Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.


INTRODUCTION
The human mouth harbours a complex microbial community containing aerobic and anaerobic bacteria. These bacteria cause polymicrobial opportunistic oral and extra oral infections. Anaerobic bacteria such as Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus, Prevotella nigrescens, Parvimonas micra, and Eubacterium nodatum cause periodontal diseases, odontogenic abscesses, orofacial infections, and have been implicated in brain abscesses 26,31 . Some of these bacteria are also isolated from sputum and ICU patients with aspiration pneumonia 17,21 . these infections. However, studies have shown that many oral anaerobic bacteria have developed 12 . has been attributed to resistance genes, present on chromosomal or plasmid DNA that can be transferred between commensal and pathogenic bacteria 30 . Studies have shown that there is a high prevalence of cfxA genes responsible for Prevotella and Capnocytophaga species isolated from periodontal pockets 6, 13 . In addition, cfxA and cfxA2 genes have been isolated from oral infection sites as well as from the causative organisms isolated from these infection sites 7,16 . It has been suggested that this cfxA/cfxA2 partition could be partly related to the genus and partly to the geographical origin of the enzyme-producing strains 9 . CfxA2 and cfxA3 genes have been detected in anaerobic bacteria isolated from patients in France, United Kingdom, Norway, Argentina, and the United States of America 11,20,25 , whereas cfxA6 have only been detected in anaerobic bacteria isolated from patients in Argentina 5 . No data are available from Africa on the production responsible for the production of these enzymes in patients with chronic periodontitis. This study was therefore conducted to isolate and identify the periodontal pockets of patients with chronic periodontitis and to detect the genes responsible for the production of these enzymes.

Study population
This study was conducted in the Oral Health Centre, Johannesburg in 2012. Forty-eight patients diagnosed with severe to moderate forms of chronic American Association of Periodontology 1 and with invited to participate in the study. Ethics clearance was obtained from the Human Research Ethics consent was obtained from all participants. Patients with a history of previous periodontal treatment, necrotizing ulcerative gingivitis, diabetes, or those who had taken systemic antimicrobials or antiwere excluded.
After careful removal of supragingival plaque, a sterile paper point was inserted into the two deepest pockets and left in place for ten seconds. Paper points were pooled in 1 mL of reduced transport ensure the viability of anaerobic bacteria.

producing bacteria
The samples were vortexed for 30 s, serially diluted in phosphate buffered saline, and 0.1 mL of 1/10 to 1/10000 dilutions were plated on 5% blood agar plates supplemented with 5 mg/L of South Africa) and 1 mg/L of menadione (Sigmadetermining the total anaerobic bacterial count. The proportion of the microbiota resistant to amoxicillin was determined by plating samples on blood agar plates supplemented with 3 μg/mL of amoxicillin with and without 0.75 μg/mL of clavulanic acid 15 . The plates were anaerobically incubated for one week at 37 o C and the number of colony forming units (cfu) was counted. The bacteria that grew on amoxicillin but not on the amoxicillin-clavulanic techniques can be used, the API 32A system was the only one available at the time.
isolates using a technique described by Handal, et al. 13 (2005). A loopful of culture was inoculated into a sterile eppendorf tube containing 10 μL of 10× PCR buffer, 15 mM MgCl 2 (Qiagen, Whitehead and 90 μL of sterile distilled water. The inoculated buffer was boiled at 95°C for 10 min, cooled on ice, and centrifuged at 5000 rpm for 10 min. The supernatant was harvested and stored at -70°C until required. Reaction (PCR) technique. A 25 μL reaction mixture containing 12.5 μL of 2× PCR Master Mix, 2.5 μL of sterile nuclease-free water, 5 μL of 5 μM primer (For bla TEM f o r b l a C f x A iCycler thermal cycler (BIO-RAD, USA) using the PCR conditions described by Handal, et al. 12 (2005) Escherichia coli ATCC 25746 (University of Copenhagen) was used as a positive control for bla TEM . The P. intermedia isolate containing the bla cfxA gene was used as the positive PCR reactions, the negative control consisted of using gel electrophoresis, sequenced, and further characterized by the BLAST option of the nucleotide -databases.

Antimicrobial susceptibility
subjected to antimicrobial susceptibility using a disk diffusion test and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) performance standards for antimicrobial disk susceptibility tests 4 . Minimum inhibitory concentrations (MICs) were performed using the microbroth dilution method according to CLSI Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria 3 . Only 17 strains were successfully revived and included in this assay.
The data were analyzed using the STATA statistical package (College Station, Texas, USA).

Demography and the prevalence of bacteria
The mean age of the patients was 52 with a range from 32 to 83 years of age. Fifty-eight percent of the patients were female. The average pocket depth of the sampled pockets was 7 mm with a range of 5 mm to 13 mm. Mean total anaerobic bacterial count on control plate was 1.8x10 6 cfu/mL. Mean total anaerobic bacterial count on the amoxicillin plate was 1.9x10 5 cfu/mL. Mean total anaerobic bacterial count on the augmentin plate was 5.9x10 4 anaerobic oral bacteria, which constitute 9.4% of the total cultivable number of bacteria. All the isolates that grew on the blood agar plate containing negative black pigmented anaerobes (Figure 1).
isolates carried the bla CfxA gene, whereas none carried bla TEM (

Antimicrobial susceptibility
subjected to the disc diffusion test for ampicillin and penicillin. The results showed that 42 to 56% of the isolates containing resistance genes were resistant more accurate, showed that 53 to 59% of isolates that carried the resistance gene were resistant and another 6% had intermediate susceptibility to

DISCUSSION
In this study, Prevotella spp. and Bacteroides spp. producing bacteria and 43% and 75% of these bacteria, respectively, carried the cfxA gene.
in the North American, French, and Norwegian population 9,13 . The cfxA genes are known to be present in Pseudomonas aeruginosa including bacteroides and oral bacteria such as Prevotella and Capnocytophaga 2,8,13 . These genes are responsible for the resistance to penicillins and cephalosporins. The presence of these genes in oral commensals is always a cause for concern because this resistance can spread to serious pathogens and cause resistance to extendedspectrum cephalosporins as well. In addition, these normal commensals do cause extraoral infections which require antibiotic treatment. For example, Capnocytophaga spp containing the cfxA3 lactamase-producing gene have been isolated from patients with leukemia and neutropenia 22,23 . Similarly, cfxA gene-containing resistant Prevotella spp have been isolated from patients with cystic 28,29 . The presence of these resistance genes in the oral microbiota could result in the commensals serving as reservoirs of antibiotic resistance 30 . producing strains carried cfxA2 genes. In the Prevotella spp, there was a predominance of the cfxA gene in North America, cfxA2 in France 20,25 , and both genes in the United Kingdom 16 . Horizontal gene transfer might explain the spread of closely related gene sequences among these periodontal species 6 . Although the cfxA3 gene has been found mainly in Capnocytophaga spp. 11,23 , in our study it was closely associated with Prevotella and Bacteroides spp. Interestingly, although cfxA6 was initially detected in Prevotella FN376426) and also found in the present study, no other studies have detected this gene. In addition, the presence of cfxA6 in Porphyromonas spp. has not been previously reported.
In this study, 10% of the periodontal pocket microbiota in 75% of the patients produced two species per producers, only 50.6% carried cfxA genes. However, which is highly stressful and competitive, are antibiotics Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes known to adapt to genetic transfer. Metagenomic oral bacteria play a major role in horizontal gene transfer 18 because it improves their chance of survival, increases virulence, and alters their metabolism and drug resistance. Both plasmid and chromosomal-borne transfer of antibiotic resistance has been shown in oral bacteria 10 . Transformation through eDNA present in the plaque 14 and membrane vesicles 24 has also been described. In addition, a highly mobile Tn916, like as transposon that facilitates conjugation, has been found in many oral bacteria such as Streptococci species, F. nucleatum, Eubacterium species, Veillonella species, and Actinobacillus species. Therefore, a potential reservoir may transfer resistance genes to other drug sensitive bacteria from these 10% of drug resistant bacteria.
values of 0.125 μg/mL. This phenomenon has also been reported by other studies 30,32 . This is bacteria, and when produced in small quantities they may contribute little to antibiotic resistance, but could play a physiological role in peptidoglycan metabolism 19 8 (2008) has also shown that cfxA gene containing Bacteroides spp. can show whereas others require induction, which suggests that the mere presence of these genes may not by more reliable broth dilution techniques (53% and 59% resistance to amoxicillin and penicillin, respectively) 27 . Nevertheless, in nonresponsive considered. Our results also showed that many fxA or Bla TEM gene, which suggests that there may be other genes responsible for the production of this enzyme (Table 2). This study only studied cfxA or Bla TEM because these genes responsible for the production bacteria. Other genes such as CepA/CblA are only 9 .
One of the limitations of our study was the on biochemical reactions and was the only technique available in our laboratory at the time of this study.
CfxA2, cfxA3, and cfxA6 genes were detected in 51% of these organisms, which comprised 10% of the total cultivable oral microbiota in our patients with chronic periodontitis. The cfxA6 gene was found in Prevotella and Porphyromonas spp, which has epidemiological implications. Fifty-six percent of the isolates that carried cfxA genes were that in nonresponsive patient antibiotics other than