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Antimicrobial susceptibility of Enterococcusfaecalis isolated from canals of root filled teethwith periapical lesions E. T. Pinheiro1, B. P. F. A. Gomes1, D. B. Drucker2, A. A. Zaia1, C. C. R. Ferraz1 &F. J. Souza-Filho11Department of Endodontic, Piracicaba Dental School, State University of Campinas, UNICAMP, Piracicaba, Brazil; and2Department of Oral Microbiology, University Dental Hospital of Manchester, Manchester, UK Results All strains were susceptible to penicillinsin vitro, however, the MICs of amoxicillin and amox- Pinheiro ET, Gomes BPFA, Drucker DB, Zaia AA, Ferraz icillin-clavulanic acid (MIC90 ¼ 0.75 lg mL)1) were CCR, Souza-Filho FJ. Antimicrobial susceptibility of Entero- lower than for benzylpenicillin (MIC90 ¼ 3.0 lg mL)1).
coccus faecalis isolated from canals of root filled teeth with All strains studied were also susceptible to vancomycin periapical lesions. International Endodontic Journal, 37, 756– and moxifloxacin, whilst 95.2% were susceptible to chloramphenicol. Amongst the isolates, 85.7% were Aim To test, in vitro, the susceptibility to different susceptible to tetracycline and doxycycline and 80.9% antibiotics of Enterococcus faecalis isolates from canals of to ciprofloxacin. The MIC of erythromycin ranged from root filled teeth with periapical lesions.
0.38 to >256 lg mL)1; only 28.5% of the strains were Methodology Twenty-one E. faecalis isolates, from susceptible (MIC £ 0.5 lg mL)1). Limited susceptibility canals of root filled teeth with persisting periapical was also observed with azithromycin which was active lesions, were tested for their antibiotic susceptibilities.
against only 14.2% of isolates. No strains produced The following antibiotics were used: benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, erythromycin, Conclusion Enterococcus faecalis isolates were com- azithromycin, vancomycin, chloramphenicol, tetracyc- pletely susceptible, in vitro, to amoxicillin, amoxicillin- line, doxycycline, ciprofloxacin and moxifloxacin. Min- clavulanic acid, vancomycin and moxifloxacin. Most isolates were susceptible to chloramphenicol, tetracyc- antimicrobial agents were determined using the E-test line, doxycycline or ciprofloxacin. Erythromycin and E. faecalis strains classified as susceptible or resistant Keywords: antimicrobial susceptibility, endodontic according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS). The strainswere also tested for b-lactamase production withnitrocefin (Oxoid, Basingstoke, UK).
Received 6 November 2003; accepted 9 June 2004 1990, Morrison et al. 1997). They have long been known to cause infections, such as enterococcal Enterococci are common inhabitants of the human bacteraemia (Murdoch et al. 2002), infective endocar- gastrointestinal and genitourinary tracts (Murray ditis (Graham & Gould 2002) and urinary tractinfections (Murray 1990, Morrison et al. 1997). Over Correspondence: Brenda P. F. A. Gomes, BDS, MSc, PhD, the last two decades, enterococci have been recognized Endodontia, Faculdade de Odontologia de Piracicaba-FOP- as the leading cause of hospital-acquired infection, UNICAMP, Avenida Limeira, 901, Piracicaba, SP, 13414-018, paralleling their increased antimicrobial resistance to Brazil (Tel.: 0055 19 3412-5215; fax: 0055 19 3412-5218;e-mail: bpgomes@fop.unicamp.br).
most currently approved agents (Mundy et al. 2000, International Endodontic Journal, 37, 756–763, 2004 Pinheiro et al. Antimicrobial susceptibility of Enterococcus faecalis Malani et al. 2002, Udo et al. 2002). Of the enterococ- However, it is important to emphasize that, because of cal species associated with colonization and infection in ecological changes in an acute situation, the microb- humans, Enterococcus faecalis is the most common iota will change. Poymicrobial infections and obligate species (Murray 1990, Mundy et al. 2000, Shepard & anaerobes are frequently found in canals of sympto- matic root filled teeth (Pinheiro et al. 2003a). There- Enterococci are also able to colonize a variety of fore, bacteria other than enterococci will often be the other sites, including the oral cavity (Smyth et al.
main target of the antibiotics in the acute infection.
1987). These microorganisms have been associated Enterococci possess a vast array of mechanisms with oral mucosal lesions in immunocompromised patients (Wahlin & Holm 1988), periodontitis (Rams antibiotics including penicillin, the drug of choice et al. 1992) and root canal infections (Molander et al.
(Hoellman et al. 1998, Shepard & Gilmore 2002).
1998, Sundqvist et al. 1998, Noda et al. 2000, Peciu- These microorganisms show intrinsic resistance to liene et al. 2000, 2001, Pinheiro et al. 2003a,b).
certain antibiotics such as cephalosporins, clindamy- Enterococci constitute a small percentage of the cin and aminoglycosides (Murray 1990, Morrison microbial species isolated from root canals of teeth et al. 1997). In addition to these intrinsic resistances, with necrotic dental pulps (Sundqvist 1992, 1994).
enterococci have acquired genetic determinants that However, they are the most commonly isolated species confer resistance to many classes of antimicrobials, from root canals of teeth with failed endodontic including tetracycline, erythromycin, chlorampheni- treatment. Enterococci are found in approximately col, and, most recently, vancomycin (Murray 1990, 50% of the canals with refractory infection (Molander Morrison et al. 1997, Mundy et al. 2000, Shepard & et al. 1998, Pinheiro et al. 2003a,b). Peciuliene et al.
(2000, 2001) have reported an isolation frequency of Clinical isolates of E. faecalis recovered from root enterococci as high as 70% when root filled teeth are canal infections can demonstrate antimicrobial resis- associated with chronic apical periodontitis. Enterococ- tance to conventional treatment regimens recom- cus faecalis is also the most common Enterococcus sp.
mended for dental procedures. Dahle´n et al. (2000) isolated from root canals; other species are rarely found have described enterococcal isolates resistant to ben- (Sundqvist et al. 1998, Peciuliene et al. 2000, 2001, zylpenicillin, ampicillin, clindamycin, metronidazole Pinheiro et al. 2003a,b). Enterococcus faecalis is usually and tetracycline; whilst Noda et al. (2000) have isolated in pure culture or as a major component of the discovered strains that are resistant to cephalosporins.
flora of previously root filled teeth with chronic apical Previous studies (Pinheiro et al. 2003b) have found periodontitis (Peciuliene et al. 2000).
E. faecalis strains which show resistance to azithromycin Antibiotics are not generally used to treat chronic and erythromycin. Thus, many antibiotics, tradition- infections, such as apical periodontitis, in root filled ally used in odontogenic infection, may prove ineffec- teeth. Chronic alveolar infections are associated with pulpless teeth which have no blood supply reaching the pulp space. Following the systemic administration of an In the case of endodontic infections associated with antibiotic, the concentration reaching the root canal is enterococci, very limited antibiotic sensitivity data are negligible and unlikely to inhibit bacterial growth.
available. The present study aimed to test, in vitro, the Therefore, systemic antibiotic therapy is neither indi- susceptibility to different antibiotics of E. faecalis isola- cated nor likely to be beneficial (Abbott et al. 1990).
ted from canals of root filled teeth with periapical Prophylactic use of antibiotics is, of course, another matter. Prophylactic use can be indicated if patients areconsidered at risk of infective endocarditis during endodontic treatment (Abbott et al. 1990, Debelianet al. 1995). In such cases, therapy should be directed primarily against the most important pathogens pre-sent.
The E. faecalis strains were isolated from canals of root Furthermore, periapical abscesses can originate from filled teeth with persisting periapical lesions as des- root filled teeth whose apical periodontitis continues cribed by Pinheiro et al. (2003a) and Gomes et al.
following treatment. Some of them need antibiotic (2004). Patients were selected from those who attended therapy prior to surgical treatment (Sousa et al. 2003).
the Piracicaba Dental School, SP, Brazil, with a need for International Endodontic Journal, 37, 756–763, 2004 Antimicrobial susceptibility of Enterococcus faecalis Pinheiro et al.
nonsurgical root canal retreatment. Patients who had that indicates the drug concentration in lg mL)1 received antibiotic treatment during the last 3 months or had a general disease were excluded from the Mueller-Hinton agar plates (Oxoid, Basingstoke, UK) 4 mm thick were inoculated using a swab thathad standardized to match the turbidity of the 0.5 McFarland standard. The surface of the plate was All coronal restorations, posts and carious defects were swabbed in three directions to ensure a complete removed. After access cavity preparation, the teeth distribution of the inoculum over the entire plate.
were individually isolated from the oral cavity with a Within 20 min of inoculation, the antimicrobial rubber dam, and disinfection was carried out using agents’ strips were applied and the plates were 5.25% sodium hypochlorite. The root filling was inverted for incubation at 35 °C in air for 16–18, removed using Gates Glidden drills (Dentsply Maillefer, 24 h for vancomycin. After incubation, the plate was Ballaigues, Switzerland) and endodontic files without examined and an elliptical zone of growth inhibition the use of chemical solvents. Irrigation with sterile was seen around the strip. The minimal inhibitory saline solution was performed in order to remove any concentration (MIC) was read from the scale on the remaining materials and to moisten the canal prior to strip at the intersection of the growth with the sample collection. For microbial sampling, a sterile E-strip. Once the MICs for the antimicrobial agents paper point was introduced into the full length of the canal (as determined with a preoperative radiograph), interpretative categories of susceptible or resistant and kept in place for 60 s. The paper point samples according to the guidelines of National Committee for from the root canal were transferred to a transport Clinical Laboratory Standards (NCCLS) (2002). All medium-VMGA III (Mo¨ller 1966, Dahle´n et al. 1993) the tests were completed in duplicate.
and taken to the microbiology laboratory for processingwithin 4 h.
Enterococcus faecalis isolates were tested for b-lactamase production with nitrocefin (Oxoid) according to the The samples were inoculated onto nonselective blood manufacturer’s instructions. Nitrocefin solution (5 lL) agar plates and incubated in aerobic and anaerobic was dropped onto a single colony of an overnight conditions. The enterococcal identification was per- culture. Development of a red colour within 60 s formed using colonial morphology, oxygen tolerance, Gram staining characteristics, and Rapid ID 32 Strep(Bio Merieux, Marcy-l’Etoile, France). In most of the cases, enterococcal strains, bile resistant, facultativelyanaerobic Gram-positive cocci, were identified as MIC range, MIC50 and MIC90 values obtained by the E-test method are shown in Table 1. Susceptibilityrates are also shown. All isolates proved susceptibleto benzylpenicillin, amoxicillin and amoxicillin-clavul- anic acid. No strains produced b-lactamase. The The susceptibility/resistance of 21 E. faecalis strains to strains studied were also completely susceptible to 11 antibiotics was measured. The following antimicro- vancomycin and moxifloxacin. The latter was the bials were tested: benzylpenicillin, amoxicillin, amoxi- most active antibiotic, in vitro, against E. faecalis with the lowest MIC values: all isolates were inhibited by vancomycin, chloramphenicol, tetracycline, doxycy- £0.5 lg mL)1. Eight strains were found to be resist- cline, ciprofloxacin and moxifloxacin.
ant to azithromycin, and two of them were also The antimicrobial susceptibility of isolates was resistant to erythromycin. Three strains were resist- investigated by means of the E-test System (AB Biodisk, ant to both tetracycline and doxycycline. One strain Solna, Sweden). The E-test uses plastic strips; one side was resistant to multiple drugs, viz. erythromycin, of the strip contains a concentration gradient of the azithromycin, tetracycline, doxycycline and chloram- antimicrobial agent; the other contains a numeric scale International Endodontic Journal, 37, 756–763, 2004 Pinheiro et al. Antimicrobial susceptibility of Enterococcus faecalis Table 1 In vitro susceptibility of 21 E. faecalis isolates from (Murray 2000, Murdoch et al. 2002, Shepard & canals of root filled teeth with periapical lesions All strains studied were susceptible to penicillins in vitro, however, the MICs of amoxicillin and amoxi- cillin-clavulanic acid were lower than for benzylpeni- cillin. These findings are in agreement with previous studies (Rams et al. 1992, Pinheiro et al. 2003b) which have found that enterococci are more sensitive to amoxicillin than to benzylpenicillin, bearing in mind that the latter can be given i.m. or i.v. not orally.
Phenoxymethyl penicillin, which can be given orally, is less active against enterococci than benzylpenicillin is (Nord & Wadstro¨m 1973). The results indicated that E. faecalis strains isolated from canals of root filled teeth with periapical lesions remain susceptible, in vitro, to MIC50, minimal inhibitory concentration including 50% of the amoxicillin. Nevertheless, the lack of enterococcal strains; MIC90, minimal inhibitory concentration including 90% resistance to penicillins in this study may be due to the limited number of strains investigated and/or recommended by NCCLS (2002): benzylpenicillin, amoxicillin geographical differences. The presence of enterococcal and amoxicillin-clavulanic acid (£8 S, ‡16 R); erythromycin (£0.5 strains resistant to penicillin and ampicillin has been S, ‡8 R); vancomycin (£4 S, ‡32 R); chloramphenicol (£8 S, ‡32 reported in endodontic infections in the USA (Matusow R); tetracycline and doxycycline (£4 S, ‡16 R); ciprofloxacin (£1S, ‡4 R). The breakpoints used for azithromycin were £2 S and 1981) and Sweden (Dahle´n et al. 2000) which under- ‡8 R (Fass 1993); and for moxifloxacin were £2 S, ‡8 R (Mather lines the need to perform susceptibility tests of these isolates. However, those authors did not provideinformation about the nature of the endodontic infec-tions, i.e. primary or secondary infections. There mostlikely is a difference in resistance pattern between enterococci from primary infections and from root filled Penicillins are the most frequently used antimicrobial teeth with continuing apical periodontitis. Further agents. Due to their historical effectiveness, minimal investigation involving enterococcal strains isolated toxicity and relatively low cost, penicillins constitute from both situations would improve knowledge about the first-choice antibiotics for odontogenic infections.
resistance pattern of enterococci in endodontic infec- Important classes of penicillins include penicillins G and V, which are highly active against susceptible Gram- Besides differences in geographical areas and origins positive cocci, and amoxicillin with an improved Gram- of infections, changes in resistance pattern of bacteria negative spectrum. b-Lactamase inhibitors such as may occur over time. Earlier studies (Zeldore & Ingle clavulanate are used to extend the spectrum of peni- 1962, Engstro¨m 1964) of enterococci isolated from root cillins against b-lactamase producing organisms (Petri canals had shown that 100% of isolates were susceptible to erythromycin. Heintz et al. (1975) found more than Bacterial resistance to penicillins has become a 90% of isolates were susceptible, whilst Stern et al.
problem of great clinical significance because of its (1990) have found 61.9% of enterococcal isolates widespread use for many years (Appelbaum et al.
susceptible to this drug. The present findings support 1990). The development of enterococcal resistance to the finding of a decrease in the enterococcal suscepti- b-lactams can be mediated by alterations in the bility to erythromycin over time. In this study, the MIC expression or binding affinities of penicillin-binding proteins. Additionally, resistance has been associated >256 lg mL)1. Two isolates were classified as resistant with the production of b-lactamase, occasionally (MIC ‡ 8 lg mL)1) and 6 (28.5%) as susceptible (Morrison et al. 1997). However, in this study, all (MIC £ 0.5 lg mL)1) according to the susceptibility isolates were negative for b-lactamase production, breakpoints determined by the NCCLS protocol; most which agrees with the findings of Udo et al. (2002).
of the isolates (65.4%) showed an intermediate pattern.
b-Lactamase production occurs only rarely in E. faecalis Similar results have been reported by Sedgley et al.
International Endodontic Journal, 37, 756–763, 2004 Antimicrobial susceptibility of Enterococcus faecalis Pinheiro et al.
(2004) who have found, amongst 12 oral enterococci, (Cotter & Adley 2001). Resistance to tetracyclines has two strains resistant to erythromycin, two (16.6%) susceptible and eight (66.6%) with an intermediate Chloramphenicol is effective against most aerobes pattern. Those studies have shown that the MIC of and anaerobes, but its potential side-effect of aplastic erythromycin, when tested against enterococcal strains, anaemia usually makes selection of another effective has increased over time; which suggests that oral and safer antibiotic a better choice (Moenning et al.
enterococci have become less susceptible to this drug.
1989). It was effective against 95.23% of the strains in Azythromycin is able to achieve higher and more this study. However, other studies have reported that sustained blood levels than erythromycin, without the 20% (Cotter & Adley 2001) to 26% (Udo et al. 2002) of gastrointestinal side effects (Grad 1997, Andrade enterococci are chloramphenicol resistant.
2000). Azythromycin was tested as a substitute for Amongst the drugs tested, vancomycin and moxifl- erythromycin and was found to be less effective against oxacin were active against all E. faecalis isolates in vitro.
enterococci than erythromycin, with only 14.2% of Vancomycin is a drug primarily active against Gram- isolates being susceptible. This finding is in accordance positive bacteria. However, it should be employed only to with those of Fass (1993). Furthermore, the latter treat serious infections (Chambers 2001). Administra- study has also reported that there is cross-resistance tion of vancomycin is an effective alternative, in patients between azithromycin and erythromycin.
who are allergic to penicillin, for the treatment of In this study, erythromycin and azythromycin endocarditis caused by viridans streptococci as well as resistance was found amongst E. faecalis isolates.
enterococci. In the latter case, penicillin or vancomycin Furthermore, E. faecalis has intrinsic resistance to is given in combination with an aminoglycoside (Murray clindamycin (Murray 1990, Morrison et al. 1997).
1990, Graham & Gould 2002). All E. faecalis strains Thus, this drug is not clinically effective for Enterococcus examined in this study were susceptible to vancomycin.
spp. Therefore, when patients are allergic to penicillins, Previous studies of the susceptibility of oral enterococci the alternative prophylactic regimens recommended for have also shown high susceptibility to vancomycin dental procedures seems to be of limited value against (Rams et al. 1992, Dahle´n et al. 2000). However, studies enterococci. Due to the predominance of E. faecalis in have highlighted the emergence of vancomycin-resist- root filled teeth with periapical lesions, alternative ant enterococci, especially amongst E. faecium and in drugs should be considered for prophylaxis in individ- lower frequency amongst E. faecalis (Murray 2000, uals at risk for endocarditis during endodontic retreat- Malani et al. 2002). These vancomycin-resistant entero- ment. Amongst the alternative drugs investigated in cocci have emerged as major nosocomial pathogens in this study, E. faecalis strains were found to be resistant hospitals, and frequently possess determinants confer- to tetracycline, doxycycline, ciprofloxacin and chlo- ring multiple drug resistance so that few therapeutic ramphenicol. Owing to geographical differences as well options remain for treating these infections (Morrison as differences over time, previously discussed in this et al. 1997, Rice 2001, Shepard & Gilmore 2002).
paper, the findings of this study are not general but Moxifloxacin and ciprofloxacin are members of the rather only applicable to the microbes tested.
quinolones. Ciprofloxacin has antimicrobial activity Tetracyclines are broad-spectrum antibiotics with against most Gram-negative bacilli and cocci., but activity against aerobic and anaerobic Gram-positive limited activity against most Gram-positive organisms.
and Gram-negative organisms. Doxycycline is one of Moxifloxacin is a new fluoroquinolone with expended the most active derivative of tetracycline. However, spectrum of activity, including anaerobes and Gram- bacterial resistance to any member of the class usually positive organisms, especially the multi-resistant ones results in cross-resistance to other tetracyclines (Cham- (Fass 1997, Oliphant & Green 2002, Speciale et al.
bers 2001), which was observed in the present study.
2002, Andersson & MacGowan 2003). In the present The strains resistant to tetracycline were also resistant study, moxifloxacin was one of the most active to doxycycline, the latter showing lower MICs against antibiotics against E. faecalis with the lowest MIC50 E. faecalis. Tetracycline resistance observed in 14.3% of and MIC90, and proved more active than ciprofloxacin, strains in this study agrees with resistance in 13.8% of which agrees with data that have been reported by isolates reported by Dahle´n et al. (2000). In contrast, several authors (Fass 1997, Mather et al. 2002, Spec- some studies have shown even higher percentages of iale et al. 2002). In addition to antimicrobial activity E. faecalis to be resistant to this antibiotic, i.e. 58% studies, the pharmacokinectic and pharmacodynamic (Rams et al. 1992), 65.1% (Udo et al. 2002) and 68.5% properties of moxifloxacin have been studied, showing International Endodontic Journal, 37, 756–763, 2004 Pinheiro et al. Antimicrobial susceptibility of Enterococcus faecalis excellent bioavailability, long half-life and good tissue Appelbaum PC, Spangler SK, Jacobs MR (1990) b-lactamase penetration of this drug. Furthermore, it has an production and susceptibilities to amoxicillin, amoxicillin- excellent tolerability (Krasemann et al. 2001).
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Microsoft word - unim study descriptions for website _1_

A Randomized Controlled Trial of Male Circumcision to Reduce HIV Incidence in Kisumu, Robert C. Bailey,1 Stephen Moses,2 Corette B. Parker,3 Kawango Agot,4 Ian Maclean,5 John N. Krieger,6 Carolyn F. M. Williams,7 Richard T. Campbell,1Jeckoniah O. Ndinya-Achola8 PI Contact Information: rcbailey@uic.edu Summary Background Male circumcision may provide significant protection against HIV-

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NAYLOR CLINIC OF CHIROPRACTIC & ACUPUNCTUREDavid K. Naylor, D.C., Dipl. Ac. (NCCAOM)612 South Church Street Burlington, NC 27215Phone: (336) 229-4226 Fax: (336) 229-6800 NUTRITIONAL EVALUATION Tests Used for Analysis: Comments: Patient Symptom Survey. Patient's comments: My concerns are fatigue and hair loss. This analysis and the recommendations are not for the purpose of t

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