In our study, 10 non-commensal pathogenic
bacteria were found in the case group and only four were noticed in the control
group. In future studies, researchers are expected to find the source of these
species in other parts of the body and record those sites as a reservoir for
systemic and oral infections.  

 

Wang et al reported that   contamination with H.
pylori increased in the HCV infected cases in comparison with
healthy controls (33).  In current
research we had only one H. pylori  positive case that were  seropositive for HCV and for better diagnosis
more cases must be evaluated

In our study, a significant association also
was noticed between the prevalence of E. coli with gender. E. coli was shown to be more prevalent among females in
comparison to males .To the best of our knowledge this
is the first report of sex differences in oral carriage of E.coli. Komiyama and
coworkers also reported a higher carriers rate
of  oral enterococcal burden among
healthy  females than males
(32) .

In current
research, H. pylori was detected in
the saliva samples of 12 disabled males. A significant association was observed
between the presence of H. pylori and
gender.Other researchers reported that H. pylori infection was
more prevalent in adult male than females(28), that these results were in line
with our
finding.  Many subjects with periodontal
pockets were demonstrated to hold H. pylori in the oral
cavity even after deracination of the microorganism from the stomach(29). But
other authors have reported that presence of H.
pylori in
the saliva may not be associated with gastric infection(30,31).

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In our study, K. pneumonia was also isolated from the saliva of
physically  disabled volunteers .In a
combined oral imprint culture of 56 elderly stroke sufferers in rehabilitation
unit of a general hospital in Hong Kong. A significant correlation was also
noted between presence of K. pneumoniae in the oral
cavity and higher risk of aspiration pneumonia in susceptible cases(27).
Stroke-related difficulty in oral hygiene maintenance and using dentures may be
associated with this pathogen carriage(27).

Many factors
can influence the bacterial colonization regarding hospitalization, immune
status alteration, inadequate or poor hygiene, salivary flow reduction and jaw
movement during normal chewing that can enhance Enterobacteriaceae colonization
(26).

The present
study, reporting a higher frequency of Enterobacteriaceae among
physically disabled people. This pathogen also was detected in mouth rinse
samples  of 32% of individual after head
and neck radiotherapy (18). Radiotherapy can alter oral cavity bacterial community and increased more subjects of Gram-negative
bacteria (23).
Evaluating oral bacteria in leukemic patients, Klebsiella was shown to
be the most isolated kind, followed by Enterobacteriaceae(19). It was reported that   gram negative bacilli  can be detected in 2.8%  of admitted patients in psychiatric nursery
home in Turkey(24) .In
an oncology hospital in Midwest of Brazil, the presence of Enterobacteriaceae was determined
in the oral cavity of the workers characterizing the phenotypic profile of the
species showing that these bacteria were colonized in the oral cavity of 18.7%
of the participants (25).

In our study,
it was found that the saliva culture in 78% of the case group and 7% of the
controls exhibited different non-oral pathogens. These bacteria may be
originated from GI tract, replicated on the dorsum of the tongue, then
contaminated the saliva. It is in line with result of studies that reported the
tongue could be a primary reservoir for non-oral pathogens such as
Enterobacteriaceae and/or Pseudomonadaceae(21,22).

In addition,
the microbiota at implants and adjacent teeth were assessed 10 years after the
placement of implants revealing that periodontitis related strains were visible
at 6.2-78.4% of the implants (15). A significant higher count of Tannerella
forsythia, Parvimonas micra, Fusobacterium nucleatum/ necrophorum,
and Campylobacter rectus has been noticed in implants in comparison with
teeth(15). Our findings are in line with these results, indicating that changes
in factors influencing general or oral hygiene can increase the risk of
colonization of different species of microbiota .

Some retention
factors such as dentures or implants were demonstrated to play a role in
harboring pathogens. In one study, S. aureus, S. pneumoniae, Haemophilus
influenza, H. parainfluenza, E. coli, K. pneumonia,
Proteus mirabilis, E. cloacae and P. aeruginosa have been
isolated from dentures and oral mucosal
surfaces of  patients suffered from  malodor (12).Saliva samples of removable denture
wearers also contaminated by a various types of non- common pathogenic bacteria
comparing to individuals without dentures (13).however in
our study none of individual have denture.

Several studies
evaluated the oral microflora of different groups such as old age subjects  , healthy removable denture users ,
dental  implants ,  cases undergoing head and neck
radiotherapy  , leukemic  cases 
,comatose patients and children with nail biting habits (12-18). However, the prevalence of non-commensal bacteria in disable cases has not
been fully examined .As
the number of studies carried on the salivary microflora of disabled
individuals is usually lower than those performed on dental plaque, there is a
controversy on similarity of microbial flora between these two sets.(20)

In the present
study, the difference in microbial population of saliva in two groups of disabled
and healthy people has been compared. A variety of non-oral pathogens were
isolated from  physically disabled
subjects including E. coli, K. pneumoniae, Shigella sp.,
Pasteurella sp., Pseudomonas sp.,Providencia sp., Serratia sp., Cedecea
sp. and E. cloacae. Disabled cases were unable to maintain the
general and oral health completely, so the microflora in this group was
different from the healthy subjects.

During the past
few years, there has been a growing awareness on the need of disabled
population to have a greater access to dental cares; while there have been a
relatively small number of dentists who have been trained to cover the special
needs of this population.

Discussion

The frequency of the detected bacteria in both groups
is presented in Table 2. In the case group, Sighella sp. (47%), Klebsiella
pneumoniae (42%) and E. coli (18%) were the main isolated strains (p=0.001).
In the control group, only K. pneumoniae (3%), E. cloacae (2%) and
Cedecea sp. (1%) were exhibited. The saliva specimens of 12 cases and
one subject of the control group showed H. pylori infection (p=0.002) (Figure 1).  . All cases
with H. pylori infection were male, indicating that the bacteria was
more prevalent among male than females (p=0.002). Two of H. pylori positive
subjects were also infected to HBV and HCV. E. coli was more prevalent
in females than males (p=0.01). However, the statistical analysis did
not show any significant correlation between gender predilection and the presence
of other bacteria in the saliva (Table 3).

The age in case group ranged from 14 to 59
years (mean age: 33.8±9.7 years) and in the control’s from 16 to 52 years (mean
age: 35.9 ±8.6 years). Among case group two were seropositive for HBV and HCV and
one subject gives history of epileptic seizure 9 months ago. Twenty two percent
of cases were negative for infection to aerobic non-commensal  pathogenic bacteria, but in 78% of their
samples, bacterial growth of these bacteria were detected. Among control group,
96% were not positive for infection to non-commensal  pathogenic bacteria (p<0.001). The saliva samples of case group exhibited more than one type of Enterobacteriaceae including E. cloacae (2%) and Cedeacea spp. (1%). Only one sample in the control group was positive for H. pylori that belonged to a male subject. Infection was totally more prevalent among males than females in both case and control groups (p=0.002). Two of H. pylori positive subjects in the case group were seropositive for HBV and HCV. E. coli was more prevalent in females than males (p=0.01), but the statistical analysis demonstrated no significant correlation between sex predilection and the presence of other bacteria in the saliva. Results All data were analyzed by SPSS software (Version 22, Chicago, IL, USA), using Chi-Square and Fisher Exact tests. Statistically significant difference was considered when p value was less than 0.05. One loop consisted of a concentrated suspension was inoculated on to blood agar(growth medium that contain trypticase soya agar enriched with 5% sheep blood  that enhanced the development of bacteria,) Eosin methylene blue(EMB ) (the selective and  proper medium for distinguishing Gram negative bacteria) and MacConkey agar media(used in the detection of coliforms  and differentiate microorganisms that ferment lactose from those that do not)using standard streak plate methods. All plates were incubated at 37°C for 24 h for bacterial growth. The suspected colonies were subjected to Gram staining. Then, the Gram negative isolates were subjected to biochemical reactions using API20E Kit .Colonies were primarily  diagnosed byAPI20E system API (Analytical Profile Index) 20E presented is a biochemical panel for detection and differentiation of types of the Enterobacteriaceae family and other gram negative rods according to the manufacturer's protocol (BioMerieuxRSA-France). Presence of one colony or more  were diagnosed as  positive   cases . All glycerol samples were aliquoted without dilution and stored at ?80°C until molecular tests were performed. Bacterial DNA was extracted from all saliva samples, using the GeneAll DNA Extraction Minikit (GeneAll, Seoul, Korea) in accordance with the manufacturer's instructions. DNA quality and quantity were checked by a spectrophotometric method (BioPhotometer; Eppendorf). The extracted DNA was stored at ?20°C for further use. Molecular diagnosis of H. pylori was conducted by PCR using gene amplification of primers glmM (glmM gene) 11. The primers used to amplify for detection  of H. pylori were assessed by conventional PCR (Table 1). PCR was performed upon a standard protocol using a 25 ml mixture containing 2.5 mlof 10x buffer (supplied with Taq polymerase), 10 pmol of each primer, 2 mM of each deoxynucleoside triphosphate, 1 U of Taq polymerase, and 200 ng of genomic DNA. The PCR was performed with BIORAD T100 thermal cycler with Micro tubes under the following conditions: denaturation for 5 min at 95°C; 35 cycles of 45 s at 95°C, 45 s at Ta°C and 1 min at 72°C; and a final extension step of 10 min at 72°C. Eosin methylene blue (EMB), MacConkey agar, blood agar, thioglycolate broth, crystal violet, safranin, Lugol's iodine acetone/alcohol reagents (for Gram staining)were purchased from Merck (Germany). API20E kit was obtained from Biomerioux (France). All other chemicals were analytic grade and were commercially available. Oral hygiene maintenance methods (flossing and brushing) were instructed to all participants by an experienced dentist, three weeks before collecting the saliva sample. Among cases who were unable to clean their teeth, plaque control methods were practiced by rehabilitation staff nurses. The unstimulated whole saliva (UWS) was collected between 10 AM to 12 PM and at least 60 minutes after the last intake of any drinks or foods. Each participant was asked to refrain from eating, drinking and smoking one hour before collecting samples. Then, their lips were cleaned and his/her mouth was rinsed with water. The subjects were instructed to spit 3 ml unstimulated saliva(10)into sterile Falcon tubes containing 3 ml thioglycholate broth (multipurpose, enriched, differential medium used primarily to determine the oxygen requirements of microorganisms). The sample collections were undertaken by a general dentist. Provided samples were centrifuged at 17000 g (12500 rpm) for 10 min and the supernatant was discarded. The remained precipitate was re-suspended in 1 ml of phosphate-buffered saline (PBS) to obtain a concentrated suspension. In a cross sectional study from October to November 2016 from one hundred disabled subjects (case group) who were kept at the Narjes Rehabilitation Center in Shiraz, southwest of Iran for at least 6 months or more, their saliva samples were collected. The case group comprised of 52 males and 48 females who had lost one or both hands in an accident.  89 participant lost their dominant hand and 11 one had lost both of their hands .The control group included 100 age- and sex-matched healthy participants who referred to various departments of Shiraz  Dental School for routine dental care. All participants were dentate and had at least 8 teeth. Patients with   clinical evidence of oral mucosal lesions, history of diabetes mellitus, pregnancy, debilitating diseases, use of antimicrobial mouth wash or treatment with antibiotics in the past two months were excluded from the study. Demographic data were obtained from the history charts of the cases. This study was carried out in accordance with the guidelines of the Declaration of Helsinki as revised in Edinburgh (1975). The study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran. A written and verbally informed consent was obtained from all participants . Methods in the bedridden patients(9). Owing to the reservoir activity of the oral cavity for several pathogens related to systemic infections, this study was designed to identify the occurrence of non-commensal  Gram-negative bacteria in the oral cavity of  physically disabled subjects in Shiraz, Iran The correlation between oral bacterial flora and the functional activity and the  health status of the elderly was evaluated showing that, the patients who stayed for a long term in the hospitals exhibited significantly less rates of commensal bacterial species in comparison to the outpatient subjects(9). However, an increasing rate of Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) and coagulase negative Staylococcus aureus (CoNS) was noted The disabled population compromise an essential part of the community estimated to be more than 1 billion subjects in the world that suffer from physical disability and can have limitations in general and oral hygiene performance.  Several factors have been shown to affect oral microbiota . It was found that opportunistic microorganism with moderate growth were present in the oral cavity of old patients, and immuno-compromised cases(8). of H. pylori is unclear, but it seems that person-to-person contact via oral-oral and faecal-oral mode are the main routes of infection(6,7). Helicobacter pylori (H. pylori) are an enteric pathogen that can cause gastritis, peptic ulcer, and gastric carcinoma. The route of transmission   Enterobacteriaceae species are transitory pathogens in the oral cavity, which may cause serious systemic conditions such as meningitis, pneumonia, food poisoning, typhoid fever and bacillary dysentery. In cases of insufficient toilet hygiene or harmful habits, these bacteria can enter the body through different origins like the mouth. It was demonstrated that the saliva samples of 25 children with nail biting habits were infected to Enterobacteriaceae(5).  It was shown that approximately 15% of the people may harbor Enterobacteriaceae in the oral cavity, while the their growth may be enhanced in older age due to xerostomia(2). found in the oral cavity of human being(4-7). desirable location for bacterial growth. In this relation, the saliva or oropharyngeal secretions act as an important source of many microorganisms that can spread the bacteria through sneezing, coughing, speaking or breathing(3). Based on the significant relationship between the oral pathogenic bacterium and several systemic diseases, it is imperative to be aware of infection to pathogenic oral and non-oral bacteria4. All bacteriologists are familiar with oral normal microflora, but recently many studies revealed isolation of certain species that are not normally The anatomical and physiological properties of the oral cavity have made it as a oral normal flora(2). The oral microbial flora is highly diverse for presence of various bacteria and fungi. Approximately 1000 bacterial species have been isolated from the oral cavity(1). Streptococci and anaerobic Gram negative bacteria have been reported as the most prevalent bacteria of human oral normal flora. Any alteration in normal flora was shown to influence the course of oral diseases as these microorganisms can play an important role in preventing colonization of pathogenic microbiota (2), while several environmental and behavioral may also affect and alter the

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