Resistance to ß-lactams and distribution of ß-lactam resistance genes in subgingival microbiota from Spanish patients with periodontitis.

OBJECTIVES: The aim of this study was to analyze the distribution of ß-lactamase genes and the multidrug resistance profiles in ß-lactam-resistant subgingival bacteria from patients with periodontitis. MATERIALS AND METHODS: Subgingival samples were obtained from 130 Spanish patients with generalized periodontitis stage III or IV. Samples were grown on agar plates with amoxicillin or cefotaxime and incubated in anaerobic and microaerophilic conditions. Isolates were identified to the species level by the sequencing of their 16S rRNA gene. A screening for the following ß-lactamase genes was performed by the polymerase chain reaction (PCR) technique: blaTEM, blaSHV, blaCTX-M, blaCfxA, blaCepA, blaCblA, and blaampC. Additionally, multidrug resistance to tetracycline, chloramphenicol, streptomycin, erythromycin, and kanamycin was assessed, growing the isolates on agar plates with breakpoint concentrations of each antimicrobial. RESULTS: ß-lactam-resistant isolates were found in 83% of the patients. Seven hundred and thirty-seven isolates from 35 different genera were obtained, with Prevotella and Streptococcus being the most identified genera. blaCfxA was the gene most detected, being observed in 24.8% of the isolates, followed by blaTEM (12.9%). Most of the isolates (81.3%) were multidrug-resistant. CONCLUSIONS: This study shows that ß-lactam resistance is widespread among Spanish patients with periodontitis. Furthermore, it suggests that the subgingival commensal microbiota might be a reservoir of multidrug resistance and ß-lactamase genes. CLINICAL RELEVANCE: Most of the samples yielded ß-lactam-resistant isolates, and 4 different groups of bla genes were detected among the isolates. Most of the isolates were also multidrug-resistant. The results show that, although ß-lactams may still be effective, their future might be hindered by the presence of ß-lactam-resistant bacteria and the presence of transferable bla genes.

The adjunctive effect of a titanium brush in implant surface decontamination at peri-implantitis surgical regenerative interventions: A randomized controlled clinical trial.

AIM: To evaluate an additional mechanical approach, a titanium brush, in the implant surface decontamination performed during the regenerative surgical therapy of peri-implantitis. MATERIAL AND METHODS: A randomized double-blinded clinical trial, with a 1-year follow-up, was carried out. After a hygienic phase, peri-implantitis-affected implants were randomly assigned to a control or to a test group. In the control group, implant surface was decontaminated both mechanically and chemically with 3% H2 O2 and plastic ultrasonic scalers, respectively, while in the test group, a titanium brush was also applied. Intrabony defects in both groups were filled with an alloplastic material (ß-tricalcium phosphate and hydroxyapatite) and covered with a collagen membrane. The primary outcome was the reduction in probing pocket depth (PPD) at the deepest site. RESULTS: Thirty patients were included, 15 in each group. At 12 months, reduction in PPD was 4.87 (standard deviation [SD] 1.55) mm and 2.85 (SD: 1.91) mm, respectively (p = 0.009). The correspondent figures for residual PPD were 3.6 (SD: 0.91) and 4.92 (SD: 1.26) mm, respectively (p = 0.007). CONCLUSIONS: The additional use of a titanium brush during regenerative treatment of peri-implantitis resulted in statistically significant benefits in terms of PPD reduction after 12 months.

Azithromycin and erythromycin susceptibility and macrolide resistance genes in Prevotella from patients with periodontal disease.

OBJECTIVES: To study oral Prevotella spp. isolated from patients with chronic periodontitis, to determine their susceptibility to azithromycin and erythromycin and to screen the presence of macrolide resistance genes therein. MATERIAL AND METHODS: Isolates with a Prevotella-like morphology were obtained from subgingival samples of 52 patients with chronic periodontitis. Each isolate was identified to the species level by sequencing of the 16S rRNA gene. In 100 Prevotella spp. isolates, azithromycin and erythromycin susceptibility was determined using the E test method, and the screening of erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q) and mef(A) genes was done by PCR. RESULTS: Prevotella intermedia and Prevotella nigrescens were the most identified species (33% each). Minimum inhibitory concentrations (MICs) ranges for both antibiotics were 0.016/0.032 to >256 µg/ml. MIC50 values for azithromycin and erythromycin were 1.5 and 1 µg/ml, respectively, and MIC90 values were >256 µg/ml for both antibiotics. Nineteen per cent of the isolates carried erm(B), and 51% carried erm(F). CONCLUSIONS: The MIC values found were high compared to previous studies. erm(F) was greatly prevalent, and we describe for the first time the erm(B) gene in Prevotella spp. The presence of either of the genes seems to be associated with a higher degree of resistance to azithromycin and erythromycin.

Association of nine pathobionts with periodontitis in four South American and European countries.

Aim: Our aim was to compare the prevalence and load of nine pathobionts in subgingival samples of healthy individuals and periodontitis patients from four different countries. Methods: Five hundred and seven subgingival biofilm samples were collected from healthy subjects and periodontitis patients in Belgium, Chile, Peru and Spain. The prevalence and load of Eubacterium brachy, Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas endodontalis, Porphyromonas gingivalis, Selenomonas sputigena, Treponema denticola, Tannerella forsythia and Treponema socranskii were measured by quantitative PCR. Results: The association with periodontitis of all species, except for T. socranskii, was confirmed in all countries but Peru, where only P. endodontalis, P. gingivalis and T. denticola were found to be significantly associated. Moreover, most species showed higher loads at greater CAL and PPD, but not where there was BOP. Through Principal Component Analysis, samples showed clearly different distributions by diagnosis, despite observing a smaller separation in Peruvian samples. Conclusions: Unlike prevalence, relative load was found to be a reliable variable to discriminate the association of the species with periodontitis. Based on this, F. alocis, P. endodontalis, P. gingivalis, T. denticola and T. forsythia may be biomarkers of disease in Belgium, Chile and Spain, due to their significantly higher abundance in periodontitis patients.

Detection and expression analysis of tet(B) in Streptococcus oralis.

Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.