A Europe-wide assessment of antibiotic resistance rates in Bacteroides and Parabacteroides isolates from intestinal microbiota of healthy subjects.

Here, we sought to assess the levels of antibiotic resistance among intestinal Bacteroides and Parabacteroides strains collected between 2014 and 2016 in Europe and also attempted to compare resistance levels between clinical and commensal isolates. Bacteroides and Parabacteroides isolates were recovered from faecal samples via the novel Bacteroides Chromogenic Agar (BCA) method. Antibiotic susceptibilities were determined by agar dilution for ten antibiotics. The values obtained were then statistically evaluated. Altogether 202 Bacteroides/Parabacteroides isolates (of which 24, 11.9%, were B. fragilis) were isolated from the faecal specimens of individuals taken from five European countries. The percentage values of isolates resistant to ampicillin, amoxicillin/clavulanate, cefoxitin, imipenem, clindamycin, moxifloxacin, metronidazole, tetracycline, tigecycline and chloramphenicol were 96.6, 4.5, 14.9, 2.0, 47.3, 11.4, 0, 66.2, 1.5 and 0%, respectively. These values are close to those reported in the previous European clinical Bacteroides antibiotic susceptibility survey except for amoxicillin/clavulanate and clindamycin, where the former was lower and the latter was higher in normal microbiota isolates. To account for these latter findings and to assess temporal effects we compared the data specific for Hungary for the same period (2014-2016), and we found differences in the resistance rates for cefoxitin, moxifloxacin and tetracycline.

Clinical Pharmacokinetics of Levornidazole in Elderly Subjects and Dosing Regimen Evaluation Using Pharmacokinetic/Pharmacodynamic Analysis.

PURPOSE: Levornidazole, the levo-isomer of ornidazole, is a third-generation nitroimidazole derivative newly developed after metronidazole, tinidazole, and ornidazole. An open-label, parallel-controlled, single-dose study was conducted for the investigation of the pharmacokinetic (PK) profile of levornidazole and its metabolites in healthy elderly Chinese subjects, and for the evaluation of 2 dosing regimens in the elderly. METHODS: Levornidazole was intravenously administered at 500 mg to healthy elderly (aged 60-80 years) or young subjects (aged 19-45 years). The PK profiles of levornidazole and its metabolites in elderly subjects were evaluated and compared with those in the young group. WinNonlin software was used for simulating the PK profile of levornidazole in the elderly population following the dosing regimens of 500 mg BID and 750 mg once daily for 7 days. Monte Carlo simulation was used for estimating the cumulative fraction of response and probability of target attainment of both dosing regimens against Bacteroides spp. RESULTS: The Cmax, AUC0-24, and AUC0-∞ values of levornidazole in the elderly group were 11.98 µg/mL, 131.36 µg·h/mL, and 173.61 µg·h/mL, respectively. The t1/2, CLt, and mean residence time from time 0 to infinity were 12.21 hours, 2.91 L/h, and 16.46 hours. The metabolic ratios of metabolites (M) 1, 2, 4, and 6 were <3.0%, and that of M16 was 17.70%. The urinary excretion values of levornidazole, M1, M2, M4, M6, and M16 over 96 hours were 10.21%, 0.92%, ~0%, 2.69%, 0.54%, and 41.98%. The PK properties of levornidazole and the urinary excretion of all metabolites were not statistically different between the 2 groups. The cumulative fraction of response was >90% against B fragilis and other Bacteroides spp, and the probability of target attainment was >90% when the minimum inhibitory concentration was ≤1 µg/mL, in both groups. IMPLICATIONS: No dosing regimen adjustment is suggested when levornidazole is used in elderly patients with normal hepatic functioning and mild renal dysfunction. The findings from the PK/PD analysis imply that both regimens may achieve satisfactory clinical and microbiological efficacy against anaerobic infections in elderly patients. Chinese Clinical Trial Registry (http://www.chictr.org.cn) identifier: ChiCTR-OPC-16007938.

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction.

BACKGROUND AND OBJECTIVE: Molecular biological methods for the detection of periodontitis-associated bacteria based on DNA amplification have many advantages over classical culture techniques. However, when it comes to assessing immediate therapeutic success, e.g. reduction of viable bacteria, DNA-based polymerase chain reaction is unsuitable because it does not distinguish between live and dead bacteria. Our objective was to establish a simple RNA-based method that is easily set up and allows reliable assessment of the live bacterial load. MATERIAL AND METHODS: We compared conventional quantitative real-time PCR (qPCR), propidium monoazide-qPCR and reverse transcription qPCR (RT-qPCR) for the detection of periodontal pathogens after antibiotic treatment in vitro. Applicability was tested using clinical samples of subgingival plaque obtained from patients at different treatment stages. RESULTS: The bacterial load was remarkably stable over prolonged periods when assessed by conventional qPCR, while both propidium monoazide intercalation as well as cDNA quantitation showed a decline according to decreasing numbers of viable bacteria after antibiotic treatment. Clinical samples of subgingival plaque were directly subjected to DNase I treatment and RT without previous extraction or purification steps. While the results of the DNA- and RNA-based methods are comparable in untreated patients, the classical qPCR frequently detected substantial bacterial load in treated patients where RT-qPCR no longer indicates the presence of those pathogens. The disagreement rates ranged between 4 and 20% in first visit patients and 8-50% in the group of currently treated patients. CONCLUSION: We propose to use RNA-based detection methods to verify the successful eradication of periodontal pathogens.

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste.

AIM: To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. DESIGN: Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. RESULTS: Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P <0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). CONCLUSION: Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

The clinical relevance of microbiologic testing: a comparative analysis of microbiologic samples secured from the same sites and cultured in two independent laboratories.

A field study using five different private periodontal practices was conducted; it compared two microbiologic culture samples simultaneously secured from the same sites within 23 individual patients and submitted for bacterial identification and antibiotic sensitivity testing to two separate laboratories. The results from the two laboratories were often different. In no instance did both laboratories agree on the presence of identical bacterial species. When only bacteria above threshold levels were compared, agreement was found in only nine of 23 cases. When examining antibiotic sensitivity, using 100% kill of all tested pathogens as the ideal, agreement between the two laboratories was poor. The laboratories agreed on the use of amoxicillin 17% of the time, tetracycline 26% of the time, and metronidazole 48% of the time. The use of amoxicillin and metronidazole in combination yielded a 78% agreement when the results of both laboratories were combined. It would appear from the data that the empirical use of amoxicillin-metronidazole combination therapy may be more clinically sound and cost effective than culturing and antibiotic selection based on the results of culture from any single microbiologic testing laboratory.

Microbiological assessment of an improved stannous fluoride dentifrice.

Recently, a new stabilized stannous fluoride (SnF2) dentifrice (SSF) has been developed. The aim of the present work was to examine the antimicrobial activity of SnF2, and to assess the long-term microbial safety of this dentifrice in a series of in vitro and clinical evaluations. Results of in vitro time-kill experiments with representative oral bacteria demonstrated that SnF2 exerts broad antimicrobial activity against both Gram-positives and Gram-negatives and, in particular, has potent activity against Streptococcus mutans. Sixty-eight subjects participated in a nine-day plaque regrowth clinical study to assess the short-term antiplaque effect of SSF. The results revealed no significant differences from the negative control, suggesting that SnF2 does not detectably or directly alter plaque microbial viability or composition. Separately, evaluation of microbial safety in a subgroup of 120 subjects participating in a six-month clinical efficacy and safety trial found no significant ecological shifts between SSF and the negative (NaF dentifrice) control among 11 supragingival microbial populations examined. The potential for development of bacterial resistance to SnF2 was assessed under both in vitro and clinical conditions. In a rigorous assessment of the ability of bacterial populations to develop either phenotypic or genotypic resistance to SnF2, representative bacteria were exposed to continuous sub-lethal concentrations of SnF2 in a laboratory chemostat for at least 9 days. Results of time-kill experiments on exposed populations revealed no significant changes in susceptibility despite exposure of over 10(12) bacteria. Based on typical spontaneous mutation rates of 10(-6) to 10(-8), these results suggested that the potential for bacteria to develop resistance to SnF2 is low. Evaluation of susceptibility to SnF2 to over 800 bacterial isolates obtained over the course of the six-month clinical trial corroborate the in vitro findings, revealing no changes in susceptibility suggestive of development of resistance to SnF2 is a microbiologically safe agent for oral use and support separate clinical observations demonstrating the safety and efficacy of this stabilized SnF2 dentifrice.