Non‐oral, aerobic, Gram‐negative bacilli in the oral cavity of Thai HIV‐positive patients on Highly‐active anti‐retrovirus therapy medication

Abstract In the present study, we identified and evaluated the antibiotic susceptibility of 96 independent, aerobic, Gram‐negative bacillus isolates from 255 Thai HIV‐positive adults who were on Highly‐active anti‐retrovirus therapy (HAART) medication. Another 46 isolates from HIV non‐HAART individuals, vertically transmitted HIV‐positive individuals, and non‐HIV controls were included for comparison. A total of 103 strains were tested for antibiotic susceptibility using disc diffusion for screening and E‐test for minimal inhibitory concentration determination, with special attention on extended‐spectrum beta‐lactamase (ESBL) isolates. Pseudomonas aeruginosa, Pseudomonas luteola, Burkholderia cepacia, Aeromonas hydrophila, Klebsiella, and Enterobacter species were the most common bacteria. All strains were resistant against penicillin, amoxicillin, clindamycin, and metronidazole. No ESBL isolates were found.

AGNB is needed, as they are frequently are multiresistant to common antibiotics used in dentistry. A major concern is the presence of the extended-spectrum beta-lactamase (ESBL) resistant isolates, which to the best of our knowledge, have not been isolated from the human oral cavity. In a previous study on the presence of opportunistic microorganisms in HIV-positive individuals, the frequency and load of such bacteria in the Highly-active anti-retrovirus therapy (HAART) group were similar to those of the non-HAART group, but significantly higher than those of HIV-negative controls. 11 In the present study, we report on the species identification and antibiotic susceptibility of AGNB isolates from the oral cavity of HIV-positive individuals on HAART medication, with special attention to the presence of ESBL isolates.

| Bacterialstrains
A total of 142 AGNB strains were used in the present study. They were originally isolated from patients attending the Infectious Diseases Clinic at the Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand. 11 Of 255 Thai HIV-positive adults on HAART medication, 96 independent AGNB strains were isolated from 87 AGNB-positive individuals; nine individuals had two different species. Another 46 species were isolated from 20 HIV non-HAART individuals (8 strains), 19 from 53 vertically-transmitted HIV-positive individuals, and 19 from 30 non-HIV controls, which were included for comparison. Sixteen individuals had two different species. The strains were originally isolated in samples taken from dorsum of the tongue, gingiva, and if present, a deep periodontal pocket or a mucosal lesion, as previously described elsewhere. 11 If the same species was present in more than one sample, the isolate from the tongue was chosen for the study. Thus, 127 strains were from the tongue and 16 from the gingiva.
The cultivation analysis followed the methods, as described in elsewhere. 11

| Antibioticsusceptibility
Routine screening for antibiotic susceptibility was performed using blood agar plates and the disc diffusion method (Oxoid, Basingstoke, UK) against six antibiotics commonly used in dentistry: penicillin V, isoxapenicillin, amoxicillin, clindamycin, erythromycin, and tetracycline. After incubation, the diameter of the inhibition zone of each strain was measured, and the strains were graded as sensitive, intermediate, and resistant. Minimal inhibitory concentration (MIC) was determined using the E-test method (Biomerieux) against doxycycline, gentamicin, ciprofloxacin, norfloxacin, ceftibuten, cefotaxime, and ceftazidime. The MIC were read from the intercept where the ellipse inhibition zone intersected with the scale. European Committee on Antimicrobial Testing recommendations cut of levels for resistance were used. 13 Screening and confirmatory test for ESBL production, and discs with ceftazidime (30 μg) and cefotaxime (30 g) were placed on the media with the test inoculum and incubated for 24 hours. Bacterial isolates showing ≤22 mm ceftazidime (corresponding to 2 μg/mL) and ≤27 mm cefotaxime (corresponding to 2 μg/mL) zones were suspected to be ESBL producers according to National Committee for

Clinical Laboratory Standards and Center for Disease Control and
Prevention. 14 For ESBL confirmation, a combined disc method was used using ceftazidime and cefotaxime with clavulanic acid discs (30 μg; Oxoid, UK).

| RE SULTS
The identification of AGNB species in the oral cavity (predominantly from the dorsum of the tongue) of the 142 strains isolated as independent strains from 117 participants is shown in Table 1 (Table 1).
All 142 strains were shown to be resistant against penicillin V, amoxicillin, clindamycin, and metronidazole using the disc diffusion test (data not shown). MIC estimation using E-test of 103 of the isolated AGNB is shown in Table 2. Ciprofloxacin showed activity against most strains, except for Stenotrophomonas spp, Burkholderia cepacia complex, and Aeromonas spp, which showed resistance in 80%, 11.1%, and 30% of the strains, respectively. A similar resistance pattern was shown for norfloxacin, ceftibuten, cefotaxime, and ceftazidime. No ESBL strains were found.

| D ISCUSS I ON
The most common AGNB species isolated in the present study were lactose non-fermenting, but oxidase-positive, strains classified as Pseudomonas spp (Pseudomonas aeruginosa and Pseudomonas luteola), Burkholderia cepacia complex, and Aeromonas spp (Aeromonas hydrophila), which are normally found in water, food, and plants, but occasionally cause nosocomial infections in immune-compromised and hospitalized patients. 7 Pseudomonas spp can occur in the transient oral microbiota, but rarely colonize the oral cavity, which is due to their strong aerobic character. Later studies using molecular biology methods have shown that their presence might be underestimated, 15 and their presence in the oral cavity has been found to be higher in complex biofilms, such as dental plaque. Pseudomonas spp are considered to be the main pathogen in chronic obstructive pulmonary disease and in biofilms on vehicles at intubation, 16 (11) NLF, non-lactose fermenting.
The remaining AGNB isolates were predominantly lactosefermenting and oxidase-negative Enterobacteriaceae, commonly referred to as "enterics," "enteric rods," or "coliform rods." They are predominantly classified into the genera of Enterobacter (Enterobacter isolated, which was somewhat surprising. To avoid misunderstandings, the designation "coliform rods" should not be used in the context of oral microbiota, because they are more likely to be something else than Escherichia coli. Enterobacter spp and Klebsiella spp are commonly related to hospital infections and affect immune-compromised patients with oral mucosal infections. 4,5 Taken together, none of these AGNB were more specifically associated to any of the HIV or non-HIV groups. They were generally present in low numbers, 11 which might indicate that their prevalence is in fact higher, but could be missed in the sampling process. Even considering presence in higher numbers (moderate/heavy loads corresponding to >10 3 colony-forming units), indicating a more colonized state of the bacteria, they did not differ between the HIV or non-HIV groups. 11 The majority of the samples (72.7%) from the gingiva also showed the same isolates from the dorsum of the tongue, indicating that AGNB might colonize mucosal surfaces in general, and are not necessarily associated to gingiva and periodontal disease specifically. 12 The increase of AGNB after antibiotic therapy could be explained by the multiresistance of AGNB. 18 The present study confirmed almost 100% resistance against antibiotics commonly used for oral infections and in periodontal therapy (penicillin, amoxicillin, clindamycin, and metronidazole). Ciprofloxacin and norfloxacin seem to be the drugs of choice once there are infections with AGNB that need antibiotic treatment. However, it is strongly recommended that antibiotics not be used for the treatment of surface infections/colonization even in immune-compromised patients, such as those who are HIV positive or immune-compromised, unless there is a risk of mortality. No ESBL strains were found in these Thai patients; however, this type of multiresistance has been reported to be frequent (>50%) in stool samples in Thailand. 18 This could be due to the fact that Escherichia coli was rarely isolated in oral samples in the present study, but were the most common ESBL isolate in the stool samples.
Some bacteria, such as Stenotrophomonas maltophila, are known to have multiresistance, which was also confirmed in the present study.
Non-oral AGNB that are isolated from the oral cavity of Thai HIVpositive individuals on HAART medication showed great genus and species diversity, and were highly resistant to common antibiotics.