Prevalence of extended spectrum beta-lactamase producing anaerobic bacteria in chronic periodontitis

Abstract

Objective:

Due to the rampant use of antibiotics bacteria are acquiring resistance to penicillin group of drugs, which results in prescription failure in clinical practice. Beta-lactamase producing organisms are not only more virulent they also cause surrounding bacteria to become resistant. Hence, this study was undertaken to know the prevalence of extended spectrum beta-lactamase (ESBL) producing anaerobic bacteria in chronic periodontitis.

Materials and Methods:

The present study was conducted for a period of 1 year from January to December 2010 at a tertiary care teaching hospital. Clinical samples were collected from the sub gingival pockets from cases of chronic periodontitis and transported to the laboratory in fluid thioglycollate medium. Gram's staining was performed and anaerobic culture put up. All the anaerobic bacteria isolated were tested for beta-lactamase production by Nitrocefin disc method.

Results:

A total of 60 samples yielded 121 isolates, out of which 26% were ESBL producers. Bacteroides fragilis was the most common organism followed by Fusobacterium species.

Conclusion:

ESBL producing anaerobic bacteria exits in chronic periodontitis cases and the present study identified 26% of the isolates to be ESBL producers. Antibiotic resistance testing is essential before starting the therapy and in emergency cases drugs should be chosen to cover ESBL producers.

INTRODUCTION

Anaerobes are the major bacterial flora of humans, however recently they are increasingly identified as pathogens in various life-threatening infections. The treatment for the same is challenging to the physician as they are increasingly becoming resistant to β lactam drugs. β-lactamase enzyme was first described by Abraham and Chain in 1940. β-lactamase enzyme was initially produced by the soil bacteria to protect self from the surrounding bacteria. Same is also been identified in pathogenic bacteria and it is seen to interfere with antibiotic activity. In the 1960s, Bacteroid penicillinase was found to produce β-lactamase enzyme, this was probably the first record of production of β-lactamase by anaerobic bacteria.[1]

Since, the identification of Penicillin by Alexander fleming, broad spectrum β-lactams remain antibiotics of choice in emergency cases.[2] Due to the rampant use of antibiotics, bacteria are acquiring resistance to penicillin group of drugs by the production of enzyme β-lactamase which hydrolyses the β-lactam ring of the drug. β-lactamase producing organisms are two edge sword as they are directly pathogenic and also release the enzyme to environment causing surrounding non-beta-lactamase-producing bacteria (BLPB) to become resistance causing antibiotic failure.[3] BLPB may be present in the oral cavity from a very early age.[4] Literature review shows that antibiotic resistance has increased in the oral flora over the last 10-15 years.[5] Since, penicillin group of drugs are still the first line of antibiotic for endodontic infections it is necessary to test for the production of β-lactamase enzyme to avoid prescription failure. Hence, this study was undertaken to know the prevalence of beta-lactamase producing anaerobic bacteria in chronic periodontitis.

Aims and objectives

• To test the anaerobic isolates from chronic periodontitis for the production of enzyme beta-lactamase.

MATERIALS AND METHODS

The study was conducted over a period of 1 year from January to December 2010 in the Department of Microbiology at a tertiary care teaching hospital which is also attached to a dental college and hospital. All the cases which are newly diagnosed as chronic periodontitis in Periodontology outpatient department were included in the study and patients who have taken antibiotic therapy or undergone any dental procedures in the past 3 months, pregnant ladies and patients with hypertension and diabetes were excluded from the study. The study comprised of 60 cases.

Subgingival plaque sample was collected with the help of sterile periodontal Gracey curette and transported to microbiology laboratory in fluid thioglycollate medium. Samples were immediately processed. Modified gram staining for anaerobes was performed and inoculated on Brucella blood agar supplemented with hemin and vitamin K, Kanamycin Vancomycin Laked Blood Agar, Bacteroid bile esculin agar and Brain heart infusion agar and incubated at 37°C in McIntosh Fildes jar. The method used for obtaining anaerobiosis in the jar was ‘internal gas generating system” described by Vaidhyalingam and Laxminarayana.[6] After 72 h of incubation culture growth was noted for morphology, pigment, pitting, hemolysis and fluorescence. And also preliminary tests such as gram stain, spot indole and catalase tests were performed. Further identification was made with the help of identification discs such as kanamycin (1 mg), vancomycin (5 μg), colistin (10 μg), sodium polynethanol sulfonate disc and nitrate disc. All the organisms isolated and identified were tested for the production of beta-lactamase by chromogenic Nitrocefin disc method.[7]

Nitrocefin disc method

Nitrocefin disc was inoculated with a small portion of growth from Brucella blood agar plate and observed for a change in color from yellow to red. Disks which did not show color change within 10 min at room temperature were additionally incubated for 60 min at 37°C. A test was considered positive when yellow color turned to pink/red [Figure 1].

Figure 1.

Nitrocefin disc showing positive and negative reaction

RESULTS

A total of 60 patients were enrolled during the study period. The patients aged 22-70 years with male to female ratio of 0.8:1. Totally 60 samples yielded 121 isolates of which 111 were anaerobes. Out of 111 organisms 29 (26%) were Extended Spectrum Beta (β) Lactamase (ESBL) producer and 82 (74%) were non beta-lactamase producing organisms. Only anaerobic Gram-negative bacilli showed ESBL production. Bacteroides fragilis was the most common organism followed by Fusobacterium spp. The ESBL producing organisms isolated in the present study are shown in Table 1.

Table 1.

Beta-lactamase producing anaerobic bacteria

DISCUSSION

The microbiological flora of oral cavity is diverse and varies in normal and disease condition. BLPB are major concern in orofacial infections. In the present study, we observed 26% of our isolates to be ESBL producers and our findings are in consistent with Iwahara et al.[8] and Walker.[5] who identified 31% and 36% respectively. While Rams et al.[9] and Patel[10] identified 52.1% and 69% ESBL producers respectively. B. fragilis was the most common ESBL producer in our study (65.5%), however as per the Clinical Laboratory Standard Institute (CLSI) guidelines B. fragilis organism should not be tested and reported as BLPB. For all practical purpose they are considered as BLPB.[7] In the present study, they were included for research purpose and all of them showed positive reaction on nitrocefin disc.

The other BLPB in our study were Fusobacterium spp (24%) followed by Fusobacterium nucleatum (3.5%), Porphyromonas spp (3.5%) and Prevotella intermedia (3.5%). While most of the studies have identified Prevotella spp as the most common BLPO.[5,8,9,10] This can be explained by the fact that resistance rates vary from hospital to hospital and also within the species of organisms. Among non-Bacteroides anaerobes penicillin resistance can be common, but is not predictable. The increasing and prevalent antibiotic resistance among anaerobes is correlated with discovery and characterization of multiple, transferable resistance determinants corresponding to their respective resistance phenotypes. In addition heavy, irrational and indiscriminate use of antibiotics may result in selection and transfer of these resistance determinants.[7] However studies of this kind from this part of India are needed in future to establish the association. Comparison of findings of our study with other studies is shown in Table 2.

Table 2.

Comparison of findings of different studies

Many different types of β-lactamase enzymes have been identified in the literature. But the main two types are Ampc β-lactamase and ESBL enzymes. Ampc β-lactamase are encoded by the genes on the chromosomes and are inducible.[11] ESBL are encoded by genes on the plasmid and are transferable.[12] Bush and Jacoby have classified β-lactamase enzyme into two groups based on the function and molecular basis depending on amino acid sequence.[13]

Chromogenic cephalosporin (nitrocefin) disc method is the recommended method by CLSI for ESBL detection.[7] Iwahara et al.[8] in their study observed 31% of Prevotella spp to be ESBL producers by nitrocefin disc method. Further, it was confirmed by detecting CfxA and CfxA2 gene by real time polymerase chain reaction for all 48 (31%) beta-lactamase positive Prevotella strains. Hence, nitrocefin disc method can be used as sensitive screening method for beta-lactamase production in developing countries where facilities for molecular methods are not available.

By molecular diagnostic methods, various susceptible genes have been identified in the literature for beta-lactamase production namely cfxA, cfxA2, cfxA3, bla (TEM), bla (SHV), bla (OXA), bla (ampC), bla (cfxA) and bla (cepA/cb1A).[8,14] The studies have also concluded that there are many different genetic determinants of beta-lactamase production which still needs to be characterized.[14]

BLPB play a key role in mixed infections. First, they cause pathogenicity by inhibiting the drugs. Secondly, they transfer the plasmid genes and render other bacteria to become resistant and lastly they release the enzymes to the environment and neutralize the drug. There are various studies available in the literature to support the mechanism of BLPB.[3] Some anaerobes are resistant to β-lactam antimicrobial agents by mechanisms other than β-lactamase production. Therefore, a negative β-lactamase test does not necessarily assure susceptibility to this class of drug.[7]

Even though, there is lot of research about BLPB in the literature, epidemiological data on BLPB in chronic periodontitis in India is still lacking. More studies of this kind are required to correctly identify these strains in different parts of India.

CONCLUSION

ESBL producing anaerobic bacteria exits in chronic periodontitis cases and the present study identifies 26% of the isolates to be ESBL producer. Antibiotic resistance testing is essential before starting the therapy and in emergency cases drugs should be chosen to cover ESBL producers.