Increased ompW and ompA expression and higher virulence of Acinetobacter baumannii persister cells.

BACKGROUND: Acinetobacter baumannii is one of the main causes of healthcare-associated infections that threaten public health, and carbapenems, such as meropenem, have been a therapeutic option for these infections. Therapeutic failure is mainly due to the antimicrobial resistance of A. baumannii, as well as the presence of persister cells. Persisters constitute a fraction of the bacterial population that present a transient phenotype capable of tolerating supra-lethal concentrations of antibiotics. Some proteins have been suggested to be involved in the onset and/or maintenance of this phenotype. Thus, we investigated the mRNA levels of the adeB (AdeABC efflux pump component), ompA, and ompW (outer membrane proteins) in A. baumannii cells before and after exposure to meropenem. RESULTS: We found a significant increase (p-value < 0.05) in the expression of ompA (> 5.5-fold) and ompW (> 10.5-fold) in persisters. However, adeB did not show significantly different expression levels when comparing treated and untreated cells. Therefore, we suggest that these outer membrane proteins, especially OmpW, could be part of the mechanism of A. baumannii persisters to deal with the presence of high doses of meropenem. We also observed in the Galleria mellonella larvae model that persister cells are more virulent than regular ones, as evidenced by their LD50 values. CONCLUSIONS: Taken together, these data contribute to the understanding of the phenotypic features of A. baumannii persisters and their relation to virulence, as well as highlight OmpW and OmpA as potential targets for drug development against A. baumannii persisters.

Copper-coated hospital surfaces: reduction of total bacterial loads and resistant Acinetobacter spp.

Healthcare-associated infections (HAIs) represent a global challenge and an even more staggering concern when related to microorganisms capable of resisting and surviving for long periods in the environment, such as Acinetobacter spp. Strategies that allow a reduction of pathogens from hospital environments represent an additional barrier in infection control protocols, minimizing transmission to hospitalized patients. Considering the antimicrobial properties of copper, here, the bacterial load and the presence of Acinetobacter spp. were monitored on high handling surfaces covered by 99.9% copper films on intensive and non-intensive care unit bedrooms in a tertiary care hospital. Firstly, copper-coated films were able to inhibit the adhesion and biofilm formation of A. baumannii strains in in vitro assays. On the other hand, Acinetobacter spp. were isolated from both copper-coated and uncoated surfaces in the hospital, although the majority was detected on surfaces without copper. All carbapenem-resistant A. baumannii isolates identified harbored the blaoxa-23 gene, while the A. nosocomialis isolates were susceptible to most antimicrobials tested. All isolates were susceptible to polymyxin B. Regarding the total aerobic bacteria, surfaces with copper-coated films presented lower total loads than those detected for controls. Copper coating films may be a workable strategy to mitigate HAIs, given their potential in reducing bacterial loads in nosocomial environments, including threatening pathogens like A. baumannii.

Antitubercular Activity of Novel 2-(Quinoline-4-yloxy)acetamides with Improved Drug-Like Properties.

Using cycloalkyl and electron-donating groups to decrease the carbonyl electrophilicity, a novel series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Structure-activity relationship studies led to selective and potent antitubercular agents with minimum inhibitory concentrations in the submicromolar range against drug-sensitive and drug-resistant Mtb strains. An evaluation of the activity of the lead compounds against a spontaneous qcrB mutant strain indicated that the structures targeted the cytochrome bc 1 complex. In addition, selected molecules inhibited Mtb growth in a macrophage model of tuberculosis infection. Furthermore, the leading compound was chemically stable depending on the context and showed good kinetic solubility, high permeability, and a low rate of in vitro metabolism. Finally, the pharmacokinetic profile of the compound was assessed after oral administration to mice. To the best of our knowledge, for the first time, a 2-(quinoline-4-yloxy)acetamide was obtained with a sufficient exposure, which may enable in vivo effectiveness and its further development as an antituberculosis drug candidate.

Osteoblastic Cell Behavior and Early Bacterial Adhesion on Macro-, Micro-, and Nanostructured Titanium Surfaces for Biomedical Implant Applications.

PURPOSE: Surface treatments may significantly affect physical-chemical properties and surface biologic responses. This study aimed to investigate the influence of alterations in the physical-chemical properties of pure titanium with different surface topographies on biocompatibility and early microbiologic response. MATERIALS AND METHODS: Titanium disks were exposed to five different surface treatments created through acid etching and anodizing methods. Surface morphology, 2D and 3D roughness, wettability, biocompatibility, and cell viability were evaluated. Osteoblast adhesion and bacterial adhesion tests were also executed. Data were statistically analyzed using analysis of variance followed by Tukey test, roughness (P < .05), and bacterial proliferation (P < .05). RESULTS: Five different surface morphologies were developed; double acid etching was shown to be significantly rougher than the others. The 2D roughness measurements were shown to be less consistent than the 3D measurements. All surfaces presented biocompatibility to allow cell behavior and differentiation. Osteoblasts presented better evolution in terms of adhesion and behavior in the nanomorphologies. High roughness significantly increased bacterial adhesion. CONCLUSION: Surface treatments may critically alter titanium properties and morphology. Therefore, roughness measurements with a wide area should be used in their evaluation. Nanotextured surfaces show a positive effect on bone cells and antibacterial response; their application is suggested when considering surface texturization for biomedical implants.

Antibacterial potential associated with drug-delivery built TiO2 nanotubes in biomedical implants.

The fast evolution of surface treatments for biomedical implants and the concern with their contact with cells and microorganisms at early phases of bone healing has boosted the development of surface topographies presenting drug delivery potential for, among other features, bacterial growth inhibition without impairing cell adhesion. A diverse set of metal ions and nanoparticles (NPs) present antibacterial properties of their own, which can be applied to improve the implant local response to contamination. Considering the promising combination of nanostructured surfaces with antibacterial materials, this critical review describes a variety of antibacterial effects attributed to specific metals, ions and their combinations. Also, it explains the TiO2 nanotubes (TNTs) surface creation, in which the possibility of aggregation of an active drug delivery system is applicable. Also, we discuss the pertinent literature related to the state of the art of drug incorporation of NPs with antibacterial properties inside TNTs, along with the promising future perspectives of in situ drug delivery systems aggregated to biomedical implants.

Bacteriophages in Dentistry-State of the Art and Perspectives.

Bacteriophages, viruses capable of killing bacteria, were discovered in 1915, but the interest in their study has been limited since the advent of antibiotics. Their use in dentistry is still very limited. The authors reviewed studies about bacteriophage structure, mode of action, uses in oral health, and possible future uses in dentistry associated with their possible action over biofilm, as well as the advantages and limitations of phage therapy.

The topical effect of chlorhexidine and povidone-iodine in the repair of oral wounds. A review.

PURPOSE: The aim of this paper was to review the current literature with regard to the use of chlorhexidine and povidone-iodine in the treatment of oral wounds. BACKGROUND: Oral mucosa is continuously subjected to physical or chemical injuries, where it becomes a common site for the occurrence of ulcerated lesions. These lesions are susceptible to infections that may delay healing. MATERIALS AND METHODS: A search of the medical and dental literature was conducted in Medline/Pubmed and Scielo using a combination of the terms oral ulcer, oral wound, wound healing, povidone-iodine and chlorhexidine, to review their mechanism of action and their use in the healing of oral wounds. RESULTS AND CONCLUSION: The use of chlorhexidine and povidone-iodine is effective in the control of local infection in a concentration-dependent manner, exerting a positive influence on the tissue repair process. Oral antiseptics appear be a good alternative in the management of these lesions, since there is a low risk of systemic toxicity and allergies, and less clinical evidence of bacterial resistance.

Antimicrobial efficacy of 0.5% peracetic acid and EDTA with passive ultrasonic or manual agitation in an Enterococcus faecalis biofilm model.

We compared the antimicrobial efficacy of EDTA and 0.5% peracetic acid (PAA), with manual agitation (MA) or passive ultrasonic irrigation (PUI) in an Enterococcus faecalis biofilm model. Fifty-five single-rooted human premolar teeth were chemo-mechanically prepared and inoculated with E. faecalis for biofilm formation. These were divided into five groups (n = 11): saline solution, PAA+MA, PAA+PUI, EDTA+MA and EDTA+PUI. Root canal sampling and scanning electron microscopy of the canal lumen and dentinal tubule areas at the different root thirds were performed. The images were ranked based on contamination level. Only the PAA groups presented with no bacterial growth, with the remaining groups not presenting significant differences among them. PAA+PUI presented with the highest median position rankings in every third and location, whereas EDTA+MA performed similarly to the saline control. No differences were found when comparing MA and PUI within the same solution, however, PUI was associated with lower contamination levels mean rankings.

Levels of persisters influenced by aeration in Acinetobacter calcoaceticus-baumannii.

AIM: To evaluate the influence of aeration on persister levels from Acinetobacter calcoaceticus-baumannii isolates exposed to meropenem or tobramycin, as well as analyze morphological and structural changes in persisters. MATERIALS & METHODS: Levels of persisters were determined after a 48-h exposure to tobramycin or meropenem under aerated or static conditions, and persisters were analyzed by scanning and transmission electron microscopy. RESULTS: The fractions of persisters varied between isolates. Aeration reduced cell survival under each antibiotic treatment, and cell survival decreased as the tobramycin concentration was increased. Interestingly, division septa were observed in persisters by electron microscopy. CONCLUSION: Aeration may have stimulated bacterial growth, providing more targets for antibiotic action and leading to increased production of reactive oxygen species, which decreased levels of persisters.

Topical chlorhexidine, povidone-iodine and erythromycin in the repair of traumatic ulcers on the rat tongue: Clinical, histological and microbiological evaluation.

OBJECTIVE: This study investigated the effect of topical application of 0.12% chlorhexidine, 10% povidone-iodine and 50% erythromycin on the optimization of healing process of traumatic ulcers made on ventral tongue of rats. DESIGN: Forty-Eight Wistar rats were randomly divided into four groups: control, chlorhexidine (Chx), povidone-iodine (PvI) and erythromycin (Er). An ulcer of 5 mm in diameter was made on the ventral tongue of the animals. After 24 h, a microbiological sample was taken and daily application of the substances started. Six animals each group were euthanized at 4 days and the others at 8 days postoperative, totaling three and seven days of treatment. Prior to euthanasia, a new microbiological collection was performed. RESULTS: The experimental groups showed less area of residual ulcer. A significant difference was seen between the PvI and Chx in relation to the control after three days of treatment (p < 0.05). Although the experimental groups displayed greater newly formed epithelial area, there was no significant difference compared to the control (p > 0.05). Er exhibed the lowest inflammation scores after seven days of treatment (p < 0.05). PvI showed reduction of microorganisms at both times and under aerobic (p < 0.01 at 3 days and p < 0.001 at 7 days) and microaerophilic (p < 0.05) conditions. Er significantly reduced the count of microorganisms in aerobic condition when compared to control group (p < 0.05 at 3 days and p < 0.01 at 7 days). CONCLUSIONS: All drugs promoted reduction of the microorganisms at the site of the injury, which may have a direct effect on the tissue repair process.

Histopathological, Microbiological, and Radiographic Analysis of Antimicrobial Photodynamic Therapy for the Treatment of Teeth with Apical Periodontitis: A Study in Rats' Molars.

OBJECTIVE: The aim of this study was to evaluate in vivo, by histological and radiographic analysis, the response of apical tissues of rats' teeth with experimentally induced apical periodontitis, after one- and two-session endodontic treatment with and without photodynamic therapy (PDT). A microbiological analysis was also performed to verify bacterial reduction after each treatment. BACKGROUND DATA: Studies carried out in recent years highlighted the antibacterial potential of PDT when associated with conventional endodontic therapy in vitro. Although the antimicrobial effect of PDT is well-established, tissue response to PDT in teeth with apical periodontitis lacks studies. METHODS: Thirty-two rats' root canals were assigned to four groups: one session/PDT-[chemomechanical preparation (CMP)+root canal filling (RCF)]; two sessions/PDT- [CMP+calcium hydroxide (CH) for 14 days+RCF]; one session/PDT+ [CMP+PDT+RCF], and two sessions/PDT+ [CMP+PDT+CH for 14 days+RCF]. For microbiological evaluation, samples were collected before and after proposed treatments. For radiographic and histological analysis, the animals were euthanized after 28 days and the mandibles surgically removed. RESULTS: PDT associated with conventional endodontic therapy was able to promote significant bacterial reduction in root canals with induced apical periodontitis, but this reduction was not significantly different to conventional endodontic therapy alone. Although radiographic evaluation showed no significant differences, histological analysis showed lower scores for neutrophils/eosinophils in PDT-treated groups and macrophages/giant cells in CH groups. CONCLUSIONS: The use of low-level laser as light source did not promote major improvement on radiographic and histological repair, but since the number of inflammatory cells slightly decreased, it may optimize repair by modulating inflammatory process. PDT may be indicated as an adjunct to conventional endodontic therapy for teeth with apical periodontitis, in association with an interappointment dressing with CH, in an attempt to produce better conditions to stimulate repair.

Effects of meropenem exposure in persister cells of Acinetobacter calcoaceticus-baumannii.

AIM: To evaluate the influence of meropenem in the Acinetobacter calcoaceticus-baumannii (ACB) persister levels. METHODS: Persister levels in planktonic and biofilm cultures from ACB isolates were evaluated after exposure to different meropenem concentrations. RESULTS: A high variability of persister fractions was observed among the isolates cultured under planktonic and biofilm conditions. Meropenem concentration did not influence persister fractions, even when far above the MIC. No correlation was found between persister levels and biofilm biomass. CONCLUSION: The magnitude of persister levels from ACB planktonic and, particularly, biofilm cultures exposed to meropenem was independent of the antibiotic concentration, dosing regimen and biofilm biomass. These findings, in a context of meropenem failure to treat chronic infections, strengthen the importance of understanding persister behavior.

Heterogeneous persister cells formation in Acinetobacter baumannii.

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.

Effectiveness of the proton pump inhibitor omeprazole associated with calcium hydroxide as intracanal medication: an in vivo study.

INTRODUCTION: The objective of this study was to evaluate the efficacy of the association of a proton pump inhibitor (omeprazole) with Ca(OH)(2) as intracanal medication in a rat model of periapical lesions. METHODS: Periapical lesions were induced on the first right mandibular molar tooth of 36 male Wistar rats (6 per group). After 28 days, the distal canal of each tooth was prepared, filled with the respective dressing (negative control group, PEG 400; positive control group, Ca(OH)(2) + PEG400; test group, Ca(OH)(2) + omeprazole + PEG 400), and sealed with amalgam for 15 or 28 days. Microbiological samples were taken in 3 periods: S1, after 28 days of lesion induction; S2, after the biomechanical preparation; and S3, after the medication (15 and 28 days). RESULTS: The radiographic and histologic analysis revealed that either Ca(OH)(2) or Ca(OH)(2) plus omeprazole dressings produced a reduction of periapical lesions at 28 days, when compared with the negative control group. The reduction of periapical lesions and inflammatory cell infiltration was visibly improved by associating omeprazole with Ca(OH)(2), with an increase of reparative bone areas. The microbiological assessment showed a significant decrease of colony-forming units count from S1 to S2 or S3 collecting times, but no differences were observed between the S2 and the S3 time-periods or among the experimental groups within the S3 period. Further bacterial characterization showed a possible selective activity of the medications. CONCLUSIONS: Our data showed that association of omeprazole with Ca(OH)(2) favored a superior repair of rat periapical lesions and seemed to display different selective activity over endodontic microbiota, in comparison with the conventional Ca(OH)(2) dressing.

Effect of ultrasonics on Enterococcus faecalis biofilm in a bovine tooth model.

INTRODUCTION: The purpose of this study was to evaluate in vitro the effect of the ultrasonic irrigation of sodium hypochlorite and EDTA in root canals of bovine teeth infected with Enterococcus faecalis. METHODS: Eighty-four bovine incisors were inoculated with E. faecalis, remaining in culture for 50 days for biofilm formation. The teeth were divided into four groups: the control group, which received no treatment; the ultrasonic + distilled water group; the conventional irrigation with sodium hypochlorite + EDTA group; and the passive ultrasonic irrigation with sodium hypochlorite + EDTA group. Microbiological tests and analysis in scanning electron microscopy (SEM) were performed. RESULTS: In microbiological testing, groups using sodium hypochlorite did not show bacterial growth. There were significant differences between the control group and the ultrasonic + distilled water group and between these groups and groups using sodium hypochlorite. In SEM analysis, at the canal wall area, there was no significant difference between the groups using sodium hypochlorite, but these were different from the others groups. The control group was significantly different from the ultrasonic + distilled water group. At the exposed tubule area, there was no significant difference between the groups. CONCLUSIONS: Passive ultrasonic irrigation can be an aid in cleaning the root canal; however, the main role in bacteria elimination is played by the irrigant.

Identification of Candida spp. by phenotypic tests and PCR.

The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans.

Phage types of Salmonella enteritidis isolated from clinical and food samples, and from broiler carcasses in southern Brazil.

272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings.