Antimicrobial Susceptibility of Porphyromonas Gingivalis Strains Isolated From Periodontal Patients by Three Testing Protocols


 Background: The main goal of antimicrobials is to eliminate microorganisms that persists despite mechanical treatment. This is the case of Porphyromonas gingivalis (Pg), frequently isolated in patients with periodontitis. Global antibiotic studies evaluated in randomized clinical trials and in vitro studies have shown mixed results regarding effectiveness and susceptibility, even with different protocols where it is not clear if the laboratory test applied can affect the results. This information is relevant in order to obtain clinical outcomes and prevent antimicrobial resistance for their over-prescription or inadequate choice. The objective of this study was to describe the antimicrobial susceptibility in vitro of Pg to metronidazole, clindamycin, amoxicillin plus clavulanate, moxifloxacin and azithromycin in periodontal patients by three testing protocols.Methods: Microbiological samples were obtained in patients with a diagnosis of generalized moderate or severe periodontitis. They were incubated in anaerobic conditions for up to 7 days, and those morphologically compatible with Pg were isolated and identified by a mass spectrometer (MALDI-TOF MS). The three most frequently protocols for antimicrobials susceptibility tests (Blood agar- McFarland 0.5- Epsilometer test; Brucella blood agar- McFarland 1.0- Epsilometer test; Brucella blood agar- McFarland 0.5-Agar dilution) were applied to the same strain describing their profile and reporting any difference between the tests. The breakpoints considered the guidelines of CLSI and previous publications.Results: 50 patients (25 women, 25 men) with periodontitis between 34-69 years were selected. Finally, 25 Pg positives strains (50%) were recovered for the susceptibility analysis and all of them were highly sensitive to all antibiotics (range 96%-100%). Only one strain was resistant to azithromycin in one protocol, and no differences were found in the susceptibility results between the three tests.Conclusion: The Pg strains were highly susceptible to the five antibiotics evaluated in this population, showed a high level of susceptibility and significant agreement between the three tests applied, therefore the type of laboratory test used had not impacted on clinical interpretation. These findings are positives in terms of susceptibility and would provide several antibiotics treatment alternatives, and its prescription could be the best choice for the patient's specific context.


Introduction
Periodontitis is a microbially-associated and host-mediated in ammatory disease that results in loss of periodontal attachment and nally the loss of teeth 1,2 . This chronic disease has become a worldwide public health problem due to its high prevalence in adults and elevated cost of treatment, if it is not treated timely generates a decrease in quality of life of affected patients 3,4 .
Periodontitis treatment is mainly mechanical, with the purpose of disrupting the established subgingival bio lm and allowing the change towards a microbiota compatible with health 5 . Evidence has shown that some patients need antibiotics as an adjunct to conventional mechanical treatment, under speci c conditions like severe cases of periodontitis or in patients with systemic diseases 6,7 . The main goal of antimicrobials is to eliminate microorganisms that persist despite the mechanical treatment. It is the case of Porphyromonas gingivalis (Pg) 8 , a highly pathogenic bacteria frequently isolated in patients with periodontitis that demonstrate a strong association in the initiation and progression of the disease 9 . This pathogen owns complex virulence factors like mbriae presence, modulation of the immune response and capacity for tissue invasion 10 . The "keystone bacteria" model showed that Pg induces subgingival bio lm dysbiosis and allows the development of some bacteria over other modulating commensal microbiota raising the pathogenicity of the entire community. Thus, individual bacteria triggers a series of collective events associated with tissue destruction by the host response 11,12 . In Chile, the prevalence of severe periodontitis in the adult population is 38.65% between 35 and 44 years and 69.35% between 65 and 74 years, mostly low socioeconomic status 13 . Several studies established a high predominance of strict anaerobic bacteria in those patients where Pg appears frequently isolated. Its prevalence differs according to the identi cation method used [14][15][16][17] .
The microbiological pro le of periodontal patients is well known, and antimicrobial treatment is an evidence-based clinical decision. Nevertheless, the antibiotics evaluated in randomized clinical trials and in vitro studies have shown mixed results regarding the effectiveness and susceptibility, even with different protocols where it is not clear if the applied laboratory test can affect the results 16,18−21 . Local susceptibility has not been evaluated, relevant information for obtaining clinical outcomes and preventing antimicrobials resistance due to their over-prescription or inappropriate choice 22,23 .
This study aimed to describe the antimicrobial susceptibility in vitro of Porphyromonas gingivalis to metronidazole, clindamycin, amoxicillin plus clavulanate, moxi oxacin and azithromycin in periodontal patients by three testing protocols.

Subjects
Microbiological samples were obtained in patients selected in the clinic of Escuela de Odontología, Ponti cia Universidad Católica de Chile, carefully selected according to inclusion and exclusion criteria (table 1). We included patients with a diagnosis of generalized moderate or severe periodontitis, according to former AAP classi cation 24 . The case de nition for moderate periodontitis was ''two or more interproximal sites with clinical attachment loss 4 mm, not on the same tooth, or two or more interproximal sites with probing depths 5 mm, not on the same tooth'' and for severe periodontitis was ''two or more interproximal sites with clinical attachment loss 6mm, not on the same tooth, and one or more interproximal sites with probing depth 5mm''. Periodontitis was considered generalized when it was present in 30% or more of the sites examined. Alveolar bone loss was evaluated with radiographs 25 .
Once patients were selected, we proceeded to explain the research protocol and requested voluntary participation in the study, and they signed an informed consent document.

Clinical periodontal parameters
Clinical records were obtained, and six sites by tooth were measured with North Carolina probes (UNC 15, Hu-Friedy Corp., Chicago IL, USA). Periodontal parameters were evaluated in all teeth presents (with exception third molars), including probing depth, clinical attachment level, plaque index (O'Leary), bleeding on probing (BOP) and suppuration.

Sampling of subgingival plaque technique
The site selected for sampling was the deepest bleeding pocket with the highest clinical attachment level loss was selected in each quadrant of the mouth. The supragingival plaque was removed with a sterile curette, and then the zone was isolated with sterile cotton rolls and air-dried. Subgingival microbiological samples were obtained, inserting two consecutively sterile paper points N o 30 (Maillefer, Switzerland) for at least 20 seconds within each site of the teeth. Samples of each patient were transported in a tube with 1 ml of RTF (reduced transport uid) at 4°C to Microbiology Laboratory within 2 hours to begin their processing 26 .

Microbiological laboratory tests
In the laboratory, the samples were vortexed for 30 seconds. Then serial dilutions were then made and plated homogeneously on blood agar (5% sterile lamb blood, 5 µg hemin/mL, 1 µg vitamin K /mL). They were incubated at 37°C in anaerobic jars for up to 7 days, and those morphologically compatible with Pg were isolated and passed to a second incubation. Finally, bacterial identi cation was con rmed through the mass spectrometer (MALDI-TOF MS; Biotyper, Bruker Daltonik, Germany).

Antimicrobial susceptibility test
We applied the three most widely used protocols for antimicrobial sensitivity tests to the same strain, described their susceptibility pro le and reported any difference detected between the tests. Table 3 shows breakpoints for interpretation of Pg susceptibility considering the guidelines of the Clinical Laboratory Standards Institute (CLSI) and previous publications.
The Epsilometer Test (E-test ® , AB Biodisk, Solna, Sweden) for metronidazole, clindamycin, amoxicillin plus clavulanate, moxi oxacin and azithromycin was applied by placing only two antibiotics strips per plate, following the manufacturer's instructions 27 .
The plates were incubated at 37°C in anaerobic atmosphere for 2 days, and quality control was Bacteroides fragilis strain (ATCC 25285) used as a reference to con rm the correct implementation. The minimum inhibitory concentration (MIC), de ned as the lowest concentration of antibiotics that inhibits microorganisms growth, was measured at the intersection of the inhibition area with the strips where was reading and interpreted according to the guidelines of the CLSI and previous publications 19,28 The isolated samples of Pg were placed inside a tube with sterile reduced thioglycolate broth in order to obtain a 1.0 McFarland standard (3.0 x 10 8 UFC/mL). After that, the nal suspension was plated on Brucella blood agar (Becton Dickinson ® ) supplemented with 5% sterile lamb blood, 5 µg hemin/mL, 1 µg vitamin K /mL. These are the culture media and turbidimetry suggested by the manufacturer (E-test ® , AB Biodisk, Solna, Sweden) 29 . Then E-test ® was applied in the same way as in the rst protocol 27 .
The plates were incubated at 37°C in anaerobic atmosphere for 2 days, and quality control was Bacteroides fragilis strain (ATCC 25285) used as a reference to con rm the correct implementation. The  The isolated samples of Pg were deposited inside a tube with reduced thioglycolate broth until to obtain a 0.5 McFarland standard. Then employing Cathra replicator were successively plated on Brucella blood agar (Becton Dickinson ® ) supplemented with 5% sterile lamb blood, 5 µg hemin/mL, 1 µg vitamin K /mL plus the individually evaluated antibiotics: metronidazole, clindamycin, amoxicillin plus clavulanate, moxi oxacin and azithromycin, in different concentrations. The plates were incubated at 37°C in anaerobic atmosphere for 2 days, and quality controls were Bacteroides fragilis strain (ATCC 25285), Clostridium di cile (ATCC 700057) and Porphyromonas gingivalis (ATCC 33277), used as a reference to con rm the correct procedure. Finally, the lowest concentration of each antibiotic that inhibits the growth of a microorganism was reported as its MIC 28 .

Data analyses
Descriptive data was recorded for the three testing protocols. The antibiotic was interpreted as susceptible when concentration was below the de ned breakpoints the strain, and it was considered resistant if it was equal or higher than the reference value (CLSI; previously publications). The concentrations at which 50% and 90% of strains were susceptible were de ned as MIC 50

Results
Microbiological samples were obtained from 50 patients (25 women, 25 men) with periodontitis, between 34-69 years (age mean 51. 4 8.19). From the total, 27 (57%) were positives to the presence of Pg; 14 male (age mean 54.1 7.38) and 13 female (age mean 47.9 9.93) patients. Table 2 describes the characteristics and clinical data for the total sample and subdivided in a positive and negative culture.
Finally, 25 strains positives to Pg (50%) were recovered for susceptibility test analysis. The breakpoints for Pg susceptibility considered the guidelines of the CLSI and previous publications and table 4 shows the quantitative results in terms of MIC range, MIC 50 and MIC 90 percentage of susceptibility, for each antibiotic in the three testing protocols.
All the selected strains were highly sensitive to metronidazole, clindamycin, amoxicillin plus clavulanate and moxi oxacin (range 96%-100%). Only one strain was resistant to azithromycin in the rst protocol, and no differences were found in the susceptibility results between the three tests.

Discussion
Previous studies identi ed the microbiological pro le of Chilean patients with periodontitis and their comparison with other countries, establishing a high Pg prevalence and a geographical difference in their results 14,15,17,30,31 . Also, some randomized clinical trials evaluated the use of some antibiotics and their impact on clinical and microbiological results 32,33 . This study is the rst that evaluates in vitro Pg susceptibility isolated from Chilean adults with periodontitis to the main antibiotics used in periodontal treatment.
Furthermore, due to the scienti c literature has reported different protocols for susceptibility testing, this study evaluated if there are differences in the results obtained through three main laboratory protocols. A descriptive methodology was used in this pilot study; therefore, 50 patients were able to obtain clinical records and microbiological samples for testing.
In a st laboratory stage, count with the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight) was an advantage because it could identify the microorganisms in a few minutes by reading their previously ionized peptides and proteins, with a high-con dence (92.9% of the isolates anaerobic bacteria), being simpler than traditional techniques 34,35 .
Anaerobic susceptibility is not frequently implemented in laboratories, but test results are relevant to direct appropriate therapy and identify anaerobic resistance, which is increasing globally 36,37 . Another important concern is that the variety of susceptibility test methods for anaerobes have signi cant advantages and disadvantages 38 and it is unclear whether the type of process could bring different results and, therefore, different clinical interpretations. For the present study, we selected the E-test® and agar dilution.
The E-test® is a simple laboratory technique that read the MICs in a strip containing a gradient of antibiotic. However, there are published different process to determine susceptibility, with several media culture as well as the concentration of colony-forming units determined by the McFarland turbidimetry. The two E-test® protocols performed in this study are the most frequently published, where brucella blood agar and blood agar were used, as well as McFarland 1 and 0.5 turbidimetry, respectively 19,29 . However, agar dilution remains the gold standard for determining susceptibility since it has the standardization by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 39 and CLSI 28 , so we also select this protocol, although it is a more complex technique and requires considerable time in the process 38 .
In general, E-test® results have a strong correlation with MICs assessed by broth or agar dilution methods; nevertheless, there is some inherent bias toward higher or lower MICs determined by some testmicroorganism-antimicrobial agent association 38 . For example, metronidazole resistance in E-test® can be overestimated if anaerobiosis is inadequate 40 or discrepancies were reported for this antibiotic when compared E-test® and agar dilution for anaerobes (13 to 14%; FDA category of very major error) 41 .
In the dilution in agar, not changing the culture media or the inoculum concentration is essential, because minor methodological adaptations to the protocols cannot guarantee reliable results and allow them to be compared with other publications 42 . Therefore, to achieve accurate and reproducible results, the test medium must accomplish a serial of requirements for certain antibiotic/species combinations. Also, the concentration of colony-forming units must be as indicated. A higher inoculum can increase MIC, speci cally if the tested bacteria produce an enzyme that destroys the antibiotic. Conversely, a lighter inoculum may arti cially lower MICs 43 . Thus, studies should be careful when comparing results because emerging mechanisms of resistance require constant vigilance about the sensitivity of each test to detect resistance accurately 44 .
In the eld of periodontal microbiology, it has been demonstrated that several antibiotics need to be much higher concentrated on reaching the MIC in a bio lm compared with microorganisms grown in a planktonic culture 45,46 , therefore in vitro test can predict the clinical e cacy (in vivo) 47 .
Despite the limitations that this study, we could interpret positives results in terms of a high level of susceptibility in all antibiotics studied against Pg, and signi cant agreement in the results between the three tests so would not have an impact on changing the clinical decision when choosing among antibiotics. These results are in accordance with previous reports where E-test® determined the susceptibility of Pg strains isolates from periodontitis. In the Netherlands and Spain periodontal population were susceptible to all antibiotics tested including metronidazole, clindamycin, azithromycin, amoxicillin plus clavulanate, amoxicillin, penicillin, tetracycline and cipro oxacin 19 . Another study, also in the Netherlands Pg was highly susceptible to metronidazole, clindamycin, amoxicillin plus clavulanate, azithromycin, amoxicillin and tetracycline 48 . In Switzerland periodontal population, they were susceptible to metronidazole, clindamycin, amoxicillin plus clavulanate, phenoxymethylpenicillin and tetracycline 20 .
For other tests, in the Swedish periodontal population, no resistance was found using disc diffusion, and agar dilution among strains of Pg isolates against the antibiotics commonly used in the treatment of periodontal disease: clindamycin, metronidazole, penicillin, amoxicillin and tetracycline 21 .
Currently, the indication of antibiotics in chronic periodontitis is performed empirically in most cases based on clinical criteria, but for this critical decision, having information about laboratory identi cation or at least recognize predominant ora would be desirable. Other clinical concern is antibiotic monotherapy prescription, if it is possible, considering the risk of resistance, adverse drug reactions and patient compliance 50 . Besides, consider choosing broad-spectrum antibiotics instead of broad-spectrum 51 and having pharmacological alternatives in case of allergies or risk of drug interactions, for example, in patients with polypharmacy.
Periodontal pathogens in the Chilean population-as in other countries-showed a marked predominance of strict anaerobic bacteria; therefore, the real need to use broad-spectrum antibiotics or their combinations should be evaluated, contemplating possible adverse reactions or risk of resistance. Therefore, a single antibiotic or a synergic combination of them must achieve an optimal result in periodontal health, but the Taking all this evidence into account, it is essential to consider the impact of antimicrobial protocols on patient care health policies and, therefore, update the clinical guidelines 54 .

Conclusions
This study suggests that Pg strains were highly susceptible to all antibiotics evaluated in this population. Through descriptive data, it was determined that the three methods to assess susceptibility in anaerobes most commonly used in periodontics found no difference in their results and provide the basis for future randomized clinical trials.   **All antibiotics showed 100% susceptibility in the tree protocols, except azithromycin in the rst protocol (96%)