Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development.

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections. Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression. Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects. Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.

Cytocompatibility and antimicrobial activity of a novel endodontic irrigant combining citric acid and chlorhexidine.

OBJECTIVES: This study aimed to evaluate the antibacterial ability and cytocompatibility of a new irrigant solution for endodontic treatment composed of 10% citric acid (CA) and 1% chlorhexidine (CHX). METHODS: Thirty-five extracted single-canal human teeth were selected and de-crowned. Canal systems (n = 7/group) were infected with Enterococcus faecalis for 4 weeks and subject to irrigation with 1% CHX; 10% CA; irrigating solution 10% CA associated with 1% CHX (CACHX); 2.5% NaOCl or sterile water (control). Microbiological samples were collected immediately and 18 h after irrigation (enriched samples). The canals were filled with culture medium post irrigation to verify the bacterial presence/absence qualitatively and quantitatively through colony counting (log10 CFU/mL). A multiparametric assay was performed after exposure of human periodontal ligament fibroblasts (HPdLF) to the test solutions. The Kruskal-Wallis test with Dunn´s post-test and Fisher's exact test were employed at the 95% confidence level to compare differences among groups. RESULTS: All tested solutions were cytocompatible with human periodontal ligament fibroblasts. No difference was observed on antibacterial activity between 1% CHX, 10% CA, CACHX and 2.5% NaOCl (p > 0.05). Eighteen hours after irrigation, CACHX samples were the only that did not present E. faecalis in the root canal system. CONCLUSIONS: The demonstrated good in vitro biocompatibility and elimination of E. faecalis suggest a potential use of 10% CA associated with 1% CHX as a solution for microbiological control during endodontic treatment. CLINICAL RELEVANCE: Irrigants play an essential role during endodontic therapy. This irrigating solution, based on the association of 10% citric acid with 1% chlorhexidine, seems viable for clinical procedures.

Antimicrobial Photodynamic Therapy Associated with Conventional Endodontic Treatment: A Clinical and Molecular Microbiological Study.

This study evaluated antimicrobial photodynamic therapy (aPDT) as an adjunct to endodontic treatment. Ten uniradicular teeth (control group (CG) = 4 (2 and test group (TG) = 6) with primary endodontic infections, from both genders, between 17 and 65 years old, were analyzed. Microbiological samples were collected before and after chemical-mechanical instrumentation (CMI), after aPDT (for the TG), and after the removal of the temporary restorations (second session). In TG, the aPDT was performed with 100 µg mL-1 methylene blue and irradiated with low power laser (InGaAIP, 660 nm; 100 mW; 40 s) with a fiber-coupled optical laser. Another irradiation (3 J; 30 s; spot size of 3 mm2 ) was performed in the gingiva close to the apical foramen. The PCR was performed, after previous whole-genome amplification, for Enterococcus faecalis, Candida genus and Bacteria domain. For TG, a positive tooth for Candida spp. before of the CMI presented negative results in subsequent samples. Additionally, E. faecalis species was present in four samples before CMI, two after CMI, in one after the aPDT and was not detected at the second session. aPDT may be an effective adjunct therapy, resulting in a reduction (P = 0.0286) of the incidence of E. faecalis before root canal obturation.

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR.

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus.

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR.

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.

Coagulase-negative staphylococci: comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin.

This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 microg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.