Volume 1, Number 1 (2016) pp 22-36 doi 10.20448/805.1.1.22.36 | Research Articles
Microbes are an often overlooked part of an ecosystem because of their small size and lack of morphological characters; however their presence is vital for every kind of known life. The human microflora is an essential and play very important role in health and disease. This study was done for isolates and identifies some samples from deferent section in the human body from Egypt. Twenty samples were collected as following: Five saliva samples of 3 children school volunteers aged (4-10 years) were collected from 3 schools in Alexandria Governorate, Egypt and 2 adult samples aged (22,30 years) were collected randomly. Five feces samples of infant babies aged (3-6 months).Ten teeth samples were collected from adult teeth roots. From our results 42 predominant colonies morphology isolated on MRS agar, BHI agar, MSA and SF agar from 12, 16 and 14 samples of saliva, teeth and faeces respectively. This found to be 24 cocci cells, 14 rods cells and 4 yeasts cells. These bacteria were identified as: 1 Staph.lentus; 5 Staph.xylosus; 1 B. brevis; 3 Lactobacillus acidophilus; 2 Lactobacillus plantarum; 8 Bacillus sp. and 18 Ec.faecalis
Keywords: Human microflora, Lactobacillus acidophilus, Ec.faecalis, B. brevis..
Citation | Fawzia M. A. El- Sakhawy; Ali Abd. El-L. Khattab; Hoda Mahrous; Atef M. Ibrahim (2016). Isolated and Identification of Some Isolated Bacterium from Human in Egypt. Journal of Biotechnological Research, 1(1): 22-36.
Copyright: This work is licensed under a Creative Commons Attribution 3.0 License
Funding : The authors declare that they have no competing interests.
Competing Interests: The authors declare that they have no competing interests.
History : Received: 9 June 2016/ Revised: 23 June 2016/ Accepted: 28 June 2016/ Published: 2 July 2016
Publisher: Online Science Publishing
The number of microbial species that exists on the earth today is unknown, and classifying them becomes a massive project because of their small size, a traditional requirement for colony isolation, a requirement for a microbiological or biochemical biotype identification, and large diversity. Molecular techniques have yielded estimates that less than 10% of the organisms in an ecosystem have been identified using traditional methods . In the 19th century the development of pure culture techniques that allowed scientists to isolate and characterize prokaryotic organisms were developed by Robert Koch who stipulated that pure cultures of the pathogenic organism must be isolated from its host and grown in pure culture .
The midgut, in contrast, showed quite a bit of microbial density. Approximately 108 colonies per midgut were counted when incubated aerobically and 3 x 108 colonies were isolated when incubated anaerobically. A dilution series isolated on medium containing carboxymethyl cellulose demonstrated that the midgut of both roach species contained 106 bacteria that grew under aerobic conditions and 107 that grew anaerobically. The most common microbes that were isolated from the midgut included anaerobes, Enterobacter agglomerans and Klebsiella oxytoca, and Citrobacter freundii. Electron microscopic examination of the midgut also showed that several types of insects harbor bacteria between the peritrophic membrane and midgut epithelium with occasional protozoa and spirochetes also present .
The characteristics of mouth are ecologically distinct from all other surfaces of the body and determine the types of microbes that are able to persist, so that not all of the microorganisms that enter the mouth are able to colonize. Moreover, distinct habitat exists even within the mouth, each of which will support the growth of a particular microbial community because of their precise biological features. Habitats with obviously different ecological conditions include mucosal surfaces (such as lips, cheeks, palate, and tongue) and teeth. The properties of mouth as a microbial habitat are dynamic and will change during the life of an individual . The oral cavity is potentially susceptible to a variety of infections due to the presence of numerous proliferative microorganisms that cause fungal, viral and bacterial infections . Dental caries can be characterized as a multifactorial infection directly related to three main factors: Microflora, substrate and host. Some carious processes may develop to the point where dental pulp is affected. Microbial involvement has been indicated as the main factor associated with the etiology of endodontic diseases .
The human GI microbiota is considered a tissue, not an organ, and is used in FMT to implant in a recipient’s GI tract. To understand the utility of FMT, it is first necessary to appreciate the compositional complexity of the GI microbiota, along with its associated functional implications. There are about 1014 bacterial cells in our body and most of these bacterial cells reside in the GI tract. Only about 30 %of the GI microbiota is detectable by culture-based techniques .
The present study isolates and identifies some deferent bacteria from deferent section in the human body from Egypt.
M.R.S. agar medium was prepared according to De Man, et al,  having following composition per liter:Peptone 10 gm
V.R.B.A. medium was prepared as described in Difco"s Manual, . The composition of the medium is:Yeast extract 3 gm
M.S.Amedium was prepared according to Difco"s Manual,  having following composition per liter:Beef extract 1 gm
B.H.I.A was obtained from Difco"s Manual, . Having the following composition:Calf brain 200.0 gm
For propagation of phathogenic cocci and other fastidious organisms associated with blood culture work and allied pathological investigations
P.C.A media was obtained from Difco"s Manual,  as the following composition:Beef extract 3 gm
S.F agar media was obtained as described in Difco"s Manual,  as the following composition:Tryptone 20 gm
Twenty samples were collected as following:
A loop of each sample was streaked on MRS Agar, Brain heart infusion agar , S.F.A , M.S.A and V.R.B.A according to Difco's Manual for determinative microbiology, then cultured at aerobic and anaerobic conditions at 37ºC for 48hrs. Abredomenant colonies were purified and examined for some bacteriological tests included gram stain, motility, catalase test, salt tolerance, oxidase test.
Representative colonies isolated throughout the present investigation were purified, kept on slants and examined microscopically using Gram staining (Huckerʼs modification).
Isolates were also tested for their ability to produce catalase.
Accordingly, the isolates were grouped into:
Strains that were found able to ferment lactose with the production of acid and gas were subjected to the four conventional IMVIC tests.
It is a complex staining method to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls, according to .
To detect catalase activity, a drop of broth culture was transferred onto a clean slide, flooded with a drop of HᴤOᴤ solution 30% and observed for the production of effervescence .
Salt tolerance test is particularly useful for presumptive identification of enterococci group D organisms, which have the specific ability to grow in the presence of 6.5% NaCl incorporated into a specific broth culture media.
Brain heart infusion and MRS supplemented with 4%, 4.5% and 6.5% and bromocresol purple as a pH indicator is used for this purpose according to the method in Biochemical tests for gram positive bacteria .
The gelatin stab method employs according to Biochemical identification tests. Nutrient gelatin deep tubes that contain 12% gelatin, heavy inoculums from a pure culture of the test organism is stabled into the media and incubated for at least 48 hrs, then placed into refrigerator for 30 min. If the organism has produced sufficient gelatinase, the tubes will remain liquid and do not solidify in the refrigerator.
The starch agar is inoculated with the pure colony and incubated at an appropriate temperature then iodine solution is added to the surface of the agar media which turned to blue-black in the presence of starch. Absence of the blue-black color indicates that starch is no longer present (Biochemical tests).
To determine if an organism is motile or non motile by inoculating an 18 to 24 hr pure culture in the center of MRS agar or B.H.I.agar medium by needle to a depth of 0.05 inch, then incubated at 35º C for 24 to 48 or more according to (the biochemical tests).
The test was made by including 10.0 ml of tryptone water (1% tryptone in distilled water) with the test organism. Tubes were incubated for 24 hr, at 37ºC, then 0.2 to 0.3 ml. of Kovacʼs reagent were added to 5.0 ml of the 24 hr. culture. The formation of a dark red colour on the surface indicated a positive test.
Kovacʼs reagent was prepared by dissolving 5.0 gm of p-dimethyl amino benzaldehyde in 75 ml .of amyl alcohol, followed by the addition of 25 ml .of concentrated hydrochloric acid.
This test was carried out by inoculating the tested isolates into 10 ml. medium which was prepared to have the following composition:Peptone 7.0 gm.
5 drops of methyl red solution were added to 5.0 ml of each culture. Positive reaction was indicated by the formation of a distinct red colour, showing the presence of acid. The indicator solution was prepared by dissolving 0.1 gm Bacto-Methyl red in 300 ml of 95% ethyl alcohol and diluting to 500 ml with distilled water.
Cultures were inoculated in tubes containing 10 ml of MR-V.P. medium. Tubes were incubated for up to 5 days. To 5 ml of the culture, 5.0 ml of a 10% KOH solution were added, mixed well, allowed to stand, exposed to the air and observed at intervals of 2, 12 and 24 hr for the formation of a pink colour.
Cultures were inoculated in tubes containing 10 ml of citrate medium which was prepared as follows:Sodium ammonium phosphate 1.5 gm.
Tubes were incubated at 37ºC for 24 hr. The positive test was detected by the presence of a uniform turbidity.
After performing the above mentioned IMViC tests, the tested strains were subjected to the Eijkman test. Cultures were inoculated into tubes containing 10 ml of the following medium:Tryptone 15.0 gm .
Tubes were incubated at 44º C for 48 hr. The presence of a uniform turbidity and gas production at the end of the incubation period was taken as a criterion for a positive test.
The API 50CH strip (Biomerieux, Marcy l’Etoile France) was used to identify Lactobacillus, Lactococcus, Leuconostoc and Streptococcus thermophilus cultures. It consists of 50 micro-tubes which contain an anaerobic zone (the tube portion), for the study of fermentation and an aerobic zone (the cupule portion), for the study of oxidation or assimilation. The first tube contains no substrate and is used as a negative control. The remaining tubes contain a defined amount of dehydrated substrate belonging to the carbohydrate family and its derivatives (heterosides, polyalcohols, uronic acids). These substrates may be metabolized by various biochemical pathways:
Ø Assimilation is indicated by growth of an organism in the cupule, when the substrate is the only source of carbon present.
Ø Oxidation is shown by a color change in the cupule portion and is due to the aerobic production of acid detected by a pH indicator included in the chosen medium.
Ø Fermentation is shown by a color change in the tube portion, and is due to the anaerobic production of acid detected by pH indicator included in the chosen medium.
Ø The API 20 strips (Biomerieux, Marcy l’Etoile France) was used to identify the Enterococcus cultures. It consists of 20 microtubes containing dehydrated substrate for the demonstration of enzymatic activity or the fermentation of sugars. A dense suspension made from a pure culture is then used to rehydrate the enzymatic substrates. The metabolic end products produced during the incubation period are either revealed through spontaneous colored reactions or by the addition of reagents.
Results from the facial hedonic scale record sheets were collated and input into a Microsoft Excel 2007 database, mean, standard deviations and p-values were calculated for each sample. P-values less than 0.05 were considered statistically significant.
Table1: shows 42 predominant colonies morphology isolated on MRS agar, BHI agar, MSA and SF agar from 12, 16 and 14 samples of saliva, teeth and faeces respectively. This found to be 24 cocci cells, 14 rods cells and 4 yeasts cells. The colonies isolated from saliva were 8, 3 and 1 cocci, rods and yeasts, respectively, while these isolated from teeth were 10, 5 and 1 respectively. The corresponding colonies isolated from faeces were 6, 6, and 2 respectively. All bacterial cells were gram positive.
As shown in table 2 the identification of 8 cocci cultures isolated from the adult and children saliva. The isolated cultures were 2, 1, 1, 1 and 3 which belong to Streptococcus pyogenes, Staphylococcus aures , Enterococcus faecalis, Lactococcus lactis and streptococcus mutans , respectively. The confirmatory test (plasma coagulase tast) has been carried at Staphylococcus cultures which were positive for this test indicating that it belongs to Staphylococcus aures.
The presence of Streptococcus pyogens normally resides in the throat and is one of the most common medical pathogens in the saliva as reported by Stjernquist-Desatnik and Orrling,  who detected streptococcus pyogens in approximately 10% of adults and 25% of children, and in as many as 60% of subjects during large outbreaks of streptococcal pharyngotonsillitis.While Tagg et al,  found Streptococcus pyogens in 5% of saliva samples of young school children in Newzealand, with a suggestion of a child-to-child transmission of the organism.
On other hand Holt et al,  reported that oral Streptococci, like Streptococcus mutans are associated with pyogenic and other infections in various sites including mouth, heart, joints, skin, muscle, and central nervous system.
This phenomenon approves the presence of Streptococcus mutans in the saliva of both adult and children. The Staphylococcus aureus presence may be due to some inflamation in the mouth while Enterococcus faecalis, Enterococcus faceium and Latctococcus lactis may be resulted from contamination.
On other hand table 3 revealed the identification of 3 rod cultures isolated also from the adult and children saliva which were found to be one lactobacillus acidophilus and 2 Lactobacillus brevis which may be come from the consumption of Dairy products.
Concerming the microflora of teeth, Table 4 revealed that the identification of 10 cocci cultures isolated from adult and children who were identified as 1, 2, 3, 2 and 2 Staphylococcus lentus, Staphylococcus xyloses, Staphylococcus aures, Streptococcus salivaries and Streptococcus mutans, respectively.
There were 5 Rods cultures in table 5 and fig 1 found as 2, 2 and 1 were belong to Bacillus brevis, Bacillus subtilis and Lactococcus acidophillus. Bacillus subtilis and Bacillus brevis were identified by PCR tecnique. Gomes et al,  and Pinheiro et al,  investigated the microbial findings of teeth with failed endodontic treatment and reported that a very limited assortment of microorganisms, with predominantly facultative anaerobic grampositive species, especially Enterococcus faecalis.
Marsh,  and Desoet et al,  notice that S. mutans and Streptococcus sobrinus have a central role in the etiology of dental caries, because these can adhere to the enamel salivary pellicle and to other plaque bacteria . Tanzer et al,  reported that Mutans streptococci and lactobacilli are strong acid producers and hence cause an acidic environment creating the risk for cavities.while Mayooran et al,  noticed that the appearance of S. mutans in the tooth cavities is followed by caries after 6-24 months.
Ruby et al,  reported that Caries is caused by indigenous oral microorganisms, mainly Streptococcus mutans, becoming a dynamic biofilm, which in the presence of fermentable sugars; produce organic acids capable of dissolving inorganic enamel and dentin followed by the proteo-lytic destruction of collagen, leaving soft infected dentin. Ercan & Tuna,  investigate the type of microorganisms isolated from teeth root canals 68% of isolates were gram positive bacteria, Streptococcus sp 14.2%, Enterococcus faecalis 9.6%, Lactobacillus acidophilus 7%, Bacillus sp 2% and E. coli 1.6%.
As shown in table 6 it revealed that the identification of 6 cocci cultures isolated from feces were found to be 3Enterococcus faecalis and 3 Enterococcus faecium
While 4 Rod cultures detected in table 7 as one of each culture Escherichia coli, Bacillus brevis, Lactobacillus brevis and Lactobacillus acidophillus.
Table 8 shown the Confirmatory identification for eleven isolates by API.
The endogenous microbial flora plays an important role in health and disease in the human. From our results 42 predominant colonies morphology isolated on MRS agar, BHI agar, MSA and SF agar from 12, 16 and 14 samples of saliva, teeth and faeces respectively. This found to be 24 cocci cells, 14 rods cells and 4 yeasts cells.