ANALYSIS OF ENTEROCOCCUS FAECALIS IN SAMPLES FROM TURKISH PATIENTS WITH PRIMARY ENDODONTIC INFECTIONS AND FAILED ENDODONTIC TREATMENT BY REAL-TIME PCR SYBR GREEN METHOD

Objective:

The aims of this study were to investigate the presence of Enterococcus faecalis in primary endodontic infections and failed endodontic treatments using real-time PCR and to determine the statistical importance of the presence of E. faecalis in a Turkish population with endodontic infections.

Material and Methods:

E. faecalis was investigated from 79 microbial samples collected from patients who were treated at the Endodontic Clinic of the Dental School of Atatürk University (Erzurum, Turkey). Microbial samples were taken from 43 patients (Group 1) with failed endodontic treatments and 36 patients (Group 2) with chronic apical periodontitis (primary endodontic infections). DNA was extracted from the samples by using a QIAamp^® DNA mini-kit and analyzed with real-time PCR SYBR Green.

Results:

E. faecalis was detected in 41 out of 79 patients, suggesting that it exists in not less than 61% of all endodontic infections when the proportion test (z= -1.645, <x= 0.05) was applied. Real-time PCR SYBR Green allowed for the detection of E. faecalis in 32 out of 43 (74.4%) in Group 1, and in 9 out of 36 (25%) in Group 2.

Conclusions:

These results suggest that E. faecalis is a frequent isolate for endodontic infections in Turkish patients, and is more often associated with failed endodontic treatments than primary endodontic infections.     

Prevalence of Enterococcus faecalis in subgingival biofilm and saliva of subjects with chronic periodontal infection

BACKGROUND AND AIMS: Enterococci are increasingly associated with nosocomial and opportunistic infections in humans. The role of the oral cavity as a reservoir for this species is unclear, particularly in the presence of oral infection. This study investigated the prevalence of Enterococcus faecalis in subgingival biofilm and saliva of patients with periodontal disease. METHODS: Samples were obtained from 56 periodontally healthy and 169 chronic periodontitis subjects. DNA was extracted from the samples and detection of E. faecalis was carried out by polymerase chain reaction. RESULTS: In general, E. faecalis was detected in 34.9% of all samples evaluated. No significant difference in the prevalence of this species between subgingival biofilm (34.6%) and saliva (35.1%) samples was observed. E. faecalis was detected significantly more often in saliva and subgingival samples of periodontitis patients (40.5% and 47.8%, respectively) compared to controls (14.6% and 17.1%, respectively; p<0.05). Moreover, significant positive correlations were observed between the presence of E. faecalis and clinical parameters of probing depth, clinical attachment level, bleeding on probing and plaque accumulation (p<0.001). CONCLUSION: The present data showed that E. faecalis is frequently detected in the oral microbiota of periodontitis patients suggesting that periodontal infection may favour the colonization by this species. Close attention should be given to these patients regarding the risk for development of E. faecalis infection in other sites of the body.     

粪肠球菌在根管内定植模式的体外研究

目的：建立体外粪肠球菌感染根管模型,观察根管内粪肠球菌的感染状态及其分布特征。方法：选择因正畸减数拔除的健康前磨牙10例,随机分为两组,将其中5例高压灭菌后分别浸泡于含有1.5 mL无菌脑心浸液(brain heart infusion,BHI)培养基和0.1 mL麦氏比浊度为5的粪肠球菌菌液的Eppendorf管中, 于CO₂孵育箱中培养21 d建成体外粪肠球菌根管感染模型,其余5例则作为对照组。将所有样本沿牙长轴近远中向劈为两半,一半作组织学Brown&Brenn染色观察,另一半置扫描电镜下观察粪肠球菌在根管内的感染状态。结果：组织学结果显示,在所有研究组样本中,粪肠球菌均不同程度地侵入牙本质小管中,测微尺测量深度可达330～1 000 μm。所有对照组样本的根管壁表面干净,牙本质小管排列有序,无细菌染色颗粒。扫描电镜结果显示,在所有研究组样本的根尖1/3段,粪肠球菌与无定形基质形成典型的生物膜结构,而在根管的冠1/3和中1/3段,粪肠球菌则成对或短链状排列在根管壁表面,呈现浮游状态。对照组的根管壁表面洁净,牙本质小管和管间牙本质结构清晰可见,未见细菌侵入。结论： 粪肠球菌可单独定植于根管内,形成生物膜并侵入牙本质小管深部。此模型可用于体外评价各种机械预备和化学消毒药物对根管内感染的清除效果,从而为临床选择治疗操作提供参考。     

肝移植术后肠球菌感染情况调查及其体外药敏分析

 目的调查肝移植患者术后肠球菌的感染状况并对其耐药性进行体外药敏检测,为肠球菌的治疗提供参考及指导.方法用法国梅里埃公司生产的VITEK32全自动细菌鉴定系统或常规生化法鉴定肠球菌到种,用K-B纸片法监测其耐药率.结果分离出的387株肠球菌中以屎肠球菌的分离率最高,为73.9%,粪肠球菌分离率21.4%,居第2位,检出氨基糖苷类高水平耐药肠球菌(HLAR)346株,占89.4%,仅检出1株万古霉素耐药肠球菌(VRE).结论肝移植患者术后感染肠球菌以屎肠球菌为主,其耐药性明显高于粪肠球菌,提示临床医师在治疗肝移植患者术后感染肠球菌时应根据分离株种类、耐药特点及药敏试验合理选用抗菌药物.     

粪肠球菌对多形核白细胞释放基质金属蛋白酶-8及凋亡影响的实验研究

目的体外研究粪肠球菌对多形核白细胞（PMNs）释放基质金属蛋白酶-8（MMP-8）及凋亡的影响。方法提取PMNs,以加入粪肠球菌悬浮液的PMNs作为实验组;加入乙酸肉豆蔻佛波醇的PMNs为阳性对照组;PMNs的PBS悬浮液为阴性对照组。培养0、20、60、120 min后,ELISA法检测各时间点MMP-8的释放量。以加入粪肠球菌裂解液的PMNs为实验组,PMNs的PBS悬浮液为对照组,培养2、5、10、15 h后,流式细胞分析仪检测PMNs的凋亡率。结果在0 min时,实验组和阳性对照组MMP-8释放量间差异无统计学意义（P＞0.01）;在60、120 min时,实验组MMP-8的释放量明显低于阳性对照组,二者间差异有统计学意义（P＜0.01）。在2、5、10、15 h,实验组凋亡率均高于对照组（P＜0.01）。结论粪肠球菌作用于PMNs后,PMNs无大量释放MMP-8现象,PMNs无凋亡延迟现象。     

Cecal ligation and puncture induced sepsis impairs host defense against Enterococcus faecium peritonitis

Purpose  Multiresistant and vancomycin resistant Enterococcus faecium (VRE) can cause serious infections in hospitalized patients with various co-morbid diseases. We investigated the course of VRE peritonitis after cecal ligation and puncture (CLP)-induced sepsis and compared this to sham operated mice. Methods  Mice were subjected to CLP or sham surgery. Forty-eight hours thereafter four groups were created by subjecting mice to peritoneal injection of either VRE or saline. Results  Mice infected with VRE after CLP were severely impaired in eliminating VRE from the peritoneal cavity and distant body sites. These mice failed to mount an early inflammatory response at the primary site of VRE infection. VRE superinfection did not influence CLP-induced organ damage or polymicrobial bacterial loads. Conclusions  Sublethal polymicrobial sepsis greatly facilitates infection and dissemination of VRE. VRE does not influence the course of CLP-induced sepsis.     

Analysis of cross infection using genomic fingerprinting in nosocomial urinary tract infection caused by Enterococcus faecalis

To evaluate cross infection and a possible outbreak of Enterococcus faecalis urinary tract infection (UTI) in our urology ward, we studied the DNA fingerprinting of E. faecalis strains isolated from nosocomial UTI patients, in the period 1982–1996, using arbitrarily primed polymerase chain reaction (AP-PCR) analysis. The serovar and amplified products of DNA extracted from clinically isolated urinary E. faecalis strains by the AP-PCR method were analyzed, and the respective isolation periods of E. faecalis-positive UTI patients were investigated. There were nine patients with E. faecalis UTI between March and May 1994 and all strains isolated from their urine specimens were serovar type 7. AP-PCR revealed that five of the nine isolates had the same pattern. It appeared that these strains had caused the outbreak of E. faecalis UTI. Cross-infection between patients with E. faecalis UTI was demonstrated by genomic fingerprinting, suggesting that cross infection had occurred via urinary catheters or by hand contact in our ward. We may, therefore, reasonably conclude that we should beware of the transmission of urinary E. faecalis and take countermeasures against its dissemination. Received: April 8, 1998 / Accepted: October 14, 1998     

Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalis

BACKGROUND/AIMS: This study aimed to investigate the biochemical mechanisms employed by the endodontic pathogen Enterococcus faecalis to confer acid- and alkali-resistance and to compare these with the mechanisms of representative oral streptococci. METHODS: E. faecalis JCM8728, Streptococcus mutans NCTC10449 and Streptococcus sanguinis ATCC10556 were used to assess both acid- and alkali-resistance by examining: (i) growth in complex media; (ii) stability of intracellular pH (pH(in)); (iii) cell durability to leakage of preloaded BCECF (2',7'-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein); and (iv) cell permeability to SYTOX-Green. RESULTS: Growth was initiated by E. faecalis at pH 4.0-11.0, by S. mutans at pH 4.0-9.0 and by S. sanguinis at pH 5.0-9.0. The pH(in) was similar to the extracellular pH in S. mutans and S. sanguinis at pH 5-10, while the pH(in) of E. faecalis was maintained at approximately 7.5-8.5 when extracellular pH was 7.5-10 and was maintained at levels equivalent to the extracellular pH when pH < 7.5. Cell membranes of E. faecalis were resistant to BCECF leakage when extracellular pH was 2.5-12 and to SYTOX-Green permeability at pH 4-10. The cell membrane durability to extracellular pH in E. faecalis was higher than that observed in the Streptococcus strains. CONCLUSION: Compared to S. mutans, E. faecalis was found to be equally resistant to acid and more resistant to alkalis. The results suggest that pH-resistance in E. faecalis is attributed to membrane durability against acid and alkali, in addition to cell membrane-bound proton-transport systems. These characteristics may account for why E. faecalis is frequently isolated from acidic caries lesions and from persistently infected root canals where calcium hydroxide medication is ineffective.     

Evaluation of the antibacterial substantivity of several intra-canal agents

The aim of this in vitro study was to compare the antimicrobial substantivity of 2% chlorhexidine gluconate (CHX), 100 mg ml(-1) doxycycline and 2.6% sodium hypochlorite (NaOCl) in bovine root dentine. Eighty dentine tubes prepared from bovine incisors were infected in vitro for 14 days with Enterococcus faecalis. The specimens were divided into five groups as follows: doxycycline HCl; CHX; NaOCl; infected dentine tubes (positive control); and sterile dentine tubes (negative control). Dentine chips were collected with round burs into tryptic soy broth. After culturing, the number of colony-forming units (CFU) was counted. In all experimental groups, the number of CFU was minimum in the first cultures, and the results obtained were significantly different from each other at any time period (P < 0.05). In the first culture, the NaOCl group and doxycycline HCl group showed the lowest and highest number of CFU, respectively. In each group, the number of CFU increased significantly by time-lapse (P < 0.05). In conclusion, the substantivity of CHX was significantly greater than NaOCl and doxycycline.