Paradoxical High-Level Spiramycin Resistance and Erythromycin Susceptibility due to 23S rRNA Mutation in Streptococcus constellatus.

Objectives: The aim of the study was to characterize phenotypically and genotypically an uncommon mechanism of resistance to macrolides, lincosamides, and streptogramins (MLS) in a Streptococcus milleri group clinical isolate. Materials and Methods: The isolate UCN96 was recovered from an osteoradionecrosis wound, and was identified using the matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the partial sequencing of the sodA gene. Antimicrobial susceptibility testing were carried out by the disk diffusion method and minimal inhibitory concentrations (MICs) were determined by the broth microdilution technique. PCR screening was performed for MLS resistance genes described in Gram-positive bacteria. Specific mutations in the ribosomal proteins L3-, L4-, and L22-encoding genes were also screened and those in domain V of the 23S rRNA gene (rrl). The number of mutated copies of the rrl gene was determined using amplification-refractory mutation system quantitative-polymerase chain reaction (qPCR) analysis. Results: The clinical isolate UCN96 was unambiguously identified as Streptococcus constellatus. It was susceptible to all macrolides and lincosamides (ML) antibiotics except spiramycin (MIC >256 mg/L) while it was also resistant to streptogramins. Screening for all acquired resistance genes was negative and no mutation was found in genes coding for L3, L4, and L22 ribosomal proteins. Of interest, a single mutation, A2062C (according to Escherichia coli numbering), was detected in the domain V of 23S rRNA. Conclusion: Mutations at the position 2062 of 23S rRNA have been detected once in Streptococcus pneumoniae, and not yet in other Streptococcus spp. This mechanism is very likely uncommon in Gram-positive bacteria because different copies of 23S rRNA operons should be mutated for development of such a resistance pattern.

The Yin and Yang of Streptococcus Lung Infections in Cystic Fibrosis: a Model for Studying Polymicrobial Interactions.

The streptococci are increasingly recognized as a core component of the cystic fibrosis (CF) lung microbiome, yet the role that they play in CF lung disease is unclear. The presence of the Streptococcus milleri group (SMG; also known as the anginosus group streptococci [AGS]) correlates with exacerbation when these microbes are the predominant species in the lung. In contrast, microbiome studies have indicated that an increased relative abundance of streptococci in the lung, including members of the oral microflora, correlates with impacts on lung disease less severe than those caused by other CF-associated microflora, indicating a complex role for this genus in the context of CF. Recent findings suggest that streptococci in the CF lung microenvironment may influence the growth and/or virulence of other CF pathogens, as evidenced by increased virulence factor production by Pseudomonas aeruginosa when grown in coculture with oral streptococci. Conversely, the presence of P. aeruginosa can enhance the growth of streptococci, including members of the SMG, a phenomenon that could be exacerbated by the fact that streptococci are not susceptible to some of the frontline antibiotics used to treat P. aeruginosa infections. Collectively, these studies indicate the necessity for further investigation into the role of streptococci in the CF airway to determine how these microbes, alone or via interactions with other CF-associated pathogens, might influence CF lung disease, for better or for worse. We also propose that the interactions of streptococci with other CF pathogens is an ideal model to study clinically relevant microbial interactions.

Pseudomonas aeruginosa Can Inhibit Growth of Streptococcal Species via Siderophore Production.

Cystic fibrosis (CF) is a genetic disease that causes patients to accumulate thick, dehydrated mucus in the lung and develop chronic, polymicrobial infections due to reduced mucociliary clearance. These chronic polymicrobial infections and subsequent decline in lung function are significant factors in the morbidity and mortality of CF. Pseudomonas aeruginosa and Streptococcus spp. are among the most prevalent organisms in the CF lung; the presence of P. aeruginosa correlates with lung function decline, and the Streptococcus milleri group (SMG), a subgroup of the viridans streptococci, is associated with exacerbations in patients with CF. Here we characterized the interspecies interactions that occur between these two genera. We demonstrated that multiple P. aeruginosa laboratory strains and clinical CF isolates promote the growth of multiple SMG strains and oral streptococci in an in vitro coculture system. We investigated the mechanism by which P. aeruginosa enhances growth of streptococci by screening for mutants of P. aeruginosa PA14 that are unable to enhance Streptococcus growth, and we identified the P. aeruginosapqsL::TnM mutant, which failed to promote growth of Streptococcus constellatus and S. sanguinis Characterization of the P. aeruginosa ΔpqsL mutant revealed that this strain cannot promote Streptococcus growth. Our genetic data and growth studies support a model whereby the P. aeruginosa ΔpqsL mutant overproduces siderophores and thus likely outcompetes Streptococcus sanguinis for limited iron. We propose a model whereby competition for iron represents one important means of interaction between P. aeruginosa and Streptococcus spp.IMPORTANCE Cystic fibrosis (CF) lung infections are increasingly recognized for their polymicrobial nature. These polymicrobial infections may alter the biology of the organisms involved in CF-related infections, leading to changes in growth, virulence, and/or antibiotic tolerance, and could thereby affect patient health and response to treatment. In this study, we demonstrate interactions between P. aeruginosa and streptococci using a coculture model and show that one interaction between these microbes is likely competition for iron. Thus, these data indicate that one CF pathogen may influence the growth of another, and they add to our limited knowledge of polymicrobial interactions in the CF airway.

In vitro/in vivo evaluation of the efficacy of gatifloxacine-loaded PLGA and hydroxyapatite composite for treating osteomyelitis.

Molten 10 wt% gatifloxacine (GLFX-loaded poly(lactide-co-glycolide) (PLGA) was introduced into three-dimensionally interconnected pores and onto the surfaces of hydroxyapatite (HA) granules. The composite granules exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 3 h to 10 days. The composite granules were implanted in bone defects created by debridement of osteomyelitis lesions in rabbit mandibles. After 4-week implantation, inflammation in the composite granule-implanted group was significantly smaller than that in the debridement group (p<0.05). Moreover, newly formed bone was observed in the pores and on the surface of HA granules of the composite. These findings show that GFLX/HA composite controls bacterial infection and supports bone regeneration for osteomyelitis treatment.

Inflammatory index and treatment of brain abscess.

This study retrospectively analyzed 12 patients with brain abscesses. Half of the patients were diagnosed inaccurately in the initial stage, and 7.2 days were required to achieve the final diagnosis of brain abscess. The patients presented only with a moderately elevated leukocyte count, serum CRP levels, or body temperatures during the initial stage. These markers changed, first with an increase in the leukocyte count, followed by the CRP and body temperature. The degree of elevation tended to be less prominent, and the time for each inflammatory index to reach its maximum value tended to be longer in the patients without ventriculitis than in those with it. The causative organisms of a brain abscess were detected in 10 cases. The primary causative organisms from dental caries were Streptococcus viridians or milleri, and Fusobacterium nucleatum. Nocardia sp. or farcinica were common when the abscess was found in other regions. The primary causative organisms of unrecognized sources of infection were Streptococcus milleri and Prolionibacterium sp. Nocardia is resistant to many antibiotics. However, carbapenem, tetracycline and quinolone were effective for Nocardia as well as many other kinds of bacteria. In summary, the brain abscesses presented with only mildly elevated inflammatory markers of body temperature, leukocyte and CRP. These inflammatory markers were less obvious in the patients without ventriculitis and/or meningitis. The source of infection tended to suggest some specific primary causative organism. It was reasonable to initiate therapy with carbapenem.

Gatifloxacine-loaded PLGA and ß-tricalcium phosphate composite for treating osteomyelitis.

Gatifloxacine (GFLX)-containing poly(lactide-co-glycolide) (PLGA) was introduced to the pores and surfaces of porous ß-tricalcium phosphate (ßTCP) granules by melt compounding whereby no toxic solvent was used. The granular composite of GFLX-loaded PLGA and ßTCP released GFLX for 42 days in Hanks' balanced solution and exhibited sufficient in vitro bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 21 days. For in vivo evaluation, the granular composite was implanted in the dead space created by the debridement of osteomyelitis lesion induced by S. milleri and B. fragilis in rabbit mandible. After a 4-week implantation, the inflammation area within the debrided area was markedly reduced accompanied with osteoconduction and vascularization in half of the rabbits, and even disappeared in one of the six rabbits without any systemic administration of antibiotics. Outside the debrided area, inflammation and sequestrum were observed but the largest of such affected areas amounted to only 0.125 times of the originally infected and debrided area. These findings showed that the granular composite was effective for the local treatment of osteomyelitis as well as an osteoconductive scaffold which supported and encouraged vascularization.

Bacteriologic epidemiology and empirical treatment of pediatric complicated appendicitis.

Preoperative samples in the context of complicated appendicitis (CA) are rarely collected, and there is no consensus regarding the optimal antibiotic therapy in children. To help optimize empirical preoperative treatment, we studied clinical and bacteriologic data from a prospective cohort of 93 children with CA in a French hospital. All the bacteria isolated from peritoneal fluids were identified, using phenotypic and/or molecular techniques. The most commonly recovered species were Escherichia coli (71%), Streptococcus group milleri (34%), anaerobes (20%), and Pseudomonas aeruginosa (19%). The association piperacillin-tazobactam is an accurate choice of empirical therapy as it is active against 97% of bacteria. A third-generation cephalosporin with metronidazole in association with an aminoglycoside is a good alternative. Although antibiotic use may be considered as an adjunct to surgical intervention of CA, the appropriate use of preoperative antibiotics is essential and must be constantly reevaluated according to the bacterial epidemiology.

Macrolide and clindamycin resistance in Streptococcus milleri group isolates from the airways of cystic fibrosis patients.

Organisms belonging to the Streptococcus milleri group (SMG) are known for their role in pyogenic infections but have recently been implicated as etiological agents of pulmonary exacerbation in adult patients with cystic fibrosis (CF). The prolonged exposure of CF patients to antibiotics prompted us to investigate the susceptibility profiles of 118 SMG isolates from the airways of CF patients to 12 antibiotics compared to 43 SMG isolates from patients with invasive infections. We found that approximately 60% of all isolates failed to grow using the standard medium for disc diffusion, Mueller-Hinton blood agar (MHBA), so we explored the usefulness of brain heart infusion (BHI) agar for susceptibility testing. Zone-of-inhibition comparisons between BHI and MHBA showed strong correlations for six antibiotics, and interpretations were similar for both medium types. For ceftriaxone and cefepime, both groups of isolates were highly susceptible. Tetracycline resistance levels were comparable between the two groups (22% in CF isolates and 17.4% in invasive isolates). However, more than half of the CF isolates were not susceptible to azithromycin, erythromycin, and clindamycin, compared to 11%, 13%, and 6.5% of invasive isolates, respectively. There were 5-fold and 8-fold increased risks of azithromycin and clindamycin resistance, respectively, for the isolates from the airways of CF patients relative to the invasive isolates. Macrolide resistance was strongly linked to chronic azithromycin therapy in CF patients. This study shows that BHI agar is a suitable alternative for antimicrobial susceptibility testing for the SMG and that SMG isolates from the airways of CF patients are more resistant to macrolides and clindamycin than strains isolated from patients with invasive infections.

Antimicrobial activity of dexamethasone and its combination with N-chlorotaurine.

OBJECTIVE: To investigate the antimicrobial effect of dexamethasone phosphate, the endogenous antiseptic N-chlorotaurine (NCT), and their combination on ear, nose, and throat microorganisms. DESIGN: In vitro study. SUBJECTS: Strains of Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus milleri, Aspergillus flavus, and Aspergillus fumigatus. INTERVENTIONS: Bacterial and fungal strains were cultured with 0.1% dexamethasone with and without a low (0.1%) or high (1%) concentration of NCT. The killing effects of dexamethasone, NCT, and the combination were monitored. RESULTS: Dexamethasone killed S. milleri and A. flavus after incubation times of 24 to 48 hours. The low concentration of NCT caused a 90% reduction of S. aureus and P. aeruginosa within 30 minutes and 99.9% reduction within 50 minutes. The high concentration of NCT reduced viable counts of S. aureus and P. aeruginosa to the detection limit within 10 minutes. The low-concentration combination (0.1% dexamethasone and 0.1% NCT) showed significant (P < .01) synergistic killing of S. aureus with 2- to 3-fold shorter killing times. The high-concentration combination (0.1% dexamethasone and 1% NCT) demonstrated more rapid killing than NCT alone in both S. aureus and P. aeruginosa. CONCLUSIONS: With short and intermediate exposure times, the combination of dexamethasone and NCT showed significantly stronger antimicrobial effects than treatment with NCT alone. Significant killing of S. milleri, A. flavus, and A. fumigatus was observed after extended exposure to dexamethasone. The combined application of dexamethasone and NCT might be a promising therapeutic option, producing high efficacy with low side effects.

In vitro susceptibility of the Streptococcus milleri group to antimicrobial peptides.

AIM: To determine the susceptibility of strains of the Streptococcus milleri group (SMG) to commercially available antimicrobial peptides. METHODOLOGY: Thirty strains of SMG from a range of sources were assessed for their susceptibility to 10 antimicrobial peptides of either human, animal or insect origin, using a double layer diffusion assay. RESULTS: The majority of the test strains were sensitive to the amidated peptides, mastoparan (100%; n = 30), magainin 2 amide (95%; n = 21) and indolicin (91%; n = 23). Some strains were susceptible to cecropin B (30%; n = 30) and histatin (10%; n = 30), whilst no activity was observed for the defensins HNP-1 and HNP-2, histatin 8, cecropin P1 and magainin 2. CONCLUSIONS: The majority of strains were resistant to the human derived peptides. The ability to resist such peptides may be a factor in the colonisation of the oral cavity and the survival and initiation of infection in the pulp and root canal environment. Interestingly, the present study indicated that amidated and alpha helical peptides exhibit antimicrobial activity against SMG. Structural modification of these peptides may allow a targeted approach for the development of these substances as preventative or therapeutic agents.

Antibacterial activity of mometasone furoate.

OBJECTIVE: To test the antibacterial properties of the topical corticoid mometasone furoate, which is used as a nasal spray. DESIGN: The activity of mometasone (0.01%, 0.1%, and 0.5%) in buffer solution against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pyogenes, and Streptococcus milleri was tested by quantitative killing assays. SETTING: In vitro study. MAIN OUTCOME MEASURE: Reduction of viable bacteria and fungi in quantitative killing assays. RESULTS: Mometasone (0.5%) reduced viable counts of S pyogenes and S milleri by 99.99% and 99.00%, respectively, after 24 hours of incubation, whereas colony-forming units (CFUs) of S aureus, P aeruginosa, and E coli were not affected by the corticoid. Mometasone (0.1%) caused a decrease in CFUs of S pyogenes of 99.90% to 99.99%, while it led to a 99.00% reduction in CFUs of S milleri, but only if low bacterial counts of 1 x 10(4) CFUs/mL were incubated. By contrast, the use of mometasone at a low concentration (0.01%) demonstrated an increase in CFUs of S milleri if the baseline bacterial count was low (1 x 10(4) CFUs/mL). CONCLUSION: Mometasone demonstrates antimicrobial activity against streptococci.

The Streptococcus milleri group--an unrecognized cause of disease in cystic fibrosis: a case series and literature review.

The "Streptococcus milleri" group (SMG) is increasingly recognized for their role in pyogenic infections including empyema and solid organ abscesses. However, SMG disease has rarely been identified in cystic fibrosis (CF). Inherent difficulties in both growing the organisms and distinguishing SMG from less virulent oropharyngeal viridans streptococci may have led to a decreased recognition of this as a CF pathogen. We report on six cases of SMG-related infection over a 4-year time-frame occurring within an adult CF clinic in Canada, and a further four cases identified through a literature review. SMG manifested disease as bronchopulmonary exacerbations in 7 of 10 patients, and 4 of 10 patients had extra-pulmonary dissemination of SMG infection. Noticeably, pulmonary exacerbations were frequently associated with atypically malodorous sputum. Furthermore, patients clinically responded to anti-microbial therapies with no anti-Pseudomonal activity. There was a consistent correlation of SMG disease and co-colonization with P. aeruginosa leading to speculation of polymicrobial interactions resulting in enhanced virulence. SMG deserves considerable attention as a potential pathogen within the airways of patients with CF.

Cervical necrotizing fasciitis: 8-years' experience of microbiology.

Cervical necrotizing fasciitis (CNF) is a life-threatening complication of pharyngeal or dental infections. The aim of this paper was to investigate whether dental or pharyngeal source result from different pathogen(s) in CNF and whether antibiotics, given before admission, influence the antimicrobial resistance of pathogens. In 152 CNF patients, Streptococcus milleri group and Prevotella species were the predominant isolates, frequently copathogens, mostly in dental CNF samples. Penicillin and clindamycin resistance were observed in 39% and 37% of cases, respectively, independently of any previous antibiotic therapy. Thus, a combined aerobe-anaerobe infection may have a synergistic effect, which allows the infection to spread in cervical tissues.

Complicated parapneumonic effusion and empyema thoracis: microbiology and predictors of adverse outcomes.

OBJECTIVES: To describe the microbiological characteristics of a cohort of patients with complicated parapneumonic effusion and empyema thoracis, and to identify the potential risk factors for adverse outcomes, with particular reference to the choice of empirical antibiotics, intrapleural fibrinolytics, adherence to management guidelines, and input from pulmonologists. DESIGN: Retrospective review. SETTING: Regional hospital, Hong Kong. PATIENTS: All patients with a diagnosis of complicated parapneumonic effusion/empyema thoracis admitted between January 2003 and June 2005. MAIN OUTCOME MEASURES: Microbiological characteristics, mortality, and surgery-free survival. RESULTS. There were 63 patients, with a mean age of 64 (standard deviation, 16) years and a male-to-female ratio of 45:18. The pleural fluid culture positivity rate was 68%; Streptococcus milleri (19%), Bacteroides (14%), Klebsiella pneumoniae (12%), and Peptostreptococcus (7%) were the most common organisms. Thirteen (21%) patients died during their index admission. Use of intrapleural fibrinolytics according to the guideline was associated with survival (P=0.001) while discordant initial antibiotic use was associated with mortality (P=0.002). Discordant initial antibiotic use was also independently associated with reduced surgery-free survival (P<0.001). Subgroup analysis showed that early intrapleural fibrinolytic use (within 4 days of diagnosis) was associated with decreased mortality (P<0.001), increased surgery-free survival (P=0.005), and shorter hospital stay (P=0.039). CONCLUSION: Organisms identified from complicated parapneumonic effusion and empyema thoracis differ from those giving rise to community-acquired pneumonia. In these patients, adherence to guidelines, early concordant antibiotic treatment, intrapleural fibrinolytics, and input from a pulmonologist were associated with improved outcomes.

Susceptibility of several macrolides and a ketolide against clinically isolated 'Streptococcus milleri' group.

The susceptibility of four macrolides (erythromycin, clarithromycin, roxithromycin and azithromycin) and a ketolide (telithromycin) was tested using 58 clinically isolated strains of the 'Streptococcus milleri' group (SMG). Among the 58 strains, 9 strains were determined to be resistant to erythromycin as well as other macrolides. Of the four macrolides and the ketolide, telithromycin was the most effective antibiotic against the SMG, including the erythromycin-resistant strains.

Management of patients with Streptococcus milleri brain abscesses.

OBJECTIVES: We evaluated the efficacy of cefotaxime in the management of brain abscesses caused by Streptococcus milleri. Twenty two patients with a S. milleri brain abscess were treated with metronidazole and cefotaxime, in accordance with recent recommendations by the British Society Of Antimicrobial Chemotherapy (BSAC). Seven patients who had Glasgow Coma Scales < or =11 also received rifampicin and high dose cefotaxime. The clinical response of the patients was determined. METHOD: A retrospective study at the Queen Elizabeth Hospital, Birmingham covering the period April 1996-March 2004 was carried out. Neurosurgical and anti-microbial therapeutic approaches were reviewed. Any evidence of improvement of clinical features and radiological disappearance of brain abscesses were determined. RESULTS: Outcome was assessed using the Glasgow Outcome Score (GOS) at 3 and 6 months from the time of surgical intervention. Eighteen patients (82%) had a good outcome by 6 months, with an outcome score of 4-5. Thirteen patients resumed normal life despite minor deficits (GOS 5), while a further five patients had moderate disability though remained independent (GOS 4). One patient had a GOS of 3 and there were three deaths (14). The minimum time to radiological resolution of the abscess was within 1 month in six cases (27) These all represented solitary lesions that required a single drainage procedure in conjunction with 4 weeks of intravenous cefotaxime and metronidazole. Ten cases (45%) had resolution within 4 months and a further three cases took at least 6 months from the time of surgery to show radiological clearance. CONCLUSIONS: This cohort of patients responded favourably to the guidelines recommended by the BSAC. This was confirmed by the Glasgow Outcome Score (GOS 4-5) at 6 months review. Cefotaxime at a higher dose with rifampicin was prescribed for patients presenting with a decreased conscious level (GCS 8-11), subsequent failure of anticipated clinical improvement or clinical deterioration. There was no clinically significant difference in GOS between the two treatment groups. An algorithm for management of brain abscess is presented, based on our clinical experience and review of the literature.

Intracranial suppuration.

OBJECTIVE: To evaluate prevalence, age, position, predisposing factors, bacteriology, clinical features and outcomes of children with subdural empyema (SDE) and brain abscess (BA). DESIGN: Retrospective hospital-based study in a tertiary children's hospital. METHODS: Clinical data were reviewed on all children classified as having SDE or BA for 10.75 years from 1 January 1992 to 31 August 2003 at the Royal Alexandra Hospital for Children, Sydney, Australia. RESULTS: Forty-six children with intracranial suppuration were identified: 26 had BA, 16 had SDE and four children had both SDE and BA. Significant differences between SDE and BA were that: sinusitis was a predisposing factor for SDE (P = 0.01), Streptococcus milleri was the main organism isolated in SDE (P = 0.02), periorbital oedema (P = 0.005) and photophobia (P = 0.02) were clinical features specifically associated with SDE, and 75% of multiple abscesses were in females (P = 0.005). The age distribution of SDE was biphasic, with peaks at <2 years and >7 years. Cases of BA peaked at age 9-11 years. Forty-eight per cent of all children were between 9 and 13 years old; 20% were <1 year old. All the children with SDE and BA were aged 1 year or less. Three of the 46 children died, all with BA. Eighteen (39.1%) returned to normal and 25 (54.3%) had neurological complications. Neurological complications were more common in the BA group. CONCLUSION: The mortality rate of intracranial suppuration is low, but morbidity remains high. A high degree of suspicion is needed to diagnose and treat intracranial infections early.

Effect of saliva on an antimicrobial tissue conditioner containing silver-zeolite.

The purpose of this study was to evaluate the influence of human saliva on the antimicrobial effect of a tissue conditioner containing an antibiotic agent, silver-zeolite. Samples of each tissue conditioner with or without silver-zeolite were prepared and a plastic disk was used as a control. Candida albicans and nosocomial respiratory infection-causing bacteria, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and the Streptococcus milleri group (S. constellatus and S. intermedius), were selected as test microorganisms. Antimicrobial effects of samples after water or saliva immersion for 28 days were evaluated by counting the number of viable cells [colony forming unit (CFU)] in each microbial suspension (100 microL). All data were statistically analysed by one-way anova and Bonferroni's test (P < 0.05). The antimicrobial effects of samples with silver-zeolite immersed in saliva against C. albicans, S. aureus and MRSA were observed while CFU of P. aeruginosa indicated no significant difference from that of the control. As for the S. milleri group, its CFU after saliva immersion showed the significantly smaller value than that of the control. It is concluded that the antimicrobial effects of samples containing silver-zeolite against all tested microbes except for P. aeruginosa and the S. milleri group are not influenced by saliva immersion for 28 days.

Apical periodontitis development and bacterial response to endodontic treatment. Experimental root canal infections in monkeys with selected bacterial strains.

In six monkeys, 160 root canals were inoculated with a combination of four bacterial strains belonging to species Streptococcus milleri, Peptostreptococcus anaerobius, Prevotella oralis, and Fusobacterium nucleatum. In two other monkeys, 24 root canals were inoculated with a five-strain combination consisting of these strains and a strain of Enterococcus faecalis. All strains were previously isolated from an infected monkey root canal. After 8-12 months, survival of the strains was recorded bacteriologically, and the reaction in the periapical region was radiographed. From 180 of 184 root canals, one or more of the bacterial strains were reisolated. The two facultative strains were more frequently reisolated than the anaerobic strains. Apical periodontitis was registered in the periapical region of more than 96% of root canals with reisolated bacteria but in none of those without reisolated bacteria. Endodontic treatment was carried out in two sessions with an interval of 14 d without interappointment dressings, and the effect was evaluated bacteriologically before and after each treatment. The chemo-mechanical treatment reduced significantly the number of strains and bacterial cells. The facultative bacteria were more resistant to the treatment than the anaerobic bacteria. The five-strain combination had a higher survival rate than the four-strain combination.