Evidence of cross-contamination of waste workers and transmission of antimicrobial resistance genes by coagulase-negative Staphylococcus isolated from dental solid waste: an intriguing study.

The aim of this study was to phenotypically and genotypically identify coagulase-negative Staphylococcus (CoNS) recovered from the nostrils of waste workers and from dental waste; 135 strains were recovered and S. epidermidis was the prevailing species. Genetic similarity (100%) was observed between the two S. epidermidis isolated from different employees on the same shift and 85% similarity between the S. epidermidis recovered from an employee's nostril and from waste. The mecA gene was found in 20 CoNS, and 20% were also found to possess the vanA gene. The blaZ gene was detected in 46.7%, and the icaA (34.8%), B and C genes (11.8% each). Our findings emphasized the biological risk to which waste workers are exposed and unprecedently confirms that it was possible to recover genetically identical bacterial species from waste and from workers' nostrils. It is important to highlight that this risk is raised by the detection of relevant antimicrobial resistance genes. The results also suggest that effective measures to correctly manage waste and promote the rational use of antimicrobials should be adopted.

Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae.

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of Escherichia coli. Here we exploited this system further under challenging conditions: by targeting the bla TEM- 1 AMR gene located on a high-copy plasmid (i.e., 100-300 copies/cell) and by directly tackling bla TEM- 1-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring bla TEM- 1 on the plasmid pSB1A2, the plasmid number and, accordingly, the bla TEM- 1 gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in bla TEM- 1 were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of E. coli, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of Galleria mellonella infected with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

BACKGROUND: New Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of ß-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the blaNDM-1 gene using the LOOP-mediated isothermal amplification technique (LAMP). METHODS: In all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were used for the blaNDM-1 standardization by LAMP. Klebsiella pneumoniae ATCC BAA-2473 and two clinical strains were used as a positive control. All results were compared to the reaction in polymerase chain reaction (PCR), considered gold standard for this detection. RESULTS: There was an excellent correlation between the two techniques employed, since all measured clinical strains were negative in both employed tests and two clinical, and a reference strains were positive. CONCLUSIONS: The lamp technique seems to be an excellent option for the rapid detection of blaNDM-1. The amplification time is much shorter than other molecular techniques, the PCR machine is not necessary, it is easy of implementation and costs is low.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Isolation and identification of Candida species in patients with orogastric cancer: susceptibility to antifungal drugs, attributes of virulence in vitro and immune response phenotype.

BACKGROUND: Because of the inherent immunosuppression of cancer patients opportunistic infections by Candida spp, occur frequently. This study aimed to identify Candida species in the oral mucosa of 59 patients with orogastric cancer (OGC) and to analyze the immunological phenotype of these patients. METHODS: The yeasts were identified by MALDI-TOF mass spectrometry (MS). For all isolates, we performed phospholipases and proteinases assays, in vitro adherence to buccal epithelial cells (BEC), minimum inhibitory concentration of antifungal drugs and determined the cytokine profile by Cytometric Bead Array flow citometry assay. RESULTS: C. albicans was the most prevalent species in OGC patients (51.6 %) and control group (66.7 %). Candida spp. strains isolated from OGC patients exhibited better adherence to BEC (p = 0.05) than did the control group. Phospholipases production by Candida strains from OGC patients was lower (51.6 %) than in the control group (61.9 %). Proteinases were detected in 41.9 % and 4.8 % of the yeasts from OGC patients and control group, respectively. Significant differences were found in the serum of OGC patients compared to the control group for IL-2, IL-10, TNF-α, IFN-γ and IL-17. CONCLUSIONS: The results of this work suggest increased virulence of yeasts isolated from OGC patients and, that this may interfere with the immune phenotype.

Solid Lipid Nanoparticles Loaded with Retinoic Acid and Lauric Acid as an Alternative for Topical Treatment of Acne Vulgaris.

Topical therapy is the first choice for the treatment of mild to moderate acne and all-trans retinoic acid is one of the most used drugs. The combination of retinoids and antimicrobials is an innovative approach for acne therapy. Recently, lauric acid, a saturated fatty acid, has shown strong antimicrobial activity against Propionibacterium acnes. However, topical application of retinoic acid is followed by high incidence of side-effects, including erythema and irritation. Solid lipid nanoparticles represent an alternative to overcome these side-effects. This work aims to develop solid lipid nanoparticles loaded with retinoic acid and lauric acid and evaluate their antibacterial activity. The influence of lipophilic stearylamine on the characteristics of solid lipid nanoparticles was investigated. Solid lipid nanoparticles were characterized for size, zeta potential, encapsulation efficiency, differential scanning calorimetry and X-ray diffraction. The in vitro inhibitory activity of retinoic acid-lauric acid-loaded solid lipid nanoparticles was evaluated against Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis. High encapsulation efficiency was obtained at initial time (94 ± 7% and 100 ± 4% for retinoic acid and lauric acid, respectively) and it was demonstrated that lauric acid-loaded-solid lipid nanoparticles provided the incorporation of retinoic acid. However, the presence of stearylamine is necessary to ensure stability of encapsulation. Moreover, retinoic acid-lauric acid-loaded solid lipid nanoparticles showed growth inhibitory activity against Staphylococcus epidermidis, Propionibacterium acnes and Staphylococcus aureus, representing an interesting alternative for the topical therapy of acne vulgaris.

Analysis of microbial load on surgical instruments after clinical use and following manual and automated cleaning.

BACKGROUND: We aimed to monitor the microbial load and identify the microorganisms recovered from surgical instruments after clinical use and following manual and automated cleaning. METHODS: This experimental study was carried out in the Laboratory of Oral Microbiology and Anaerobes at the Federal University of Minas Gerais in Brazil. Microbial samples were taken from 125 surgical instruments used in 25 types of gastrointestinal surgeries. RESULTS: The average microbial load was 93.1 CFU/100 mL after clinical use and 41 CFU/100 mL and 8.24 CFU/100 mL on instruments following 2 sequential steps of manual cleaning, respectively, and 75 CFU/100 mL and 16.1 CFU/100 mL on instruments after automated cleaning. Surgical wound classification significantly affected the microbial load recovered on instruments. Coagulase-negative Staphylococcus, Escherichia coli, Pseudomonas spp, Stenotrophomonas maltophilia, and Acinetobacter baumannii complex were recovered. CONCLUSIONS: The average microbial load observed after the cleaning steps decreased, and the decrease in microbial load was more pronounced using the manual method compared with that observed using the automated method.

Proteomic changes in Bacteroides fragilis exposed to subinhibitory concentration of piperacillin/tazobactam.

Bacteroides fragilis is the anaerobe most frequently isolated from clinical specimens and piperacillin/tazobactam is among the drugs that can be used to treat polymicrobial infections in which this bacteria is often involved. During antibiotic therapy, inhibitory concentrations of antibiotics are always followed by subinhibitory concentrations which can generate phenotypic changes in bacteria. So, in this study we aimed to evaluate changes in the proteomic profile of B. fragilis grown in a sub-MIC of PTZ, using 2-D electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time of-flight. Analysis of the 2-DE gels showed 18 spots with significantly different volume percentages between experimental conditions and 12 were successfully identified by MS/MS. Two proteins with decreased abundance in sub-MIC condition were involved in the glycolysis (glyceraldehyde-3-phosphate dehydrogenase and triose phosphate isomerase), others two involved in amino acid metabolism (Oxoacyl-(acyl-carrier protein) synthase II and dihydrodipicolinate reductase), and finally, one protein involved in fatty acid metabolism (UDP-N-acetylglucosamine acyltransferase). Among the proteins with increased abundance, we founded three ATP synthase (alpha, beta, and alpha type V), which could be involved in antibiotic bacterial resistance by efflux pump, one protein involved in glycolysis (enolase), and one involved in protein degradation (aminoacyl-histidine dipeptidase). In conclusion, our data show overall changes in the proteome of B. fragilis conducted by sub-MIC of PTZ, whose consequences on bacterial physiology deserve further investigation.

Count, identification and antimicrobial susceptibility of bacteria recovered from dental solid waste in Brazil.

In Brazil, few studies on microbial content of dental solid waste and its antibiotic susceptibility are available. An effort has been made through this study to evaluate the hazardous status of dental solid waste, keeping in mind its possible role in cross-infection chain. Six samples of solid waste were collected at different times and seasons from three dental health services. The microbial content was evaluated in different culture media and atmospheric conditions, and the isolates were submitted to antibiotic susceptibility testing. A total of 766 bacterial strains were isolated and identified during the study period. Gram-positive cocci were the most frequent morphotype isolated (48.0%), followed by Gram-negative rods (46.2%), Gram-positive rods (5.0%), Gram-negative-cocci (0.4%), and Gram-positive coccobacillus (0.1%). Only two anaerobic bacteria were isolated (0.3%). The most frequently isolated species was Staphylococcus epidermidis (29.9%), followed by Stenotrophomonas maltophilia (8.2%), and Enterococcus faecalis (6.7%). High resistance rate to ampicillin was observed among Gram-negative rods (59.4%) and Gram-positive cocci (44.4%). For Gram-negative rods, high resistance was also noted to aztreonam (47.7%), cefotaxime (47.4%), ceftriaxone and cefazolin (43.7%), and ticarcillin-clavulanic acid (38.2%). Against Gram-positive cocci penicillin exhibit a higher resistance rate (45.0%), followed by ampicillin, erythromycin (27.2%), and tetracycline (22.0%). The present study demonstrated that several pathogenic bacteria are present in dental solid waste and can survive after 48 h from the waste generation time and harbor resistance profiles against several clinical recommended antibiotics.

Composition analysis of dental solid waste in Brazil.

When developing proper waste management strategies, it is essential to characterize the volume and composition of solid waste. The aim of this work was to evaluate the composition of dental waste produced by three dental health services in Belo Horizonte, Minas Gerais State, Brazil. Two universities, one public and one private, and one public dental health service were selected. Waste collection took place from March to November 2007. During this period, three samples were collected from each dental health service. The total amount of dental waste produced in one day of dental work was manually separated into three categories: infectious and potentially infectious waste, accounting for 24.3% of the total waste; non-infectious waste, accounting for 48.1%; and domestic-type waste, accounting for 27.6% (percentages are for mean weights of solid waste). Our results showed that most of the waste considered as biomedical may be misclassified, consequently making the infectious waste amount appear much larger. In addition, our results suggest that the best waste minimization method is recycling, and they help to define an appropriate waste management system in all three of the dental health services involved in this study.

Antimicrobial susceptibility of microorganisms recovered from intraabdominal infections at Belo Horizonte, Brazil.

We evaluated antimicrobial susceptibility patterns of microorganisms isolated from intraabdominal infection of Brazilian patients, by agar dilution, agar diffusion, and E test. Among the strictly anaerobes, 57.7% were resistant to penicillin, 28.2% to clindamycin, and 9.9% to metronidazole. The majority of Escherichia coli and Staphylococcus were sensitive and resistant to almost all drugs, respectively. Half of Candida samples were resistant to itraconazole. Our data reinforce the importance of this kind of study to support rational antimicrobial therapy.