Bioinhbition of Diarrhogenic Gram-negative Bacteria using the Tukey-HSD Test

A variety of probiotics show promise as effective therapies in the control of diarrhoea, however, diets and geographical locations affect probiotic therapy. In this pilot study, four indigenous probiotic candidates, Lactobacillus acidophilus AAOOL4, L. reuteri AAOOCH1, L. plantarum AAOO25NN and L. delbrueckii AAOOT20 were investigated for their in vitro bactericidal effects on bacterial pathogens implicated in infantile diarrhoea using the Tukey test. The inoculum levels were between 5 log10 cfu g −l at 1% (inoculum per ogi sample) for 96 h at 35°C. The bactericidal effect of the probiotic candidates on the microbial load of the inoculated samples was determined by plate counts 24 hourly. Significant differences (p<0.05) were observed between the control (4.79-5.28 log10 cfu ml) and the inoculated samples AAOOL4 (<1.00-4.61 log10 cfu ml ), AAOON25 (<1.00-4.71 log10 cfu ml), AAOOCH1 (<1.00-4.67 log10 cfu ml ) and AAOOT20 (<1.00-4.78 log10 cfu ml ), especially the mixed probiotic culture-inoculated samples, MLC (<1.00-4.56 log10 cfu ml ). The diarrhogenic candidates were not within detectable limits between 72 and 96 h in most of the samples, indicating their in vitro bactericidal effects. The indigenous probiotic candidates can therefore, serve in the control of infantile diarrhoea.


INTRODUCTION
For more than a century, researchers have suggested that live bacterial cultures, such as those found in yogurt, might be useful in the prevention and treatment of gastrointestinal disorders [11] . The development of innovative biodrugs by using live microorganisms that are active in the human digestive environment has also been considered by various workers [2,3,5,8,9,12,18,20,22] and potential medical applications in few developed countries are numerous. However, studies on probiotics in Nigeria are still very scanty and yet mostly undocumented. This preliminary study is therefore, undertaken to investigate the in vitro bactericidal effects of locally developed probiotics on diarrhogenic bacteria implicated in infantile diarrhoea.

MATERIALS AND METHODS
Bacterial species: Diarrhogenic stool and vomitus specimens were obtained from pediatric patients at Oni Memorial Children Hospital, Ibadan, and from epidemic patients in Abia, Benue and Eboyin states of Nigeria. Final microbiological analyses were carried out at the Nigeria Institute of Medical Research (NIMR), Lagos and Department of Botany and Microbiology, University of Ibadan laboratories.
Lactobacillus strains were obtained from infantile faecal samples of healthy children, and indigenous fermented foods and beverages. Standard phenotypic taxonomic tools were employed for the identification of the bacterial species used in this study [1,7,14] .

Survival of diarrhogenic bacteria in ogi:
The diarrhogenic bacteria and the Lactobacillus strains were separately grown using standard procedures and appropriate culture media. For each set up, the inoculum was prepared by separately washing the bacterial strains several times with sterile phosphatebuffer saline. The microbial load of the freshly prepared inoculum was adjusted to give about log 10 5 cfu ml −1 of the diarrhogenic bacteria and 10 7 cfu ml −1 of the Lactobacillus strains.
The method of Svanberg et al. [17] was adopted for the preparation of ogi. The appropriate volume of tap water was brought to a vigorous boil and the corresponding quantity of the ogi paste was added while stirring to avoid lumps. Cooking continued until the ogi paste became boiled. The probiotic candidates were then introduced at concentrations of 10 7 cfu ml −1 into the cooled ogi (supplemented with 1% glucose) in the culture flasks (10% v/v) and left for about 6 h. Ten ml of the diarrhogenic cell suspensions (10 5 cfu ml −1 ) was then added to culture flasks and the contents (1% v/v) were thoroughly homogenised to ensure even distribution of the inoculum. Incubation was at 32-35°C for 96 hours. Both the control samples (no single or mixed probiotic cultures) and the probiotic culture samples contained same inoculum levels at 0 h and growth of the diarrhogenic indicator bacteria was monitored throughout the experimental period. The quantitative determination of the surviving diarrhogenic bacteria was the plate count method.

Statistics:
The bactericidal rates of the potential probiotic candidates were analysed with the chi-square test (2×3 Table) Tukey (Statview; SAS institute Inc., Cary, NC, USA). p<0.05 was considered significant. The results are presented as the median and range, based on the groups in homogenous subsets and on Type III sum of squares unless otherwise indicated. Table 1-8 show the growth kinetics (log cfu ml −1 ) of the diarrhogenic bacteria inoculated into ogi already containing single and mixed probiotic candidates. Both the control samples (uninoculated cultures) and the probiotic culture samples contained same inoculum levels at 0 h and growth of the indicator bacteria was monitored throughout the experimental period. Although there were minor differences, a similar decline trend was observed among the growth kinetics of the diarrhogenic indicator isolates. Inoculation of the test diarrhogenic isolates, Pseudomonas aeruginosa 23S, Klebsiella sp. 24S, Klebsiella pneumoniae 33S, Klebsiella pneumoniae 411, Citrobacter aerogenes 55S, Klebsiella pneumoniae 57S and two reference strains, Escherichia coli NCTC11560 and V157 into laboratory prepared ogi previously inoculated with the probiotic candidates (L. acidophilus AAOOL4, L. reuteri AAOOCH1, L. plantarum AAOO25NN and L. delbrueckii AAOOT20) as single and mixed cultures showed significant inhibition of the various diarrhogenic bacterial isolates between 48 and 96 h, with the highest inhibition mostly produced by the mixed probiotic cultures. There was a sharp decrease in the number of the surviving diarrhogenic bacteria in the probiotic-inoculated ogi.

RESULTS AND DISCUSSION
Within 72 h of incubation, all the diarrhogenic bacterial strains were within the limit of detection of less than 1.00 in the culture samples containing the mixed probiotic candidates (MLC) except for Ps. aeruginosa 23S, while at 96 h of incubation, in the single and mixed cultures, all the diarrhogenic bacterial isolates were within the limit of detection of less than Table 1: Growth kinetics (log cfu mL −1 ) with standard deviation of Pseudomonas aeruginosa 23S inoculated into laboratory prepared ogi preinoculated with single and mixed probiotic cultures .00 k Values represent mean scores (n = 3) with standard deviations. Sample means having the same letters for each incubation period are not significantly different. UNS = unseeded ogi sample; AAOOL4 = Lactobacillus acidophilus; AAOON25 = Lactobacillus plantarum; AAOOCH1 = Lactobacillus reuteri; AAOOT20 = Lactobacillus delbruecki; MLC = Mixed Lactobacillus cultures; <1.00 = less than 1.0 (limit of detection). Table 2: Growth kinetics (log cfu mL −1 ) with standard deviation of Klebsiella aerogenes 24S inoculated into laboratory prepared ogi preinoculated with single and mixed probiotic cultures     .00 a Values represent mean scores (n = 3) with standard deviations. Sample means having the same letters for each incubation period are not significantly different. UNS = unseeded ogi sample; AAOOL4 =Lactobacillus acidophilus; AAOON25 = Lactobacillus plantarum; AAOOCH1 = Lactobacillus reuteri; AAOOT20 = Lactobacillus delbruecki; MLC = Mixed Lactobacillus cultures; <1.00 = less than 1.0 (limit of detection).   1.00. It was noticeable that the diarrhogenic indicator isolates in the probiotic pre-inoculated ogi reached significantly lower levels faster than the uninoculated ogi samples (Table 1-8).
The anti-diarrhoeal properties of lactobacilli flora of the human intestine known as probiotics (healthpromoting bacteria) have been investigated since the 1960s by various workers [2,8,13,19,21] . The beneficial properties of a probiotic however, should not only be based on theoretical aspects. Some scientific proof should be given that the probiotic improves the properties of the indigenous microflora and/or beneficially affects the host. The results of this present study therefore, showed a significant in vitro decline trend among the growth kinetics of the diarrhogenic bacterial indicator strains that were inoculated into boiled, supplemented ogi already containing single and mixed probiotics. The viability of the diarrhogenic bacteria, Pseudomonas aeruginosa 23S, Klebsiella aerogenes 24S, Klebsiella pneumoniae 33S, Klebsiella pneumoniae 411S, Citrobacter sp. 55S, Klebsiella pneumoniae 57S and two reference strains, Escherichia coli NCTC11560 and V157 inoculated into ogi already seeded with single and mixed selected probiotic candidates reduced significantly between 12 and 96 h of incubation, however, most of the indicator isolates were within the limit of detection of less than 1.00 by 96 h of incubation.
About 2.2 million children die from diarrhoeal diseases each year in developing countries, nearly all Table 6: Growth kinetics (log cfu mL −1 ) with standard deviation of Klebsiella pneumoniae 57S inoculated into laboratory prepared ogi preinoculated with single and mixed probiotic cultures .00 a Values represent mean scores (n = 3) with standard deviations. Sample means having the same letters for each incubation period are not significantly different. UNS = unseeded ogi sample; AAOOL4 =Lactobacillus acidophilus; AAOON25 = Lactobacillus plantarum; AAOOCH1 = Lactobacillus reuteri; AAOOT20 = Lactobacillus delbruecki; MLC = Mixed Lactobacillus cultures; <1.00 = less than 1.0 (limit of detection) Table 7: Growth kinetics (log cfu mL −1 ) with standard deviation of Escherichia coli NCTC11560 inoculated into laboratory prepared ogi preinoculated with single and mixed probiotic cultures     from dehydration as a result of persistent diarrhoea that is often aggravated by malnutrition [6] . The greatest mortality from diarrhoeal diseases and enteric infections occur in infants and small children, thus, over thirteen percent of the children born in certain parts of Latin America die before their fifth birthday with diarrhoea-associated diseases as the major cause of their death. In some cases, children of 36 months of age experience as many as 4 episodes of severe diarrhoea annually and, in this age group, gastroenteritis is reported to be a major cause of death [10] . Studies in Nigeria have also shown diarrhoea as the commonest cause of death among hospitalized children under-5 years of age [4] , while the transmission of the pathogens can occur mostly through contaminated food or water or by person-to-person contact [10] . This study has demonstrated that Lactobacillus species with the ability to produce antimicrobial compounds against diarrhogenic bacteria are wide spread in Nigerian indigenous fermented foods and beverages as well as in infantile faecal samples of healthy children.
The development of rationally based applications of probiotic therapy could have very useful clinical contributions and their impact could be of very great importance in Nigeria, especially if they result in effective but low-cost ways of prevention and treatment of infantile diarrhoea. The basal food sample was studied through simulations of ogi, the most popular non-industrial infant weaning food in Nigeria, and was shown to have good performances in the support of the probiotic bacterial candidates. In this study, none of the selected Lactobacillus strains, selected as potential probiotic candidates, L. acidophilus AAOOL4, L. delbrueckii AAOOT20, L. plantarum AAOON25 and L. reuteri AAOOCH1 gave any inhibitory activity towards other Lactobacillus strains, indicating possible lack of adverse effects in their selection as probiotic candidates against other closely related selected probiotic candidates. This research therefore, reports that some indigenous, non strain-specific probiotic candidates of Nigerian fermented foods and beverages as well as of infantile faecal specimen' origin were inhibitory against infantile diarrhogenic bacterial pathogens in vitro.