain were also investigated. Eighty‐four percent of the strains were classified as C. jejuni and 16 percent as C. coli. All of the strains were adherent to HEp‐2 cells, 39% were invasive and 31.5% enterotoxigenic. We found significantly more invasive strains in the non‐enterotoxigenic group than in the enterotoxigenic one. There would seem to be no correlation between enterotoxigenicity or invasiveness and serogroup. The results of this study suggest the existence of multiple mechanisms for C. jejuni‐ and C. coli‐induced diarrhoea and that the mechanisms may differ from one strain to another.

Inhibition of enterotoxin production by, and growth of enteropathogens in a lactic acid-fermenting cereal gruel

Growth and enterotoxin production of three strains of Campylobacter jejuni or Escherichia coli known to produce heat labile enterotoxins were evaluated when added into a lactic acid-fermenting cereal gruel, togwa. A single strain of each of the enteropathogens was simultaneously inoculated with a lactic acid starter culture (sc) to reach a level of about 107 c.f.u./ml and was left to ferment for 48h. Gruels without sc (control gruel), pure cholera toxin in fermenting or control gruel and the test bacteria inoculated into nutrient broth were used as positive toxin controls; gruel without inoculated test bacteria was the toxin-negative control. Viable colonies were counted by spread plating 0.1ml of gruel subsamples collected at intervals during the fermentation period onto different selective media and the heat labile enterotoxin production was evaluated using an assay on Chinese hamster ovary (CHO) cells. In the fermenting gruels, no viable cells of the C. jejuni and E. coli strains were detected after 8 and 24h incubation, respectively, but inocula increased in number or remained at the initial level in the control gruel and in the nutrient broth. After 24h incubation, all supernatants of control gruels with inoculated bacteria showed enterotoxicity to the CHO cells (indicated by elongation of 20–50% of the cells). No toxin activity was observed in the fermenting gruels with or without added bacteria or in control gruel alone. Pure cholera toxin added to control gruel caused the enterotoxigenic effect in 70–100% of the CHO cells, but no activity was detected when it was added to the fermenting gruel. The CHO cells were affected instead by a low pH level but were not elongated. Adjusting the pH of fermented gruels to approximately neutral levels restored the toxin effect when cholera toxin was added but not in the presence of added test bacteria. We conclude that lactic acid fermented cereal gruels with pH ≤4 have a high potential to inhibit the growth of enteropathogenic bacteria of the genera C. jejuni and E. coli and to inhibit production of heat labile enterotoxins. Regular consumption of fermented cereal weaning foods will therefore reduce transmission of enterotoxin-producing bacteria, and ingestion of enterotoxins.