In our study, 10 non-commensal pathogenicbacteria were found in the case group and only four were noticed in the controlgroup. In future studies, researchers are expected to find the source of thesespecies in other parts of the body and record those sites as a reservoir forsystemic and oral infections.

  Wang et al reported that   contamination with H.pylori increased in the HCV infected cases in comparison withhealthy controls (33).  In currentresearch we had only one H. pylori  positive case that were  seropositive for HCV and for better diagnosismore cases must be evaluated In our study, a significant association alsowas noticed between the prevalence of E. coli with gender. E. coli was shown to be more prevalent among females incomparison to males .To the best of our knowledge thisis the first report of sex differences in oral carriage of E.

coli. Komiyama andcoworkers also reported a higher carriers rateof  oral enterococcal burden amonghealthy  females than males(32) . In currentresearch, H. pylori was detected inthe saliva samples of 12 disabled males. A significant association was observedbetween the presence of H.

pylori andgender.Other researchers reported that H. pylori infection wasmore prevalent in adult male than females(28), that these results were in linewith ourfinding.  Many subjects with periodontalpockets were demonstrated to hold H. pylori in the oralcavity even after deracination of the microorganism from the stomach(29).

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Butother authors have reported that presence of H.pylori inthe saliva may not be associated with gastric infection(30,31). In our study, K.

pneumonia was also isolated from the saliva ofphysically  disabled volunteers .In acombined oral imprint culture of 56 elderly stroke sufferers in rehabilitationunit of a general hospital in Hong Kong. A significant correlation was alsonoted between presence of K. pneumoniae in the oralcavity and higher risk of aspiration pneumonia in susceptible cases(27).Stroke-related difficulty in oral hygiene maintenance and using dentures may beassociated with this pathogen carriage(27).

Many factorscan influence the bacterial colonization regarding hospitalization, immunestatus alteration, inadequate or poor hygiene, salivary flow reduction and jawmovement during normal chewing that can enhance Enterobacteriaceae colonization(26). The presentstudy, reporting a higher frequency of Enterobacteriaceae amongphysically disabled people. This pathogen also was detected in mouth rinsesamples  of 32% of individual after headand neck radiotherapy (18). Radiotherapy can alter oral cavity bacterial community and increased more subjects of Gram-negativebacteria (23).Evaluating oral bacteria in leukemic patients, Klebsiella was shown tobe 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 nurseryhome in Turkey(24) .Inan oncology hospital in Midwest of Brazil, the presence of Enterobacteriaceae was determinedin the oral cavity of the workers characterizing the phenotypic profile of thespecies 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 thecontrols exhibited different non-oral pathogens.

These bacteria may beoriginated from GI tract, replicated on the dorsum of the tongue, thencontaminated the saliva. It is in line with result of studies that reported thetongue could be a primary reservoir for non-oral pathogens such asEnterobacteriaceae and/or Pseudomonadaceae(21,22).In addition,the microbiota at implants and adjacent teeth were assessed 10 years after theplacement of implants revealing that periodontitis related strains were visibleat 6.2-78.4% of the implants (15).

A significant higher count of Tannerellaforsythia, Parvimonas micra, Fusobacterium nucleatum/ necrophorum,and Campylobacter rectus has been noticed in implants in comparison withteeth(15). Our findings are in line with these results, indicating that changesin factors influencing general or oral hygiene can increase the risk ofcolonization of different species of microbiota .Some retentionfactors such as dentures or implants were demonstrated to play a role inharboring pathogens. In one study, S. aureus, S. pneumoniae, Haemophilusinfluenza, H. parainfluenza, E.

coli, K. pneumonia,Proteus mirabilis, E. cloacae and P. aeruginosa have beenisolated from dentures and oral mucosalsurfaces of  patients suffered from  malodor (12).Saliva samples of removable denturewearers also contaminated by a various types of non- common pathogenic bacteriacomparing to individuals without dentures (13).however inour study none of individual have denture.Several studiesevaluated the oral microflora of different groups such as old age subjects  , healthy removable denture users ,dental  implants ,  cases undergoing head and neckradiotherapy  , leukemic  cases ,comatose patients and children with nail biting habits (12-18). However, the prevalence of non-commensal bacteria in disable cases has notbeen fully examined .

Asthe number of studies carried on the salivary microflora of disabledindividuals is usually lower than those performed on dental plaque, there is acontroversy on similarity of microbial flora between these two sets.(20)In the presentstudy, the difference in microbial population of saliva in two groups of disabledand healthy people has been compared. A variety of non-oral pathogens wereisolated from  physically disabledsubjects including E. coli, K.

pneumoniae, Shigella sp.,Pasteurella sp., Pseudomonas sp.,Providencia sp., Serratia sp., Cedeceasp. and E. cloacae.

Disabled cases were unable to maintain thegeneral and oral health completely, so the microflora in this group wasdifferent from the healthy subjects. During the pastfew years, there has been a growing awareness on the need of disabledpopulation to have a greater access to dental cares; while there have been arelatively small number of dentists who have been trained to cover the specialneeds of this population. DiscussionThe frequency of the detected bacteria in both groupsis presented in Table 2. In the case group, Sighella sp. (47%), Klebsiellapneumoniae (42%) and E. coli (18%) were the main isolated strains (p=0.

001).In the control group, only K. pneumoniae (3%), E. cloacae (2%) andCedecea sp. (1%) were exhibited.

The saliva specimens of 12 cases andone subject of the control group showed H. pylori infection (p=0.002) (Figure 1).  . All caseswith H. pylori infection were male, indicating that the bacteria wasmore prevalent among male than females (p=0.002). Two of H.

pylori positivesubjects were also infected to HBV and HCV. E. coli was more prevalentin females than males (p=0.01). However, the statistical analysis didnot show any significant correlation between gender predilection and the presenceof other bacteria in the saliva (Table 3).

The age in case group ranged from 14 to 59years (mean age: 33.8±9.7 years) and in the control’s from 16 to 52 years (meanage: 35.9 ±8.6 years). Among case group two were seropositive for HBV and HCV andone subject gives history of epileptic seizure 9 months ago. Twenty two percentof cases were negative for infection to aerobic non-commensal  pathogenic bacteria, but in 78% of theirsamples, bacterial growth of these bacteria were detected.

Among control group,96% were not positive for infection to non-commensal  pathogenic bacteria (p<0.001). Thesaliva samples of case group exhibited more than one type of Enterobacteriaceaeincluding E. cloacae (2%) and Cedeacea spp. (1%). Only onesample in the control group was positive for H. pylori that belonged toa male subject.

Infection was totally more prevalent among males than females inboth case and control groups (p=0.002). Two of H. pylori positivesubjects in the case group were seropositive for HBV and HCV. E. coli wasmore prevalent in females than males (p=0.

01), but the statisticalanalysis demonstrated no significant correlation between sex predilection andthe presence of other bacteria in the saliva.ResultsAll 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 wasless than 0.05.One loop consisted of a concentrated suspensionwas inoculated on to blood agar(growth mediumthat contain trypticase soya agar enriched with 5% sheepblood  that enhanced the development of bacteria,) Eosin methylene blue(EMB ) (the selectiveand  proper medium fordistinguishing Gram negative bacteria) and MacConkey agarmedia(used in the detection of coliforms and differentiate microorganisms that ferment lactose from those that do not)using standard streak plate methods. All plateswere incubated at 37°C for 24 h for bacterial growth.

The suspected colonieswere subjected to Gram staining. Then, the Gram negative isolates weresubjected to biochemical reactions using API20E Kit .Colonies were primarily  diagnosed byAPI20E systemAPI (Analytical Profile Index) 20E presented is a biochemical panelfor detection and differentiation of types of the Enterobacteriaceae family andother gram negative rods according to the manufacturer’s protocol (BioMerieuxRSA-France).

Presence of one colony or more  werediagnosed as  positive   cases . All glycerol samples were aliquotedwithout dilution and stored at ?80°C until molecular tests were performed. BacterialDNA was extracted from all saliva samples, using the GeneAll DNA Extraction Minikit(GeneAll, Seoul, Korea) in accordance with the manufacturer’s instructions.

DNAquality and quantity were checked by a spectrophotometric method (BioPhotometer;Eppendorf). The extracted DNA was stored at ?20°C for further use. Moleculardiagnosis of H. pylori was conducted by PCR using gene amplification ofprimers glmM (glmM gene) 11. Theprimers used to amplify for detection  ofH. pylori were assessed by conventional PCR (Table 1).

PCR was performedupon 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 deoxynucleosidetriphosphate, 1 U of Taq polymerase, and 200 ng of genomic DNA. The PCR wasperformed 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 1min 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 (forGram staining)were purchased from Merck (Germany). API20E kit was obtained fromBiomerioux (France). All other chemicals were analytic grade and werecommercially available.

Oral hygiene maintenance methods (flossing and brushing)were instructed to all participants by an experienced dentist, three weeksbefore collecting the saliva sample. Among cases who were unable to clean theirteeth, plaque control methods were practiced by rehabilitation staff nurses. Theunstimulated whole saliva (UWS) was collected between 10 AM to 12 PM and atleast 60 minutes after the last intake of any drinks or foods. Each participantwas asked to refrain from eating, drinking and smoking one hour beforecollecting samples. Then, their lips were cleaned and his/her mouth was rinsedwith water. The subjects were instructed to spit 3 ml unstimulated saliva(10)intosterile Falcon tubes containing 3 ml thioglycholate broth (multipurpose, enriched, differential mediumused primarily to determine the oxygen requirements of microorganisms). The samplecollections were undertaken by a general dentist. Provided samples were centrifugedat 17000 g (12500 rpm) for 10 min and the supernatant was discarded.

Theremained precipitate was re-suspended in 1 ml of phosphate-buffered saline(PBS) to obtain a concentrated suspension.In a cross sectional study from October toNovember 2016 from one hundred disabled subjects (case group) who were kept atthe Narjes Rehabilitation Center in Shiraz, southwest of Iran for at least 6months or more, their saliva samples were collected. The case group comprisedof 52 males and 48 females who had lost one or both hands in an accident.  89 participant lost their dominant hand and 11one had lost both of their hands .The control group included 100 age- andsex-matched healthy participants who referred to various departments of Shiraz  Dental School for routine dental care. Allparticipants 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 antimicrobialmouth wash or treatment with antibiotics in the past two months were excludedfrom the study. Demographic data were obtained from the history charts of thecases.This study was carried out in accordance withthe guidelines of the Declaration of Helsinki as revised in Edinburgh (1975).The study protocol was approved by the Ethics Committee of Shiraz University ofMedical Sciences, Shiraz, Iran. A written and verbally informed consent wasobtained from all participants .Methodsin the bedridden patients(9). Owing to thereservoir activity of the oral cavity for several pathogens related to systemicinfections, this study was designed to identify the occurrence of non-commensal Gram-negative bacteria in the oralcavity of  physically disabled subjectsin Shiraz, IranThe correlation between oral bacterial floraand the functional activity and the health status of the elderly was evaluated showing that, the patientswho stayed for a long term in the hospitals exhibited significantly less ratesof commensal bacterial species in comparison to the outpatient subjects(9).However, an increasing rate of Pseudomonas aeruginosa,methicillin-resistant Staphylococcus aureus (MRSA) and coagulasenegative Staylococcus aureus (CoNS) was notedThe disabled population compromise an essentialpart of the community estimated to be more than 1 billion subjects in the worldthat suffer from physical disability and can have limitations in general andoral hygiene performance.

 Severalfactors have been shown to affect oral microbiota . It was found thatopportunistic microorganism with moderate growth were present in the oralcavity of old patients, and immuno-compromised cases(8).of H. pylori is unclear, but it seemsthat person-to-person contact via oral-oral and faecal-oral mode are the mainroutes of infection(6,7).

Helicobacter pylori (H. pylori) are an enteric pathogenthat can cause gastritis, peptic ulcer, and gastric carcinoma. The route oftransmission Enterobacteriaceae species aretransitory pathogens in the oral cavity, which may cause serious systemicconditions such as meningitis, pneumonia, food poisoning, typhoid fever andbacillary dysentery. In cases of insufficient toilet hygiene or harmful habits,these bacteria can enter the body through different origins like the mouth. Itwas demonstrated that the saliva samples of 25 children with nail biting habitswere infected to Enterobacteriaceae(5).  It was shown that approximately 15% of thepeople may harbor Enterobacteriaceae in the oral cavity, while the their growthmay be enhanced in older age due to xerostomia(2).found in the oral cavity of human being(4-7).desirable location for bacterial growth.

Inthis relation, the saliva or oropharyngeal secretions act as an importantsource of many microorganisms that can spread the bacteria through sneezing, coughing,speaking or breathing(3). Based on the significant relationship between theoral pathogenic bacterium and several systemic diseases, it is imperative to beaware of infection to pathogenic oral and non-oral bacteria4. Allbacteriologists are familiar with oral normal microflora, but recently manystudies revealed isolation of certain species that are not normallyThe anatomical and physiological properties ofthe oral cavity have made it as aoral normal flora(2).The oral microbial flora is highly diverse forpresence of various bacteria and fungi.

Approximately 1000 bacterial specieshave been isolated from the oral cavity(1). Streptococci and anaerobicGram negative bacteria have been reported as the most prevalent bacteria ofhuman oral normal flora. Any alteration in normal flora was shown to influencethe course of oral diseases as these microorganisms can play an important rolein preventing colonization of pathogenic microbiota (2), while severalenvironmental and behavioral may also affect and alter the