Occurrence and antimicrobial susceptibility of enteric rods and pseudomonads isolated from the dental prostheses biofilm

ABSTRACT Aspiration of oral bacteria leads to cardiac and respiratory infectious diseases and dentures can act as a reservoir for pathogenic microorganisms. Objective: To determine the occurrence and the in vitro antimicrobial susceptibility of enteric rods and pseudomonads from the denture biofilm of 52 subjects at the Center for Dental Specialties of Sobral/ Ceara, Brazil. Material and Methods: Denture biofilm was collected and samples plated on MacConkey agar. The isolated bacterial colonies were identified using the BBL Crystal enteric/non-fermenter system. Antibiotic bacterial susceptibility was assessed by the disc diffusion method of amoxicillin, amoxicillin/clavulanic acid, doxycycline, tetracycline, tobramycin, imipenem, cefotaxime, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) of cefotaxime, tobramycin, doxycycline, imipenem, and ciprofloxacin was determined for 40 species by E-test. Results: 34 subjects (65.4%) harbored enteric rods in their prostheses. Klebsiella pneumoniae (26.5%), Escherichia coli (23.5%), and Enterobacter aerogenes (23.5%) were the most prevalent species. All organisms were susceptible to ciprofloxacin and most species were resistant to amoxicillin or amoxicillin/clavulanic acid, demonstrating variable sensitivity patterns to other antimicrobials. However, the MIC showed the emergence of strains with reduced sensitivity to ciprofloxacin (MIC90≥3 μg/ mL) and cefotaxime (MIC90≥2 μg/mL). Conclusion: The findings show high prevalence of nosocomial diseases-related bacterial species and low susceptibility to antimicrobial drugs. Therefore, these results imply caution against the indiscriminate use of broad spectrum antibiotics in dental practice.


INTRODUCTION
Variations in the oral microbiota are directly related to age and have been attributed to the use of dentures 16 . The oral health status declines as a result of the aging process and individuals culminate with the need of dental prostheses, affecting their health, functional activities, and self-esteem 8 . Worldwide, 810 million people are aged 60 or over, which is predicted to increase to at least two billion by 2050, 22% of the entire global population, and challenges for oral health care delivery, to an increasingly aged population with declining oral health 13,19,31 . These individuals may not be able to clean their dentures properly, which in turn could result in exposing these individuals to the possibility of systemic severe infections. Currently, denture liners are available as silicone-based and acrylic resinbased. The adhesion to these materials depends on the properties of the surface of the microbial cells complex three-dimensional architecture. One of the problems directly related to these materials is still turn has received little attention by patients and general clinicians as its dental counterpart 32 .
The presence of enteric rods and pseudomonads of their pathogenic potential and ability to adhere to solid surfaces 2,17 . In addition, infections caused by of the bacterial resistance to a variety of antibiotics, cephalosporins, aminoglycosides, carbapenems, chloramphenicol, aztreonam, trimethoprim/ sulfametaxazol, tetracycline, and doxycycline 5,24 .
The clinical importance of the non-fermenters gram-negative bacilli presence in the denture biofilm has significantly increased because of infections, high rates of morbidity, mortality in hospitalized subjects, and high levels of resistance. The dissemination of gram-negative bacteria with acquired antimicrobial drugs resistance is becoming a global problem. The propagation and dissemination of these microorganisms have 11,25 . Enteric microorganisms and pseudomonads have However, there are current reports of resistant enteric rods to carbapenems 30 . Among bacterial isolates resistant to ampicillin or amoxicillin, the production of these hydrolytic enzymes seems to be the major mechanism of resistance to resistance to tetracycline was widely disseminated in the microbial enteric rods, and many of tested microorganisms were resistant 25 .
Having known such information, our hypotheses are that wearing dental prostheses older people may harbor superinfecting microorganisms in their the surface of these appliances as well as these bacterial species could possess antimicrobial resistance. Therefore, we consider relevant to determine the prevalence of Enterobacteriaceae and Pseudomonadaceae species isolated from in vitro susceptibility to antimicrobial drugs, since these opportunistic microorganisms were previously 15,28,29 .

Study population
Enteric microorganisms were isolated from the inner surface of dentures of 52 subjects within a 2-year follow-up period (2007)(2008) at the Center for Dental Specialties -Sobral, State of Ceará, Brazil. The requirements for inclusion in this study were: no history of diabetes, no use of antimicrobials in the past three months, nor other medication that could affect their systemic or local immune system. All subjects signed a written consent form that was approved by the Institutional Review Board of Federal University of Ceará (COMEPE nº 258/07).

Data collection
All subjects answered a questionnaire about their systemic health and a dental and soft tissue examination was performed to assess their oral in the medical record of each patient. Initially, the surfaces of the dentures were thoroughly dried with sterilized gauze to avoid contamination by dentures was collected with the aid of a sterile swab, and the samples were immediately placed in sterile phosphate buffered saline (PBS, pH 7.5, 0.8% NaCl) to the Department of Microbiology and Parasitology, Federal University of Ceará, in Sobral, and processed within a maximum of 2 hours after collection. Samples were dispersed by agitation (30 seconds) and serially diluted (10 -1 and 10 -2 ) in PBS. Aliquots of 100 μL of the solutions obtained were plated on MacConkey agar (Acumedia, Lansing, Michigan, USA). Then, the plates were incubated at 36°C±1°C for 24 hours.
Gram staining, colony morphology on MacConkey agar plates (Acumedia, Lansing, Michigan, USA), production of oxidase (Oxidase Newprov Strips, Enterobacteriaceae enteric bacilli fermenters, oxidase-negative bacilli, and non-fermenter bacilli was carried out by the system BBL Crystal Enteric/Nonfermenter (Becton Dickinson Systems, Cockeysville, Maryland, USA) according to the manufacturer's instructions. The sample Escherichia coli ATCC 25922 was used as control.

Antimicrobial susceptibility test
previously collected were stored in broth heart infusion medium (Acumedia, Lansing, Michigan, USA) with glycerol (3:1) at -80°C before performing the antimicrobial susceptibility tests. Then, each strain was seeded in broth heart infusion medium (Acumedia, Lansing, Michigan, USA) and incubated in a microbiological greenhouse at 36°C±1°C for 24 2016;24(5):462-71 hours. Thereafter, in order to perform the sensitivity test, it was used the disk diffusion method of the Clinical & Laboratory Standards Institute manual 6 . The antimicrobial drugs tested were amoxicillin (10 the medium used for this test. After seeding and incubating the samples in a microbiological greenhouse at 36°C±1°C for 24 hours, the zones of inhibition were measured and the organisms were according to CLSI references 6,7 . A total of 52 strains of enteric rods and pseudomonads were submitted to susceptibility tests. For multidrug resistant species, the Minimum Inhibitory Concentration (MIC) was determined for the following drugs: cefotaxime (CT), tobramycin (TM), doxycycline methodology used was E-test ® (AB Biodisk, Solna, Sweden) and interpretations were made according to the CLSI references 6,7 .

Statistical analysis
The Mann-Whitney test was used to determine differences regarding age groups for men and women, and the chi-square test was used to access differences between genders, age groups, and time of prostheses use correlated between the presence and absence of the studied microorganisms. Differences of p<0.05 were considered statistically
According to Tables 2 and 3, 34 (65.4%) subjects harbored enteric bacilli and/or pseudomonas species on dental prostheses. Most of these subjects (91.2%) aged over 50. Also, the studied microorganisms were found in 15 (75%) subjects in the group of subjects who wore dental prostheses over a 10-year period of time (Table 4)

DISCUSSION
Enteric bacilli and pseudomonads are opportunistic pathogens at different human body sites. The oral cavity functions as a reservoir for pathogenic species 3,17 . As a result of the aging process, the oral health status declines, often leading to defective oral/denture hygiene in geriatric subjects, being the reasons for this fact the lack of hygiene education of these subjects or of those caring for them. Consequently, oral hygiene is often neglected, resulting in poor oral health and in an increase in the presence of local or general infections that can be related with the presence of enteric microorganisms in the oral cavity 4,9 . Here we show that dental prostheses can harbor this study; in addition, these microorganisms can be even resistant to antimicrobial drug therapy as demonstrated here. This is really alarming because, albeit many of these subjects might not be systemically compromised by any disease at the moment they house such pathogens in their oral cavity, they could suffer from any illness that could dampen and compromise their immune system, making them, in turn, more susceptible to be infected by the oral opportunistic pathogens they are harboring. Thus, a proper denture hygiene protocol is essential, since it could prevent these individuals from exposition to these bacterial species, considering that respiratory pathogens are more capable of colonizing teeth and dentures instead of soft tissues, and pneumonia is the main cause of death related to infection in older people 8 . By wearing dental prostheses, individuals are at a higher risk of aspirating such pathogens from the oral appliance and lungs, being reported a high prevalence of respiratory pathogens on the denture 21 . Furthermore, not only are such pathogens involved in the development of aspiration pneumonia, but also they have been     * CT (0.002-32 μg/mL), TM (0.016-256 μg/mL), DC (0.016-256 μg/mL), IP (0.002-32 μg/mL), CI (0.002-32 μg/mL) **μg/mL in the city of Sobral. As opposed to a similar study in a Japanese population, in which the prevalence of potential pathogens on dentures was 18% for E. cloacae and 16% for K. pneumoniae, our research found that K. pneumoniae was the most prevalent at 26.5%, followed by Escherichia coli and E. aerogenes, placed after 23.5% 28 . Another study showed that denture plaque in patients with chronic obstructive pulmonary disease was colonized by pathogens of the respiratory tract, including: E. coli, Pseudomonas spp., and Klebsiella spp. Over 33% of the isolated pathogens are part of rod-shaped and gram-negative Enterobacter spp. The isolation of the mentioned bacteria from denture plaque proves that dental prostheses might become a source of infection of the respiratory tract or may exacerbate chronic respiratory diseases 25 .
Contrary to the data we report, another group described a much lower (20.3%) prevalence of enterobacteriaceae in the oral cavity of older people from Greece that used dental prostheses 14 , whereas a different group reported that 48% of edentulous subjects harbored enteric rods in their oral cavity, being K. oxytoca, E. cloacae, and K. pneumoniae species more prevalent 12 . According to these authors, the prevalence of such microorganisms in the denture biofilm may be related to the ability of these species to adhere to the polymer of the denture and to aggregate in the presence of ammonium sulfate. This could explain the high prevalence of K. pneumoniae reported in our study.
The classic literature shows that the prevalence of these organisms in the oral cavity of systemically healthy individuals can vary among different populations 1,26,27 . Thus, the high prevalence (65.4%) we observed in this study could be attributed to disadvantaged health infrastructure and educational issues that could have led to ingestion of contaminated food or water. Also, poor hygiene habits and indiscriminate use of antibiotics may play a role in this high prevalence 4,11 .
In fact, it was observed that the prostheses encountered when conducting a properly cleaning, we can include the lack of adequate patient guidance, characteristics of the prostheses, decreased motor ability, and lack of proper products in the market to carry out the correct denture cleaning. The habit of brushing the prostheses with toothpaste and the use of common brushes may not be the best way the prolonged use of the same prostheses for many years might contribute to the colonization process by potential pathogens 4 . It is relevant to highlight that this information shows a major limitation of these studies, since there are many factors that can lead to microbial colonization of dentures. In our study, we found prostheses being worn for 40 years, and most of the subjects wearing contaminated dentures have been doing so for over 10 years.
Regarding the antimicrobial susceptibility of microorganisms, our data show that 86.5% of anaerobic facultative gram-negative bacilli were resistant to amoxicillin and over half of them had resistance to amoxicillin with clavulanic acid contrary to other studies that showed the association amoxicillin/clavulanic acid being active on less than half of ampicillin or amoxicillin resistant isolates from detected in 28.3% of the targeted microorganisms and it was particularly frequent in E. cloacae, genera Klebsiella, Serratia, and Pseudomonas 11 .
The analysis of the MIC proved that the antimicrobial drug with the highest inhibitory activity against the enteric bacilli and pseudomonads 90 we observed a decreased susceptibility to this antimicrobial agent in two (22.2%) strains of K. pneumoniae respectively. Although it is a much lower frequency than that observed previously 20 , decreased 90 >2 μg/ mL) among clinical isolates of Enterobacteriaceae species is a particular concern, since enteric rods were able to colonize the denture surface of non-hospitalized subjects, which could spread in the community. Fluoroquinolones are the most widely used antibiotics worldwide, and are the drugs of choice for empirical therapy for urinary tract infection. Fluoroquinolone resistance in members of the Enterobacteriaceae family has until recently been attributed to mutations in the gyrase and topoisomerase genes quinolone resistancedetermining regions. Given their transferability and the possibility that they cause increases in resistance that might affect the clinical response to treatment, the detection of quinolone resistance should be routinely performed. There are several surveillance or retrospective studies with clinical isolates, and these studies showed the characteristic features observed in drug-resistant strains in addition to epidemics caused by them 33 . support to the prevalent view that ESBL plasmids are conjugative, may be borne on transposons, and that the genes may have high mutation frequencies. Treatment of infections caused by these ESBL-producing bacteria has become challenging. In addition to being resistant to commonly used usually resistant to other classes of antibiotics is the practice of irrational usage of antibiotics, leading these microorganisms to exhibit a unique microorganism resistant pattern, which hugely impacts on clinical choice of a correct antibiotic once antimicrobial drug resistance develops, making 15,22,23 . Antimicrobial resistance surveillance programs susceptibility of clinically relevant enteric bacteria and pseudomonads from nosocomial infections and the environment 5,18 . However, they can be found in of the individuals to systemic infections or even worsening them. Thus, it is crucial to emphasize that dentures can harbor such pathogens and this is the reason why a correct hygiene protocol must be carried out in order to avoid such ultimately, would increase the risk of one to be exposed to enteric bacilli.

CONCLUSION
K. pneumoniae, E. coli, and E. aerogenes were the predominant species found on the denture biofilm. Most enteric bacilli and Pseudomonas spp. were resistant to amoxicillin and amoxicillin clavulanate, with variable susceptibility patterns to other antimicrobial drugs. The antibiotic that showed the highest inhibitory activity against them was from the results obtained in this study, we suggest is important to avoid the colonization of dental prostheses by multidrug resistant bacteria as well as avoiding the indiscriminate prescription of antibiotics will help diminish the multidrug resistance insurgence.