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

Tetracycline and multidrug resistance in the oral microbiota : differences between healthy subjects and patients with periodontitis in Spain

Introduction : Antibiotic resistance is widely found even among bacterial populations not having been exposed to selective pressure by antibiotics, such as tetracycline. In this study we analyzed the tetracycline-resistant subgingival microbiota of healthy subjects and of patients with periodontitis, comparing the prevalence of tet genes and their multidrug resistance profiles. Methods : Samples from 259 volunteers were analyzed, obtaining 813 tetracycline-resistant isolates. The prevalence of 12 antibiotic resistance genes was assessed, and multidrug profiles were built. Each isolate was identified by 16S rRNA sequencing. Differences in qualitative data and quantitative data were evaluated using the chi-square test and the Mann-Whitney-U test, respectively. Results : tet (M) was the most frequently detected tet gene (52.03%). We observed significant differences between the prevalence of tet (M), tet (W), tet (O), tet (32) and tet (L) in both populations studied. Multidrug resistance was largely observed, with resistance to kanamycin being the most detected (83.64%). There were significant differences between the populations in the prevalence of kanamycin, chloramphenicol, and cefotaxime resistance. Resistant isolates showed significantly different prevalence between the two studied groups. Conclusion : The high prevalence of multidrug resistance and tetracycline resistance genes found in the subgingival microbiota, highlights the importance of performing wider and more in-depth analysis of antibiotic resistance in the oral microbiota

A two-directional approach to pyrrolizidines: total syntheses and biological evaluation of alkaloid cis-223B and (+/-)-xenovenine

Total syntheses of alkaloid cis-223B and xenovenine are reported in 3 and 4 steps respectively using a two-directional synthesis/triple reductive amination strategy, and their neurotoxic properties assessed

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

peer reviewed[en] 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

Prevalencia y caracterización de genes de resistencia a antibióticos en bacterias orales

En aquesta tesi es va estudiar la resistència de la microbiota oral a 3 de les classes d'antibiòtics més utilitzades en la pràctica dental: les tetraciclines, els macròlids i els β-lactàmics. En primer lloc, es va analitzar la resistència a tetraciclina de la microbiota subgingival d'individus sense periodontitis, per tal de conèixer quin era l'abast de la resistència a un antibiòtic de molt baixa exposició entre la població actual. A aquest treball el va seguir una rèplica en la que es va estudiar la microbiota subgingival d'individus amb periodontitis. En ambdós tipus de mostres es va fer un screening de gens de resistència a tetraciclina i a altres gens que podien indicar la presència d'elements genètics mòbils. Ambdós estudis van mostrar alts nivells de microorganismes resistents a tetraciclina a les mostres obtingudes, i van ser comparades entre elles al tercer estudi, on es van observar diferències entre la microbiota obtinguda i la prevalencia dels gens de resistència en les dues poblacions. La transferència genètica horitzontal a l'ambient oral va ser investigada al quart estudi, en el qual es va demostrar que el gen de resistencia a tetraciclina tet(B), el qual no s'havia descrit abans en grampositius, s'havia integrat al genoma de dos aïllats d'Streptococcus oralis. La resistència a macròlids va ser analitzada al cinqué estudi, en el que es va investigar la susceptibilitat de bacteris del gènere Prevotella a eritromicina i azitromicina, sent el segon un macròlid l'ús del qual ha anat creixent degut a la seva eficacia demostrada i als seus interessants efectes beneficiosos. Per conèixer si la resistència a macròlids en aïllats de Prevotella estava asociada a la presència de gens de resistència a macròlids, es va fer un screening per a aquests gens, així com un anàlisi estadístic que va confirmar l'increment de resistència a macròlids en presència del gen erm(F). Per últim, al sisé estudi es va analitzar la resistència a β-lactàmics, la classe d'antibiòtics més utilitzada en la pràctica dental, en mostres obtingudes de pacients amb periodontitis. La resistència a amoxicilina i cefotaxima, els dos β-lactàmics estudiats, va ser trobada a la majoria de les mostres analitzades juntament amb gens que codifiquen per a β-lactamases i β-lactamases d'espectre estès.En esta tesis se estudió la resistencia de la microbiota oral a 3 de las clases de antibióticos más utilizadas en la práctica dental: las tetraciclinas, los macrólidos y los β-lactámicos. En primer lugar, se analizó la resistencia a tetraciclina de la microbiota subgingival de individuos sin periodontitis para conocer cual era el alcance de la resistencia a un antibiótico de muy baja exposición actualmente. A este estudio le siguió una réplica en la que se trabajó con la microbiota subgingival de individuos con periodontitis. En ambos tipos de muestras se hizo un screening de genes de resistencia a tetraciclina y a otros genes que podían indicar la presencia de elementos genéticos móviles. Ambos estudios mostraron altos niveles de microorganismos resistentes a tetraciclina en las muestras obtenidas, y fueron comparados entre sí en el tercer estudio, donde se observaron diferencias en la microbiota obtenida y la prevalencia de los genes de resistencia en una y otra población. La transferencia genética horizontal en el ambiente oral fue estudiada en el cuarto estudio, en el que se demostró que el gen de resistencia a tetraciclina tet(B), nunca antes descrito en grampositivas, se había integrado en el genoma de dos aislados de la especie Streptococcus oralis. La resistencia a macrólidos fue analizada en el quinto estudio, en el que se investigó la susceptibilidad de bacterias del género Prevotella a eritromicina y azitromicina, siendo el segundo un macrólido cuyo uso ha ido creciendo debido a su eficacia demostrada y sus efectos beneficiosos. Para conocer si la resistencia a macrólidos en aislados de Prevotella estaba asociada a la presencia de genes de resistencia a macrólidos, se hizo un screening para dichos genes, así como un análisis estadístico que confirmó el incremento de resistencia a macrólidos en presencia del gen erm(F). Por último, en el sexto estudio se analizó la resistencia a β-lactámicos, la clase de antibióticos más utilizada en la práctica dental, en muestras obtenidas de pacientes con periodontitis. La resistencia a amoxicilina y a cefotaxima, los dos β-lactámicos estudiados, fue encontrada en la mayoría de las muestras analizadas junto con genes que codifican para β-lactamasas y β-lactamasas de espectro extendido.The oral environment is widely colonised by bacteria, which grow in a multispecies biofilm structure that confers many benefitial properties to the microorganisms, including better resistance to mechanical and chemical stress and a greater comunication and genetic exchange among them. The undisturbed growth of the oral biofilm can lead to certain pathologies such as caries, gingivitis or periodontitis. Antibiotics can be necessary as adjuvants in the treatment of these pathologies. However, antimicrobials treatments could fail in the presence of genetic determinants that confer resistance to the antimicrobial given. Resistance to antibiotics is a growing problem of modern medicine. The use of antibiotics is widespread and they are essential for the treatment of many infectious diseases. International organizations, such as the World Health Organization and the European Centre for Disease Prevention and Control, have underlined the importance of surveillance of antibiotic resistance and their causes as an essential mesure to avoid its propagation and to design strategies to fight them. In this thesis, we studied the resistance of the oral microbiota to the 3 main classes of antibiotics used in the dental practice: tetracyclines, macrolides and β-lactams. In the first place, we analised tetracycline resistance in the subgingival microbiota of healthy individuals without periodontitis in order to know which was the extent of the resistance to an antibiotic with a low level of exposition among the current population. A replica to this study followed, in which subgingival microbiota of patients with periodontitis was used. Screening of tetracycline resistance genes and other genes, that could indicate the presence of mobile genetic elements, was performed in both types of samples. Both studies showed high numbers of microorganisms resistant to tetracycline, and both were compared between them in a third study, where differences were observed in the microbiota obtained and the prevalence of the resistance genes. Horizontal genetic transfer in the oral environment was investigated in the fourth study, in which the integration of the tetracycline resistance gene tet(B), that has never before been described in grampositive bacteria, in the genome of two Streptococcus oralis isolates was proven. Resistance to macrolides was analised in the fifth study, in which the susceptibility of bacteria of the Prevotella genus against erythromycin and azythromycin, being the latter a macrolide which use has been growing due to its proven effectiveness and its interesting beneficial effects, was also tested. In order to know if resistance to macrolides in Prevotella isolates could be related to the presence of macrolide resistance genes, an screening for such genes was performed, together with a statistical analysis which confirmed the increase of macrolide resistance in the presence of the gen erm(F). Finally, resistance to β-lactams, the most used class of antibiotics in the dental practice, was analised in the sixth study in samples obtained from patients with periodontitis. Resistance to amoxicilin and cefotaxime, the two β-lactams studied, was found in most of the analised samples together with genes that code for β-lactamases and Extended Spectrum β-lactamases

Whole Genome Sequencing and Phenotypic Analysis of Antibiotic Resistance in <i>Filifactor alocis</i> Isolates

There is scarce knowledge regarding the antimicrobial resistance profile of F. alocis. Therefore, the objective of this research was to assess antimicrobial resistance in recently obtained F. alocis clinical isolates and to identify the presence of antimicrobial resistance genes. Isolates were obtained from patients with periodontal or peri-implant diseases and confirmed by sequencing their 16S rRNA gene. Confirmed isolates had their genome sequenced by whole genome sequencing and their phenotypical resistance to nine antibiotics (amoxicillin clavulanate, amoxicillin, azithromycin, clindamycin, ciprofloxacin, doxycycline, minocycline, metronidazole, and tetracycline) tested by E-test strips. Antimicrobial resistance genes were detected in six of the eight isolates analyzed, of which five carried tet(32) and one erm(B). Overall, susceptibility to the nine antibiotics tested was high except for azithromycin in the isolate that carried erm(B). Moreover, susceptibility to tetracycline, doxycycline, and minocycline was lower in those isolates that carried tet(32). The genetic surroundings of the detected genes suggested their inclusion in mobile genetic elements that might be transferrable to other bacteria. These findings suggest that, despite showing high susceptibility to several antibiotics, F. alocis might obtain new antimicrobial resistance traits due to its acceptance of mobile genetic elements with antibiotic resistance genes in their genome

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

Elaboration of an aluminum oxide thin film deposited by magnetron sputtering on boron nitride substrates: role of the physical and chemical properties of BN substrates

International audienceBoron nitride is a non-oxide ceramic exhibiting excellent thermal and chemical stability. This compound exists in various crystallographic structures, the most common being hexagonal (h-BN) and cubic (c-BN). The surface of this material can be protected from wear by a hard-ceramic compound like aluminum oxide.In this study, aluminum oxide films were deposited on boron nitride substrates by magnetron sputtering. The aim of this work is to evaluate the quality of the interface between these two materials. To do so, the first step was to control the process parameters like the target power, the working pressure and the substrate bias, in order to determine their influence on the deposition rate, on the thin film structure, and on the chemical composition.The use of the two different substrates (h- and c-BN) has also allowed studying the role of the physical and chemical properties of BN substrates on the adhesion of the alumina thin film. The roughness and the surface state of the substrates have been characterized by XPS, optical profilometry, XRD and SEM. It was found that the roughness average of the surface is around 1.48 μm ± 0.12 μm and the elements detected by XPS were boron (37.6 ± 1.3 at.%), nitrogen (47.2 ± 0.9 at.%), oxygen (4.9 ± 0.2 at.%) and carbon (10.3 ± 2.0 at.%).Finally, the role of an annealing treatment on the structural properties of the alumina coating and on its adherence has been investigated. This heat treatment has been performed at 1200°C in vacuum. After annealing, the Al2O3 – BN system has been evaluated using XRD, SEM, XPS, TEM. The interface between the BN substrate and the Al2O3 film was observed by TEM. The corresponding cross section of the h-BN – Al2O3 system clearly shows that there is no interface formation between the substrate and the coating