Inclusion complexes of 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin with 2-hydroxypropyl-(-cyclodextrin: solubility and antimicrobial activity

The aim of the present study is to improve the solubility and antimicrobial activity of 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin by formulating its inclusion complexes with 2-hydroxypropyl-ß-cyclodextrin in solution and in solid state. The phase solubility study was used to investigate the interactions between 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin and 2-hydroxypropyl-ß-cyclodextrin and to estimate the molar ratio between them. The structural characterization of binary systems (prepared by physical mixing, kneading and solvent evaporation methods) was analysed using the FTIR-ATM spectroscopy. The antimicrobial activity of 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin and inclusion complexes prepared by solvent evaporation method was tested by the diffusion and dilution methods on various strains of microorganisms. The results of phase solubility studies showed that 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin formed the inclusion complexes with 2-hydroxypropyl-ß-cyclodextrin of AP type. The solubility of 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin was increased 64.05-fold with 50% w/w of 2-hydroxypropyl-ß-cyclodextrin at 37 oC. The inclusion complexes in solid state, prepared by the solvent evaporation method, showed higher solubility in purified water and in phosphate buffer solutions in comparison with 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin alone. The inclusion complexes prepared by solvent evaporation method showed higher activity on Bacillus subtilis and Staphylococcus aureus compared to uncomplexed 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin due to improved aqueous solubility, thus increasing the amount of available 3-(3-(2-chlorophenyl)prop-2-enoyl)-4-hydroxycoumarin that crosses the bacterial membrane.

Caracterização molecular de Staphylococcus aureus isolados de queijos artesanais da Serra da Canastra / Molecular characterization of Staphylococcus aureus isolated from artisanal cheeses from Serra da Canastra

O queijo Minas Artesanal da Canastra é produzido na Serra da Canastra (MG), utilizando leite cru, coalho e pingo, que é uma cultura endógena natural de cada queijaria. Devido ao uso de leite cru, o produto pode veicular microrganismos causadores de doenças veiculadas por alimentos, como Staphylococcus aureus. A caracterização molecular é uma ferramenta importante para avaliar a população microbiana do alimento e direcionar a aplicação de medidas de controle na produção. Este estudo caracterizou a diversidade genética, o potencial de virulência e determinou o perfil de susceptibilidade a antimicrobianos de S. aureus isolados de queijos produzidos na Serra da Canastra. Para o estudo transversal foram analisados 248 isolados de queijos que tinham um tempo de maturação de 22 dias, provenientes de 83 propriedades. Por outro lado, no estudo longitudinal foram analisados outros 197 isolados coletados ao longo do processo de maturação, provenientes de três propriedades. Os isolados foram submetidos a testes bioquímicos para confirmação do gênero e para a confirmação da espécie de S. aureus, foi identificado o gene nuc por meio da técnica de PCR. Além disso, foi pesquisado o gene mecA para a detecção de S. aureus Resistente a Meticilina (MRSA). Após os testes de confirmação, 144 isolados do estudo transversal e 159 do estudo longitudinal foram positivos para o gene nuc, específico para S. aureus. Posteriormente, o perfil clonal foi determinado por Eletroforese de Campo Pulsado (PFGE) utilizando a enzima SmaI e tipagem do locus agr por PCR multiplex. A análise por PFGE foi realizada no programa BioNumerics. A técnica PCR foi realizada para identificar a presença de genes que codificam a produção de hemolisinas, toxina TSST-1, enterotoxinas SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), formação de biofilme e Componentes Microbianos de Superfície que Reconhecem a Matriz de Moléculas Adesivas (MSCRAMMs). Os isolados foram submetidos ao teste de susceptibilidade a antimicrobianos por disco de difusão. Por último, a formação de biofilme em microplaca de 96 poços, em caldo TSB a 37°C, foi verificada pela metodologia de Cristal Violeta. O gene mecA foi detectado em 1,9% dos 445 isolados. A tipagem agrrevelou que 83 (27,4%) dos isolados são do tipo agr-I, 95 (31,4%) agr-II e 43 (14,2%) agr-III, sendo que não foram detectados isolados classificados como agr-IV. A tipagem por PFGE revelou um total de 54 perfis. Assim, um isolado representativo de cada perfil foi utilizado nos demais testes que mostraram a presença dos genótipos spa mais frequentes t127 e t605 (20,58%); t002 (14,70%), seguidos pelos tipos t267 (8,82%); t1234 e t693 (5,8%) e t021, t177, t306, t321, t359, t442, t521, t693 e t5493 (2,9%). Além disso, encontramos a presença dos genes do grupo SEs, sea 1 (1,8%), seh 11 (20,3%), sei 10 (18,5%), sej 7 (12,9%), seg e seo 14 (25,9%), sem 8 (14,8%), e os genes seb, sec, sed, see e tst não foram detectados. Para os genes das hemolisinas, hla foi positivo em todos os isolados e hlb foi positivo em 53 (98,1%) isolados. Os genes positivos para MSCRAMMS foram fnbA, fnbB 18 (33,3%), clfA, clfB e eno 53 (98,1%), fib 44 (81,4%), bbp 4 (7,4%), cna 17 (31,4%) e ebps 10 (18,5%). Por último, os genes de formação de biofilme icaA e icaD estiveram presentes em 38 (70,3%) e 25 (46,2%) dos isolados, respectivamente. Na avaliação de susceptibilidade a antibióticos dos 54 isolados escolhidos, 25 (46,3%) apresentaram maior resistência a penicilina e 13 (24,0%) a tetraciclina. Em menor porcentagem (1,8%), 1 isolado cada foi resistente a eritromicina, cefoxitina, clindamicina, gentamicina, cotrimazol, azitromicina e trimetropim. Além disso, 8 isolados (14,8%) apresentaram resistência intermediaria a tetraciclina, 3 (5,5%) a gentamicina e 1 (1,8%) a tobramicina. No teste para a determinação da formação de biofilme por cristal violeta, 13,7%, foram classificados em isolados não formadores, 60,8% em fracamente formadores, 25,5% moderadamente formadores e nenhum como fortemente formador. A alta diversidade de cepas de S. aureus observada neste estudo mostrou que existem vários tipos de linhagens circulando na região da Canastra. A caracterização revelou uma elevada frequência de genes de virulência e que mais estudos são necessários para avaliar o potencial de produção de enterotoxinas nos queijos artesanais. A melhora dos procedimentos de higienização durante todas as etapas de produção pode ser uma solução para a redução dos níveis de contaminação por S. aureus

Canastra Minas Artesanal cheese is produced in Serra da Canastra (MG), using raw milk, rennet and a natural endogenous culture called pingo. Due to the use of raw milk, the product can carry microorganisms that cause foodborne diseases, such as Staphylococcus aureus. Molecular characterization is an important tool to assess the microbial population of food and guide the application of control measures in production. This study characterized the genetic diversity, virulence potential and determined the antimicrobial susceptibility profile of S. aureus isolated from cheeses produced in Serra da Canastra. A total of 248 isolates from 22 days ripened cheeses were obtained from 83 properties (cross sectional study). Another 197 isolates were collected during maturation (longitudinal study), in three properties. The isolates were submitted to biochemical tests to confirm the genus and to confirm the S. aureus species, the nuc gene was identified by PCR. In addition, the detection of mecA gene was performed for the detection of Methicillin Resistant S. aureus (MRSA). After confirmation tests, 144 isolates from the cross-sectional study and 159 from the longitudinal study were positive for the nuc gene, specific for S. aureus. Subsequently, the clonal profile of the isolates was determined by Pulsed Field Gel Electrophoresis (PFGE) using the SmaI enzyme and typing of the agr locus by multiplex PCR. PFGE analysis was performed using the BioNumerics program. PCR was performed to identify the presence of genes encoding the production of hemolysins, TSST-1 toxin, enterotoxins SEs (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEO, SEM), biofilm formation and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The isolates were submitted to the antimicrobial susceptibility test by disc diffusion. Finally, biofilm formation in a 96-well microplate in TSB broth at 37°C was verified by the Cristal Violeta method. The mecA gene was detected in 1.9% of the 445 isolates. Agr typing revealed that 83 (27.4%) of the isolates are agr-I, 95 (31.4%) agr-II and 43 (14.2%) agr-III, and no isolate was classified as agr-IV. PFGE typing revealed a total of 54 profiles. Thus, a representative isolate of each profile was used in the other tests that showed the presence of the most frequent spagenotypes t127, t605 (20.58%); t002 (14.70%), followed by types t267 (8.82%); t1234, t693 (5.8%) e t021, t177, t306, t321, t359, t442, t521, t693 and t5493 (2.9%). In addition, we found the presence of the genes of the SEs group: sea 1 (1.8%), seh 11 (20.3%), sei 10 (18.5%), sej 7 (12.9%), seg and seo 14 (25.9%), sem 8 (14.8%), while seb, sec, sed, see and tst genes were not detected. For hemolysin genes, hla was positive in all isolates and hlb was positive in 53 (98.1%) isolates. The positive genes for MSCRAMMS were: fnbA, fnbB 18 (33.3%), clfA, clfB e eno 53 (98.1%), fib 44 (81.4%), bbp 4 (7.4%), cna 17 (31.4%) and ebps 10 (18.5%). Finally, the biofilm formation genes icaA and icaD were present in 38 (70.3%) and 25 (46.2%) of the isolates, respectively. In the evaluation of antibiotic susceptibility of the 54 isolates, 25 (46.3%) showed greater resistance to penicillin and 13 (24.0%) to tetracycline. In a lower percentage (1.8%), 1 isolate each was resistant to erythromycin, cefoxitin, clindamycin, gentamicin, contrimazole, azithromycin and trimethoprim. In addition, 8 isolates (14.8%) showed intermediate resistance to tetracycline, 3 (5.5%) to gentamicin and 1 (1.8%) to tobramycin. In the test for the determination of biofilm formation by crystal violet, 13.7% were classified as non-forming isolates, 60.8% as weakly forming, 25.5% moderately forming and none as strongly forming. The high diversity of S. aureus strains observed in this study showed that there are several types of strains circulating in the Canastra region. The characterization revealed a high frequency of virulence genes and that further studies are needed to assess the potential for enterotoxin production in artisanal cheeses. The improvement of hygiene procedures during all stages of production can be a solution for reducing the levels of contamination by S. aureus

Characterization, Antioxidant and Antibacterial Potentials of Tamarindus indica L. Fruit Pulp Extract Loaded O/W Nanoemulsions

Abstract The main purposes of the current study were to formulate o/w nanoemulsions as a carrier for Tamarindus indica (tamarind) fruit pulp extract and to study the antioxidant and antibacterial potentials of nanoemulsions containing tamarind extract, focusing on cosmetic/hygiene applications. The o/w nanoemulsions using a mixture of Tween 80 and Span 80 as an emulsifier (5%w/w) were prepared by a high pressure homogenization process. Two concentrations of sweet tamarind extract, 3.3 and 6.6%w/w, based on the bioactivity study, were incorporated into the blank nanoemulsions to produce loaded nanoemulsions, F1-3.3TE (3.3%) and F1- 6.6TE (6.6%). As compared with the unloaded nanoemulsion, both tamarind extract loaded nanoemulsions showed reduced pH and significantly increased viscosity. Overall, the loaded nanoemulsions had droplet sizes of approximately 130 nm, zeta potential around -38 mV and polydispersity index (PDI) values less than 0.2. The nanoemulsion F1-3.3TE had better stability (e.g. significantly greater % tartaric acid content and lesser PDI value) than the nanoemulsion F1-6.6TE did. The antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl assay revealed that the nanoemulsions F1-3.3TE and F1-6.6TE had scavenging activities of 81.66 ± 0.77% and 63.80 ± 0.79%, respectively. However, antioxidant activity of these two formulations decreased under stress conditions (heating-cooling cycles). Such incidence did not occur for their antibacterial properties investigated by agar well diffusion technique. The two formulations exhibited inhibition zones of approximately 24.0-27.7 mm against Staphylococcus aureus and Staphylococcus epidermidis, responsible for malodor of underarms. The results suggest the potential of using sweet tamarind pulp extract loaded nanoemulsions as hygiene products.

Prophages encoding human immune evasion cluster genes are enriched in Staphylococcus aureus isolated from chronic rhinosinusitis patients with nasal polyps.

Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund-Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, lukE/D, sea. Intact prophages were more frequently identified in CRSwNP than in CRSsNP (P=0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP (P=0.0008) and intact phiNM3 were exclusively found in CRSwNP patients (P=0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.

Bloodstream infections in head and neck cancer patients after curative-intent radiotherapy: a population-based study from the Danish Head and Neck Cancer Group database.

BACKGROUND: Patients with head and neck squamous cell carcinoma (HNSCC) undergoing radiotherapy (RT) or chemoradiation (CRT) may become immunocompromised. In this population-based study, we aimed to investigate the risk factors, microbiological aetiologies, prognosis and impact on early non-cancer mortality of bloodstream infections (BSIs) after RT/CRT. METHODS: Patients with HNSCC of the pharynx, larynx and oral cavity treated with curative-intent RT/CRT in Denmark between 2010 and 2017 and subsequent BSI episodes occurring within 18 months of RT/CRT initiation were identified in national registries. RESULTS: We included 5674 patients and observed 238 BSIs. Increasing age, stage and performance status were significantly associated with an elevated BSI risk, while sex, smoking and high-grade mucositis were not. Human papillomavirus-positive oropharyngeal cancer patients had a decreased risk. Staphylococcus aureus accounted for 34% of episodes occurring during the first 3 months. The 30-day post-BSI mortality rate was 26% (95% confidence interval: 19-32) and BSIs were involved in 10% of early non-cancer deaths. CONCLUSION: The risk of BSI development is associated with several patient- and disease-related factors and BSIs contribute considerably to early non-cancer mortality. Empiric antibiotic treatment regimens should prioritise coverage for S. aureus when treating suspected systemic infection in this population.

Increasing prevalence of hypervirulent ST5 methicillin susceptible Staphylococcus aureus subtype poses a serious clinical threat.

Staphylococcus aureus (S. aureus) is a clinical pathogen of great significance causing metastatic or complicated infections. ST5 clonotype isolates have dominated S. aureus infections for more than 10 years in Shanghai, China, and the proportion of methicillin-susceptible S. aureus (MSSA) has remarkably increased in the past decades. By whole-genome sequencing (WGS) 121 ST5 clonotype S. aureus isolates using next-generation sequencing (NGS) platforms and characterizing the evolutionary dynamics of ST5 linages, we found that MSSA evolved independently, making it a subtype differed from other MRSA clones. Drug resistance gene analysis by using the NGS data demonstrated that ST5 clonotype MRSA might be more tolerant under the threat of antimicrobials, which was confirmed in further in vitro susceptibility tests. However, MSSA subtype isolates exhibited relatively high virulence upon the analysis of virulence factors. Furthermore, MSSA subtype isolates displayed higher hemolysis capacity and higher ability to adhere to epithelial cells including A549 human alveolar epithelial cells and HaCaT human skin keratinocytes, caused more severe infections in murine abscess model. With its high virulence and enhanced magnitude in the past decades, the ST5 MSSA subtype poses a serious clinical threat hence more attention should be paid to the prevention and control.

Presence of egc-positive major clones ST 45, 30 and 22 among methicillin-resistant and methicillin-susceptible oral Staphylococcus aureus strains.

The oral cavity may comprise a significant reservoir for Staphylococcus aureus but the data on molecular epidemiology and clonal distribution of oral strains are really scarce. This study aimed to evaluate the clonal relatedness in S. aureus isolated from oral cavity and their relationship with carriage of virulence genes, and antimicrobial resistance profiles. A total of 139 oral S. aureus isolates were obtained from 2327 analysed oral samples of dental patients. Antimicrobial susceptibility testing was performed. Isolates were characterized using protein A gene (spa) typing, spa-CC clonal complexes, toxin genes and SCCmec typing for MRSA. High resistance rates for penicillin, tetracycline and gentamicin were detected, respectively 58.3%, 42.4%, and 35.2%. Twelve (8.6%) S. aureus isolates were identified as MRSA. All of MRSA isolates were mecA-positive and mecC-negative. SCCmec IV was the most common type (66.7%), which was typical for community-acquired MRSA (CA-MRSA). Overall, the enterotoxin gene cluster (egc) was the most frequent detected virulence factor (44.9%), both in MSSA and MRSA isolates. Presence of genes encoding for the enterotoxins (sea, seb, sec, seh, sek), exfoliative toxin A (eta), and toxic shock syndrome toxin-1 (tst) was also observed. Strains carrying lukS-PV/lukF-PV genes belonged to SCCmecV- spa type t437. The most prevalent spa types were t091, t015, t084, t002, t571, and t026 among all 57 identified. Spa types, including 3 new ones, grouped in 6 different spa-CC clonal complexes, with four major dominated; CC45, CC30, CC5, and CC15. This study demonstrated that both methicillin-susceptible and methicillin-resistant major European clones of S. aureus could be isolated from the oral cavity of dental patients, with the emergence of PVL-positive CA-MRSA strains. The oral cavity should be considered as a possible source of toxigenic egc-positive S. aureus strains, in terms of potential risk of cross-infection and dissemination to other body sites.

MRSA Prevalence in Preoperative S. aureus Nasal Culture Isolates is Significantly Different From a Traditional Hospital-wide Antibiogram.

Hospital antibiograms, because they are typically derived from samples obtained from hospitalized patients, may overestimate the prevalence of methicillin resistance in S. aureus in individuals presenting to the hospital for surgery. Because hospital antibiograms are commonly used to justify empiric perioperative prophylactic antibiotic selection prior to surgery, this may lead to unnecessary treatment with broad-spectrum antibiotics such as vancomycin. In a single-institution study, we observed that in our hospital antibiogram the proportion of S. aureus that are methicillin-resistant (MRSA) was significantly higher (45%) than isolates in preoperative nasal cultures obtained at the same hospital in outpatients prior to their lower extremity joint replacement surgery (13%): mean difference 0.32, [95% CI 0.25, 0.39], p <0.0001. These data suggest that hospital antibiograms may overstate the true prevalence of MRSA in those at risk for MRSA surgical site infections who present from the outpatient setting.

Multilocus Sequence Typing of Staphylococcus aureus.

Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacterial species and pathogens that cause outbreaks or epidemics of infectious diseases. MLST provides a powerful solution for molecular epidemiological characterization of bacterial strains, including Staphylococcus aureus, by using the sequences of the internal region of seven housekeeping genes. In previous studies, we utilized MLST to analyze the genotypes of S. aureus isolates from pediatric patients with cystic fibrosis and revealed three prevalent ST types, including ST5, ST30, and ST8 in these isolates. In this chapter, we describe a detailed procedure of MLST for genotyping S. aureus.

Derivation and validation of an early diagnostic score for mediastinitis after cardiothoracic surgery.

OBJECTIVES: The aim of this study was to elaborate on and validate a score for the early diagnosis of mediastinitis after cardiothoracic surgery. METHODS: Between 2007 and 2017, patients who experienced thoracic surgical-site infection after cardiothoracic surgery were enrolled. Laboratory, clinical, and chest CT findings were retrospectively analyzed. Patients were followed up until hospital discharge or intra-hospital death. Univariate and multivariate regression analyses were performed. RESULTS: 950 surgical-site infections were found and analyzed (131 mediastinitis, 819 superficial/deep infections). Of the 131 mediastinitis episodes, 88% required surgical thoracic debridement,Staphylococcus aureus was identified in 43%, and overall mortality was 42%. The following variables were related to mediastinitis diagnosis: sternal diastasis (OR=2.5; 95% confidence interval [95%CI]: 1.2-5.3; P=0.012), bilateral pleural effusion (OR=1.9; 95%CI: 1.0-3.6; P=0.04), leukocyte count ≥14,000cells/mm3 (OR=2.5; 95%CI: 1.3-4.7; P=0.006), male sex (OR=2; 95%CI: 1.11-4; P=0.022), and positive blood culture (OR=3.0; 95%CI: 1.6-5.6; P=0.001). The score predicted with reasonable accuracy mediastinitis in the derivation cohort (AUC-ROC, 0.7476) and the validation cohort (AUC-ROC, 0.7149). Groups with high (31%) and low (5%) risk of mediastinitis were identified. CONCLUSIONS: An early diagnostic score in patients with surgical-site infection after cardiothoracic surgery identified groups with a low and high risk for mediastinitis.

Significance of Infections in Implant Loss After Breast Reconstruction in the Course of Breast Cancer Treatment.

The aim of the study was to analyze the reasons for removing implants after breast reconstruction in the course of treatment of breast cancer. The study involved 428 patients, who underwent a total of 648 breast reconstruction procedures using artificial implants. 47 out of 648 cases (7.3%) were identified in which the implant had to be removed. Of the 47 cases, 57.4% had undergone deferred reconstruction, and 42.6% immediate reconstruction; 27.7% had undergone pre-operative chemotherapy and radiotherapy, 27.7% pre-operative chemotherapy, and 2.1% pre-operative radiotherapy; 6.4% were diabetic, 4.3% active smokers, and more than 50.0% had BMI greater than 25 kg/m2. In 83.0% of the analyzed cases, the reason for removal of the implant was infection, in 8.5% it was local recurrence of breast cancer, in 4.3% it was damage (leakage) of the implant, and in 2.1% it was post-operative pain. About 87.0% of infections appeared within one year of implantation; however, less than a half developed within 90 days of the reconstructive surgery, and up to 30 days only about 13.0% had appeared. Among the etiological agents of infections were: coagulase-negative Staphylococcus (31.3%), Staphylococcus aureus (18.7%), Enterococcus faecalis (9.4%), Enterobacter cloacae (18.8%), Pseudomonas aeruginosa (12.5%), Acinetobacter lwoffii (3.1%), and other Gram-negative fermenting rods accounted for 6.2%. Infections were the most common reason for removing the implant after breast reconstruction. and occurred most often as late infections (>30 days after surgery). The time of observation for infectious complications should be at least 1 year.The aim of the study was to analyze the reasons for removing implants after breast reconstruction in the course of treatment of breast cancer. The study involved 428 patients, who underwent a total of 648 breast reconstruction procedures using artificial implants. 47 out of 648 cases (7.3%) were identified in which the implant had to be removed. Of the 47 cases, 57.4% had undergone deferred reconstruction, and 42.6% immediate reconstruction; 27.7% had undergone pre-operative chemotherapy and radiotherapy, 27.7% pre-operative chemotherapy, and 2.1% pre-operative radiotherapy; 6.4% were diabetic, 4.3% active smokers, and more than 50.0% had BMI greater than 25 kg/m2. In 83.0% of the analyzed cases, the reason for removal of the implant was infection, in 8.5% it was local recurrence of breast cancer, in 4.3% it was damage (leakage) of the implant, and in 2.1% it was post-operative pain. About 87.0% of infections appeared within one year of implantation; however, less than a half developed within 90 days of the reconstructive surgery, and up to 30 days only about 13.0% had appeared. Among the etiological agents of infections were: coagulase-negative Staphylococcus (31.3%), Staphylococcus aureus (18.7%), Enterococcus faecalis (9.4%), Enterobacter cloacae (18.8%), Pseudomonas aeruginosa (12.5%), Acinetobacter lwoffii (3.1%), and other Gram-negative fermenting rods accounted for 6.2%. Infections were the most common reason for removing the implant after breast reconstruction. and occurred most often as late infections (>30 days after surgery). The time of observation for infectious complications should be at least 1 year.

Antibiotic-resistant Staphylococcus aureus strains of swine origin: molecular typing and susceptibility to oregano (Origanum vulgare L.) essential oil and maqui (Aristotelia chilensis (Molina) Stuntz) extract.

AIM: The molecular typing and the susceptibility of Staphylococcus aureus strains of swine origin to antibiotics, oregano (Origanum vulgare L.) essential oil (EO) and Chilean blackberry maqui (Aristotelia chilensis (Molina) Stuntz) extract were determined. METHODS AND RESULTS: Twenty S. aureus strains of swine origin were subjected to molecular typing, of which six strains were selected for antimicrobial susceptibility testing. The epsilon test (Etest) was used to determine the antibiotic susceptibility. The susceptibility to natural antimicrobials (NAs): oregano EO, maqui extract, thymol (Thy) and carvacrol (Carv), was carried out using the disk diffusion method. The S. aureus strains were genetically diverse. All strains were resistant to at least one class of antibiotic, and two strains were multidrug-resistant. The minimum inhibitory concentration of oregano EO, Thy and Carv was 0·01-0·04%. Maqui extract did not show antistaphylococcal activity. CONCLUSIONS: Natural antimicrobials extracted from oregano have an inhibitory activity against S. aureus strains from swine origin, with no effect using maqui extract. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the characteristics of S. aureus strains of swine origin, and about the potential use of NAs from oregano to enhance the control of antibiotic-resistant S. aureus strains in the pork supply chain.

Acute Respiratory Illness in Rural Haiti.

OBJECTIVES: Acute Respiratory Infection (ARI) is the most common cause of childhood morbidity and mortality in developing countries, including Haiti. Our objective was to detect pathogens found in children with ARI in rural Haiti to help develop evidence-based guidelines for treatment and prevention. METHODS: Retrospective study of students with ARI at four schools in rural Haiti. Viral and/or bacterial pathogens were identified by qPCR in 177 nasal swabs collected from April 2013 through November 2015. RESULTS: Most common viruses detected were Rhinovirus (36%), Influenza A (16%) and Adenovirus (7%), and bacteria were Streptococcus pneumoniae (58%) and Staphylococcus aureus (28%). Compared to older children, children aged 3-5 years had more Influenza A (28% vs. 9%, p=0.002) and Adenovirus detected (14% vs. 3%, p=0.01). Similarly, S. pneumoniae was greatest in children 3-5 years old (71% 3-5yrs; 58% 6-15 years; 25% 16-20 years; p=0.008). Children 3-10 years old presented with fever more than children 11-20 years old (22% vs 7%; p=0.02) and were more often diagnosed with pneumonia (28% vs 4%, p<0.001). CONCLUSIONS: Younger children had increased fever, pneumonia, and detection of Influenza A and S. pneumoniae. These data support the need for influenza and pneumococcus vaccination in early childhood in Haiti.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital

Abstract INTRODUCTION Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. METHODS We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. RESULTS The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. CONCLUSIONS Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.

Molecular characterization and antimicrobial susceptibility of Staphylococcus aureus isolated from children with acute otitis media in Liuzhou, China.

BACKGROUND: There have been few studies focused on the prevalence, bacterial etiology, antibiotic resistance, and genetic background of Staphylococcus aureus (S. aureus) in children with acute otitis media (AOM) in China. METHODS: A retrospective study was conducted in Liuzhou Maternity and Child Healthcare Hospital. Patients younger than 18 years diagnosed with AOM were enrolled in the study. Middle ear fluid specimens were collected and cultured for bacterial pathogens. The antibiotic susceptibility, virulence genes, macrolide resistant genes and sequence types of S. aureus were identified. RESULTS: From January 1, 2013 to December 31, 2015, a total of 228 cases of AOM were identified. Pathogenic bacteria were positive in 181 (79.4%) of 228 specimens. Streptococcus pneumoniae was the most common bacteria (36.4%), followed by S. aureus (16.2%). Among the 37 S. aureus isolates, 12 (23.5%) were methicillin-resistant S. aureus (MRSA), and 25 (77.5%) were methicillin-susceptible S. aureus (MSSA). A total of 23 isolates (62.2%) were resistant to erythromycin, 40.5% of isolates were resistant to clindamycin, and 37.8% isolates were resistant to tetracycline. Twenty-three isolates were multi-drug resistant (MDR) S. aureus. Eighteen isolates carried the pvl gene. Up to 22 (59.4%) isolates expressed ermA gene, 8 (21.6%) isolates expressed both ermA and ermC genes, and only 8.1% expressed ermB. Among all S.aureus isolates, 7 sequence types (STs) were identified by multilocus sequence typing (MLST). The most common ST was ST59 (16/37, 43.2%), followed by ST45 (7/37, 18.9%) and ST30 (7/37, 18.9%). The predominant MSSA isolates were ST59-t437-MSSA (5/25, 20.0%), the prevailing MRSA isolates were Taiwan related strains ST59-SCCmec-IVa/V (5/12, 41.6%). CONCLUSIONS: S. aureus was the second most common cause for AOM in children in Liuzhou. Most of the S. aureus was MDR which carried a high proportion of ermA and ermC gene. CA-MRSA (ST59-SCCmec-IV/V-t437) is circulating in children with AOM. These findings support continued surveillance of S. aureus infections in children with AOM in both communities and hospitals.

Detection of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in Brazilian mastitic milk goats by multiplex-PCR / Detecção de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em leite caprino mastítico no Brasil, por multiplex-PCR

This study evalueted the prevalence of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in milk samples from 257 goats (513 half-udders) and ten bulk tanks, from ten dairy goat farms of São Paulo State, Brazil, by multiplex-PCR. The samples were screened by microbiological culture (gold-standard), and tested by different multiplex-PCR protocols for the detection of each bacterium. A total of 178 half-udders resulted positive by microbiological culture, with coagulase-negative staphylococci (70%), S. aureus (13.5%), S. intermedius (7.9%), and Enterobacteriaceae (4%) the prevalent pathogens. In other way, multiplex-PCR detected 173 pathogens in 151/523 (28.9%; CI95% 25.2-32.9%) milk samples 144/513 (28.1%) half-udders and 7/10 (70%) bulk tanks, with E. coli (86/162, 51.9%) and S. aureus (50/162, 30.9%) the prevalent ones in half-udders, and S. aureus (6/10, 60%) and E. coli (4/5, 36.4%) in bulk tanks. Multiplex-PCR showed a high performance for the detection of three bacteria at a time in mastitic goat milk direct from half-udders or bulk tanks. Thus, this multiplex-PCR protocol proved to be an adequate tool for the identification of the most common mastitis pathogens, independent of their phenotypic characteristics in the diagnosis of clinical mastitis in goats, allowing a continuous and better vigilance and monitoring the herd, being included in quality programs.(AU)

Este estudo avaliou por multiplex-PCR a prevalência de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em amostras de leite de 257 caprinos (513 tetos) e dez tanques de expansão, em dez fazendas leiteiras do estado de São Paulo, Brasil. As amostras foram triadas por cultura microbiológica (padrão-uro) e testadas por diferentes protocolos multiplex-PCR para a detecção de cada bactéria. Um total de 178 amostras de leite foram positivos na cultura microbiológica, com estafilococos coagulase-negativos (70%), S. aureus (13,5%), S. intermedius (7,9%) e Enterobacteriaceae (4%) como patógenos prevalentes. Por outro lado, a PCR multiplex detectou 173 patógenos em 151/523 (28,9%, IC95% 25,2-32,9%) amostras de leite, 144/513 (28,1%) amostras de tetos e 7/10 (70%) em tanques de expansão, E. coli (86/162, 51,9%) e S. aureus (50/162, 30,9%) foram identificados nas amostras de tetos e S. aureus (6/10, 60%) e E. coli (4/5, 36,4%) em tanques expansão. Multiplex-PCR mostrou um alto desempenho para a detecção das três bactérias em leite de cabra com mastite ou em tanques de expansão. Dessa forma, este protocolo multiplex-PCR provou ser uma ferramenta adequada para a identificação dos patógenos mais comuns da mastite, independentemente de suas características fenotípicas no diagnóstico de mastite clínica em caprinos, permitindo uma vigilância contínua e melhor acompanhamento do rebanho, sendo incluído em programas de qualidade.(AU)

The Prevalence and Significance of Staphylococcus aureus in Patients with Non-Cystic Fibrosis Bronchiectasis.

RATIONALE: Staphylococcus aureus is commonly cultured from the sputum of patients with bronchiectasis; however, little is known about the prevalence of the organism in these patients, the characteristics of patients who have grown the organism, or its implications. OBJECTIVES: Determine the relationship between S. aureus and pulmonary function, frequency of exacerbations, and frequency of hospitalization in patients with bronchiectasis Methods: The Bronchiectasis Research Registry is a database of adults with non-cystic fibrosis bronchiectasis identified from 13 sites within the United States. Baseline and follow-up demographic, spirometric, microbiologic, and therapeutic data were entered into a central web-based database. Patients were grouped into three cohorts based on their previous respiratory cultures at the time of entry into the Registry: 1) no prior S. aureus or glucose-nonfermenting gram-negative bacilli (NF-GNB) (Pseudomonas, Stenotrophomonas, or Burkholderia spp.); 2) prior S. aureus at least once; or 3) no prior S. aureus but prior NF-GNB at least once. The association between S. aureus isolation and pulmonary function and frequency of exacerbations and hospital admissions was assessed, both at baseline and after 1 year of follow-up. RESULTS: S. aureus was cultured from 94 of 830 patients (11.3%) included in the analysis. Patients who had grown S. aureus before entry into the Registry had a frequency of prior exacerbations and baseline pulmonary function that was between that of patients who had grown NF-GNB and those who had grown neither NF-GNB or S. aureus. Similarly, at the first follow-up visit after study entry, patients who had grown S. aureus had a frequency of exacerbations and hospitalizations that was between those of patients who had grown NF-GNB and those who had grown neither NF-GNB nor S. aureus. However, in multivariate analysis, S. aureus was not associated with pulmonary function, frequency of exacerbation, or hospital admissions. There were no significant differences in patient characteristics or outcomes between patients who had methicillin-sensitive and methicillin-resistant S. aureus. CONCLUSIONS: Staphylococcus aureus does not appear to be an independent risk factor for severe disease in patients with bronchiectasis enrolled in the Bronchiectasis Research Registry.