6Journal of Dental Research and Scientific Development | 2014 | Vol 1 | Issue 1

Evaluating the antibiotic susceptibility of bacteria isolated from the pyogenic abscess of dental origin

Aditi Mahalle, Revati Deshmukh1, Apurv Mahalle2

improvement in the morbidity and mortality associated with infectious diseases, microorganisms have proved to be quite adaptable, displaying an unsettling ability to reemerge in continuing cycles of disease. This ability has been shown over the past decade in the number and variety of so‑called emerging infections not previously recognized in humans and also in the reemergence of diseases, sometimes in slightly different forms, caused by well‑known microorganism such as Staphylococci, Streptococci, Escherichia coli, and Mycobacterium tuberculosis.[5] Reemergence is partly a result of acquisition of antibiotic resistance mechanism either through mutation or transfer of genetic information from other bacteria.[6]

The knowledge of common pathogens and resistance status can guide the clinician towards appropriate selection of empirical antibiotics. But on reviewing the available literature it has been observed that not many studies are available which have tried to assess the common pathogens causing orofacial infection, the bacterial isolates from pyogenic abscess of dental origin, and the antibiotic susceptibility of these bacterial isolates in particular.[7‑10]

Correspondence to: Dr. Aditi Mahalle, Department of Oral Pathology and Microbiology, D Y Patil University's, Dr. D Y Patil Dental College and Hospital, Maheshnagar, Pimpri, Pune ‑ 411 018, Maharashtra, India. E‑mail: aditi_pravin@yahoo.co.uk

Introduction

The human oral cavity is a biological system that supports life for many species of microorganisms, but when these microorganisms penetrate into the deeper tissues or in case of compromised host resistance, bacterial infection results. The presentation of bacterial infection of dental origin is constantly changing and is a measurable reflection of modern evolution of oral flora.[1,2] The bacteriological studies have shown variations in the microbiology of acute dental infections. According to Yoris et al.,[3] this difference undoubtedly reflects variations in the bacteriological techniques employed for their isolation, identification, and gradual changes in flora due to injudicious use of antimicrobials.

The availability of “wonder drugs” in the years immediately after World War II ushered in the era of complacency in infectious disease treatment. Antibiotics were prescribed when symptoms first appeared without determining either the cause of the disease or the chemotherapeutic susceptibility of the microbes.[4] Although there has been recent dramatic

Original Research

Department of Oral Pathology and Microbiology, DYP University's Dr. D Y Patil Dental College and Hospital , Pimpri , Pune, 1Oral Pathology and Microbio logy, Bharati Vidyapeeth Dental College and Hospital, Pune, 2Department of Anaesthesiology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharashtra, India

AbstractAim: To evaluate the antibiotic susceptibility of bacteria isolated from the pyogenic abscess of dental origin. Objective: To assess the bacterial isolates of the pyogenic abscesses of dental origin and evaluate the antibiotic susceptibility of the same.Study Design: The study was an in-vitro experimental open clinical trial. Surgical aspiration of pus was done in 30 patients suffering from pyogenic abscesses of dental origin. The pus sample was subjected to standard methods for isolation and identification of microorganisms as per Topley and Wilson method and antimicrobial susceptibility by the method of Kirby‑Bauer was done. Results: Aerobes were isolated in 22 instances (70%), of which facultative anaerobes were isolated in 4 instances. Pure isolates of anaerobes were isolated in 4 instances (13.3%). In four cases no organisms were isolated. In this study Staphylococcus aureus was isolated in 13 instances (43.3%), Streptococcus viridians in 4 instances (13.3%) followed by anaerobic Streptococci in 4 instances (13.3%). The susceptibility and resistance of bacteria to 12 antibiotic panels most recommended for Gram positive organism showed 100% susceptibility for Ceftriaxone, Amoxyclav and Amikacin, while 95% for Cefuroxime and Sparfloxacin. Maximum resistance was noted with Amoxycilin in 7 cases (37%) followed by azilides group. The Gram negative isolates showed 100% sensitivity for the 9/10 antibiotic panel. 2 cases were resistant to Ampicillin. Anaerobic isolates tested against panel of 10 drugs showed 100% susceptibility to all. Conclusion: Within the limits of the present study, it can be concluded that pyogenic abscesses of dental origin has isolates of mixed nature. The study results can act as a guide for the clinician while prescribing antibiotics.

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Key words: Antibiotic sensitivity test, microbes, pyogenic abscess

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Mahalle, et al.: Bacterial isolates and antibiotic susceptibility

Journal of Dental Research and Scientific Development | 2014 | Vol 1 | Issue 1

with tap water at the end of the 30 s. Then the smears were counterstained with 0.25% safranin for 30 s, washed, drained, blotted, and examined under oil immersion to determine the morphology of microbes.

Method for aerobic organism identificationThe pus sample was aseptically plated on blood agar plate (Yogiraj Micromedia Limited, Pune, India) and on MacConkey’s agar plate (Yogiraj Micromedia Limited, Pune, India) and incubation was done under aerobic conditions at 37°C for 24 h. The colonies on both the plates were noted. Blood agar plate was used for all organisms and MacConkey’s agar plate was for gram negative organisms. Blood agar and MacConkey’s agar plate were examined especially for the presence of following organisms: Staphylococcus aureus, Streptococci species, Klebsiella species, and Pseuomonas aeruginosa. The morphology was confirmed with gram staining. The identification of organism was done by biochemical tests. Assay time/turnaround time ‑ 2 days.

Method for anaerobic organism identificationThe pus sample in thioglycollate broth was inoculated in the same and incubated at 37°C overnight. Subculture was done on blood agar and it was immediately put in an anaerobic biobag with indicator (Equitron Limited, Mumbai, India). The bag was sealed with parafilm and incubation was done at 37°C for 48 h. Gram smear and aerotolerance testing was done. If the colony grows on aerobic culture, it is unlikely to be an obligate anaerobe (except Clostridium septicum and Actinomycetes). If no growth occurs on aerobic culture, the organism is presumptively identified by means of Gram’s reaction, colony characters, and available biochemical tests. Assay time/turnaround time‑5 days.

Steps in antibiotic sensitivity testA suitable dilution of broth culture or broth suspension of test bacterium was flooded on the surface of Mueller‑Hinton agar (Yogiraj Micromedia Limited, Pune, India). The plate was entitled to ensure uniform spreading and excess of broth was pipetted off. After drying the plate at 37°C for 30 min, antibiotic discs (Himedia Laboratories Limited, Mumbai, India) four or five per 10 cm plate were applied with sterile forceps. After overnight incubation, the degree of sensitivity was determined by measuring the zones of inhibition of growth around the discs. Growth was inhibited around the disc containing antibiotics to which the bacteria were susceptible but not around those to which it was resistant.

Data analysisThe collected data was analyzed in Microsoft Excel 2007. A descriptive analysis was performed.

Results

The pus sample from 30 orofacial abscess cases were studied using culture techniques for both aerobic and anaerobic

Hence, this study was undertaken firstly, to assess the bacterial isolates of the pyogenic abscesses of dental origin and secondly, to evaluate the antibiotic susceptibility of these bacteria.

Materials and Methods

This was an in vitro experimental open clinical trial. Thirty patients who fulfilled the inclusion and exclusion criteria were recruited for the study. Informed consent was obtained from the subjects for aspiration and use of aspirated fluid for analysis.

The categorization of patients with abscess into mild, moderate, and severe was based on the clinical sign, symptoms, and radiographic investigations. Inclusion criteria: Dentoalveolar abscesses (which includes periapical abscess, gingival abscess, periodontitis, and pericoronitis), without any prior surgical decompression or drainage. Patients who were severely debilitated, who were receiving antibiotic therapy more than 5 days, who not did not give any consent and unable to cooperate were excluded from the study.

In the present study, the standard methods for isolation and identification of microorganisms as per Topley and Wilson method and antimicrobial susceptibility by the method of Kirby‑Bauer[11] were followed.

Method for obtaining aspirateThe overlying skin was decontaminated with the betadine solution and depending on the clinical situation the general or local anesthesia was administered. A surgical drainage was done intra‑ or extraorally to collect the specimen with an 18 guage needle and divided into three parts each. Two parts were injected into the bottles containing nutrient broth (Yogiraj Micromedia Limited, Pune, India) for aerobic microflora and thioglycolate media (Yogiraj Micromedia Limited, Pune, India) for anaerobic microflora, respectively. Third part was used for gram staining. Complete decompression or surgical drainage was done after collection of specimen. The findings of aspiration were recorded as pus/straw‑colored fluid/soft cheesy material/blood/no aspiration. The aspirate obtained was subjected to gram staining, aerobic culture, anaerobic culture, antimicrobial susceptibility, and resistance testing. The two bottles and the syringe were sent to the laboratory at the earliest (within 1 h), for microbiological study.

Microbiological procedureGram stainingSmears were prepared and heat‑fixed for 10 s. Crystal violet stain was flooded for 1 min on heat fix smear and excess dye was poured off and washed gently in tap water and the slide was drained with blotting paper. Gram’s iodine was used as a mordant by washing with iodine, and then adding more iodine and leaving it on the smear until 1 min is over. Smears were first washed with tap water and drained carefully and then decolorized with 95% alcohol for 30 s. Lastly, they were washed

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isolates. The antimicrobial susceptibility of the isolates to 32 panel antibiotics was examined. The patients were in the age range of 31‑40 years (mean age 35.4 ± 1.7 years). Twenty were females (66.3%) and 10 males (33.33%).

Table 1 shows the instances of occurrence of various bacteria isolated from pyogenic abscesses of dental origin. Staphylococcus aureus was the predominant microorganism reported in 13 cases (43.3%). Aerobes were isolated in 22 instances (70%), of which facultative anaerobes were isolated in four instances. Pure isolates of anaerobes were isolated in four instances (13.3%). In four cases no organisms were isolated.

Table 2 shows the susceptibility and resistance of bacteria to 12 antibiotic panels most recommended for gram positive organism. Hundred percent susceptibility was reported for ceftriaxone, amoxyclav, and amikacin, while 95% for cefuroxime and sparfloxacin. Maximum resistance was noted with amoxycilin in seven cases (37%) followed by azilides group.

The gram negative isolates showed 100% sensitivity for the 9/10 antibiotic panel. Two cases were resistant to ampicillin [Table 3].

For anaerobic isolates a panel of 10 drugs was tested [Table 4]. Hundred percent susceptibility to all the antibiotics was noted.

Gram positive and gram negative cocci and/or bacilli were isolated in all the 30 patients on the basis of Gram staining done to determine the morphological characteristics. Overall the gram positive bacteria are present among 28 cases (93.3%) and gram negative bacteria among 10 cases (33.3%). Gram positive cocci (28, 93.3%) and gram negative bacilli (10, 33.3%) showed maximum growth percentage in this study. Whereas gram positive bacilli were found only in four patients (13.3%) and no gram negative cocci was isolated (0%).

Discussion

The oral cavity is a cradle for various types of microorganisms. The conducive environment present in the oral cavity helps in growth and nurturing of these microorganisms.[2] The dental abscess leading to severe sepsis and mortality rate of 10‑40% has been known since antiquity.[12] Facultative anaerobes were frequently and predominantly found in samples of 1‑2 days of clinical symptoms suggestive of cellulitis like infection at this stage.[13,14]

Culturing of microorganisms in abscesses is being done way back from 1913.[4,15]

In the present study, both aerobic and anaerobic microorganisms were isolated which showed that the pyogenic infections of dental origin are mixed in nature. This result is also in concordance with studies done by Grigsby et al., (1976),[5] Dymock et al., (1996),[6] Labriola et al., (1983),[16] Robert (1980),[17] and Kuriyama et al., (2000).[18] In this study Staphylococcus aureus was isolated in 13 instances (43.3%), Streptococcus viridians in four instances (13.3%), followed by anaerobic Streptococci in four instances (13.3%) which is in concordance with studies done by Mahabaleshwara et al., (2005),[11] Munson et al., (2005),[19] and Anthony et al., (2006).[20] The isolation of Staphylococcus aureus has clinical significance, as this has high resistance rate to many known antibiotics.

The second most predominant isolates were Streptococcus viridians (13.3%) and anaerobic Streptococci (13.3%). This finding is in concordance with the study carried out by Kuriyama et al., (2000).[18] In the present study, the isolation of pure aerobic, facultative anaerobes and pure anaerobes indicate polymicrobial nature of bacteria in the pyogenic abscess of dental origin.[1,16,21] The percentage of isolation of anaerobic bacteria (13.3%) was less in the present study as compared to the studies done by Grigsby et al., 1976,[5] but it is similar to the study conducted by Aderhold et al., 1987.[9] The isolation of high number of gram positive cocci and gram negative bacilli are in accordance to other reported studies.[19,20,22‑24]

Antibiotic susceptibilityIn the present study, different panel of drugs were used against gram positive, gram negative, and anaerobic isolates. Amoxycillin showed the maximum resistance (37%); whereas, 100% sensitivity was given by the cephalosporin group. The study results are in concordance with the other studies.[8,11,18] Labriola et al., 1983[16] showed that more organisms were resistant to penicillin in his study (21 specimens out of 50) and doubted the use of penicillin as drug of choice in orofacial infections. Moenning et al., (1989)[1] reviewed the literature to conclude that despite mounting evidence of microorganisms becoming increasingly resistant to penicillin, it would seem presumptuous to state that penicillin is currently not effective against most odontogenic infections.

The small number of samples for gram negative (n = 3) and anaerobic isolates (n = 4) for susceptibility testing was a lacunae in the study. This limits drawing any definitive conclusion

Table 1: The instance of occurrence of the various bacterial isolates from 30 casesInstance of occurrence; n (%) Total

Aerobes Streptococcus viridans; 4 (13.3) Non‑hemolytic Streptococci; 1 (3.3) Streptococcus pyogenes 1 (3.3) 22/30Aerobe and facultative anaerobes

Klebsiella species; 1 (3.3)Klebsiella pneumoniae; 1 (3.3)

Escherichia coli; 1 (3.3) Staphylococcus aureus 13 (43.3)

Anaerobes Anaerobic Streptococci; 4 (13.3) 4/30Nil 4/30 4/30

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about the antibiotic susceptibility. Further study with larger sample size is needed for valid conclusion.

Other than culture, microbial identification can be done by immunologic methods and newer molecular techniques

by different polymerase chain reaction (PCR) methods, deoxyribonucleic acid (DNA) microarrays, fluorescent in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) analysis, restriction fragment length polymorphism (RFLP) analysis, etc.[25]

Conclusion

In the study Staphylococcus aureus, Streptococcus viridans, and anaerobic Streptococci were the prevalent microorganisms. Gram positive aerobes showed highest resistance to amoxicillin, whereas the Gram negative isolates showed highest resistance to ampicillin. The anaerobes isolated showed 100% sensitivity to the panel of antibiotics. A hundred percent sensitivity was noted to the panel of drugs by the anaerobes isolated in this study.

Within the limits of the present study, it can be concluded that pyogenic abscesses of dental origin has isolates of mixed nature. The study results can act as a guide for the clinician while prescribing antibiotics.

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Table 2: The percentage of susceptibility and resistance obtained for gram positive isolates (aerobes) for the mentioned drugsName of drug Susceptibility Resistance

Instances Percentage Instances PercentageAzithromycin 15 79 4 21Roxithromycin 15 79 4 21Clindamycin 15 79 4 21Cephalothin 17 89 2 11Cefaclor 17 89 2 11Cefuroxime 18 95 1 5Ceftriaxone 19 100 0 0Amoxyclav 19 100 0 0Amikacin 19 100 0 0Ofloxacin 16 84 3 16Sparfloxacin 18 95 1 5Amoxycillin 12 63 7 37Only in 19 /30 cases gram positive aerobes were detected

Table 3: The percentage of sensitivity and resistance obtained for gram negative isolates for the mentioned drugsName of drug Sensitivity Resistance

Instances Percentage Instances PercentageCiprofloxacin 3 100 0 0Gentamycin 3 100 0 0Tobramycin 3 100 0 0Amikacin 3 100 0 0Cefuroxime 3 100 0 0Cifoperazone 3 100 0 0Cefotaxime 3 100 0 0Amoxyclav 3 100 0 0Co‑trimoxazole 3 100 0 0Ampicillin 1 33 2 67Only in 3/30 cases gram negatives aerobes were detected

Table 4: The percentage of sensitivity and resistance obtained for anaerobe isolates for the mentioned drugsName of drug Sensitivity Resistance

Instances Percentage Instances PercentageCefotaxime 4 100 0 0Ceftriaxone 4 100 0 0Clindamycin 4 100 0 0Chloramphenicol 4 100 0 0Amoxyclav 4 100 0 0Sparfloxacin 4 100 0 0Metronidazole 4 100 0 0Ampicillin 4 100 0 0Penicillin 4 100 0 0Co‑trimoxazole 4 100 0 0Only in 4/30 cases anaerobes were detected

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18. Kuriyama T, Nagakawa K, Karasawa T, Saiki Y, Yamamoto E, Nakamura S. Bacteriologic features and antimicrobial susceptibility in isolates from orofacial odontogenic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:600‑8.

19. Munson MA, Pitt‑Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002;81:761‑6.

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22. Khateery SM, Parker MI. A retrospective analysis of orofacial infections

requiring hospitalization. Saudi Dent J 2001;13:96‑100.23. Storoe W, Haug RH, Lillich TT. The changing face of odontogenic

infections. J Oral Maxillofac Surg 2001;59:739‑48.24. Szyszkowska AM, Koszel UO. Microbiologic profile of acute dental

infections and the resistance of causative species to the antibiotics. Ann Marie Curie Univ 2007;72:401‑6.

25. Siqueira JF Jr, Rocas IN. Exploiting molecular methods to explore endodontic infections: Part 1‑‑current molecular technologies for microbiological diagnosis. J Endod 2005;31:411‑23.

How to cite this article: Mahalle A, Deshmukh R, Mahalle A. Evaluating the antibiotic susceptibility of bacteria isolated from the pyogenic abscess of dental origin. JDRSD 2014;1:6-10

Source of Support: Nil. Conflict of Interest: No conflict of interest.

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