Antibiotic resistance, biofilm formation, and molecular epidemiology of Staphylococcus aureus in a tertiary hospital in Xiangyang, China.

Staphylococcus aureus is a common clinical pathogen that causes various human infections. The aim of this study was to investigate the antibiotic susceptibility pattern, molecular epidemiological characteristics, and biofilm formation ability of S. aureus isolates from clinical specimens in Xiangyang and to analyze the correlation among them. A total of 111 non-duplicate S. aureus isolates were collected from the Affiliated Hospital of Hubei University of Arts and Science. All isolates were tested for antibacterial susceptibility. Methicillin-resistant S. aureus (MRSA) was identified by the mecA gene PCR amplification. All isolates were analyzed to determine their biofilm-forming ability using the microplate method. The biofilm-related gene was determined using PCR. SCCmec, MLST, and spa types of MRSA strains were performed to ascertain the molecular characteristics. Among the 111 S. aureus isolates, 45 (40.5%) and 66 (59.5%) were MRSA and MSSA, respectively. The resistance of MRSA strains to the tested antibiotics was significantly stronger than that of MSSA strains. All isolates were able to produce biofilm with levels ranging from strong (28.9%, 18.2%), moderate (62.2%, 62.1%), to weak (8.9%, 19.7%). Strong biofilm formation was observed in MRSA strains than in MSSA strains, based on percentages. There were dynamic changes in molecular epidemic characteristics of MRSA isolates in Xiangyang. SCCmecIVa-ST22-t309, SCCmecIVa-ST59-t437, and SCCmecIVa-ST5-t2460 were currently the main epidemic clones in this region. SCCmecIVa-ST5-t2460 and SCCmecIVa/III-ST22-t309 have stronger antibiotic resistance than SCCmecIVa-ST59-t437 strains, with resistance to 6 ~ 8 detected non-ß-lactam antibiotics. The molecular epidemic and resistance attributes of S. aureus should be timely monitored, and effective measures should be adopted to control the clinical infection and spread of the bacteria.

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.

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

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.

Multilocus sequence analysis reveals genetic diversity in Staphylococcus aureus isolate of goat with mastitis persistent after treatment with enrofloxacin.

Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering.

Methicillin-resistant Staphylococcus aureus from infected skin lesions present several virulence genes and are associated with the CC30 in Brazilian children with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease and colonization by Staphylococcus aureus may affect up to 100% of these patients. Virulent and resistant isolates can worsen AD patient clinical condition and jeopardize the treatment. We aimed to detect virulence genes and to evaluate the biofilm production of S. aureus isolates from infected skin lesions of children with AD. Methicillin resistance was detected by phenotypic and molecular tests and the virulence genes were detected by PCR. Biofilm formation was assessed by bacterial growing on microtiter plates and later stained with safranin. Genotyping was performed by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. Among 106 AD patients, 55 (51.8%) had developed S. aureus cutaneous infections and 23 (41.6%) were methicillin-resistant (MRSA). All 55 isolates carried the fnbA, hla, icaA, sasG, and seu genes, and more than 70% presented cna, eap, ebpS, hlg, and pvl genes. Clonal complex (CC) 30 was the main lineage found (34.5%), especially among MRSA isolates (52.2%). The egc cluster and the bbp gene were significantly the most frequent in MRSA isolates and in USA1100/ST30/CC30 lineage. Most of the isolates (74.5%) were non-biofilm producers and many of them only started to produce it in the presence of fibrinogen. There was no significant association between S. aureus isolates features and the AD severity. This study demonstrated a high frequency of CC30 MRSA isolates presenting several virulence genes in infected skin lesions of AD children in Brazil, that may influence the severity of the disease and the treatments required.

Molecular characterization of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from human milk samples in Brazil.

Human milk is the best nutrient for infants. The donor human milk is stored in a milk bank before pasteurization. However, the human milk is not sterile and could be colonized with different types of bacteria. Many studies have shown S. aureus to be the most prevalent potential pathogen detected in human milk. This study characterized 22 methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from raw human milk for the presence of virulence genes and agr type. Moreover, the genotypic as identified characterization was realized. The presence of virulence genes sei, seg, sec, seh, and etb was identified in resistant and sensitive strains. We observed the predominance of agr type II. The presence of SCCmec IV (67%, 4/6) and V (33%, 2/6) characterized resistant strains as CA-MRSA. Endemic lineages detected (ST1635/CC5-t002, ST5/CC5-t002, ST72/CC5-t126, ST1/CC1-t127, ST45/CC45-t065, and ST398/t1451) could be related to epidemic clones, such as USA800/ST5, USA700/ST72, USA400/ST1, USA600/ST45, and ST398. This study made it possible to understand the characteristics of virulence and clonality of some strains that circulate in breast milk in our region. The discovery of human milk colonization by MSSA and MRSA strains with molecular characteristics similar to infectious clones spread globally demonstrates the importance of monitoring strains that can spread and cause serious infections.

Global prevalence and distribution of vancomycin resistant, vancomycin intermediate and heterogeneously vancomycin intermediate Staphylococcus aureus clinical isolates: a systematic review and meta-analysis.

Vancomycin-resistant Staphylococcus aureus (VRSA), Vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) are subject to vancomycin treatment failure. The aim of the present study was to determine their precise prevalence and investigate prevalence variability depending on different years and locations. Several international databases including Medline (PubMed), Embase and Web of Sciences were searched (data from 1997 to 2019) to identify studies that addressed the prevalence of VRSA, VISA and hVISA among human clinical isolates around the world. Subgroup analyses and meta-regression were conducted to indicate potential source of variation. Publication bias was assessed using Egger's test. Statistical analyses were conducted using STATA software (version 14.0). Data analysis showed that VRSA, VISA and hVISA isolates were reported in 23, 50 and 82 studies, with an overall prevalence of 1.5% among 5855 S. aureus isolates, 1.7% among 22,277 strains and 4.6% among 47,721 strains, respectively. The overall prevalence of VRSA, VISA, and hVISA before 2010 was 1.2%, 1.2%, and 4%, respectively, while their prevalence after this year has reached 2.4%, 4.3%, and 5.3%. The results of this study showed that the frequency of VRSA, VISA and hVISA after 2010 represent a 2.0, 3.6 and 1.3-fold increase over prior years. In a subgroup analysis of different strain origins, the highest frequency of VRSA (3.6%) and hVISA (5.2%) was encountered in the USA while VISA (2.1%) was more prevalent in Asia. Meta-regression analysis showed significant increasing of VISA prevalence in recent years (p value ≤ 0.05). Based on the results of case reports (which were not included in the calculations mentioned above), the numbers of VRSA, VISA and hVISA isolates were 12, 24 and 14, respectively, among different continents. Since the prevalence of VRSA, VISA and hVISA has been increasing in recent years (especially in the Asian and American continents), rigorous monitoring of vancomycin treatment, it's the therapeutic response and the definition of appropriate control guidelines depending on geographical regions is highly recommended and essential to prevent the further spread of vancomycin-resistant S. aureus.

Profiles of Staphyloccocus aureus isolated from goat persistent mastitis before and after treatment with enrofloxacin.

BACKGROUND: Staphylococcus aureus is one of the main causative agents of mastitis in small ruminants. Antimicrobial use is the major treatment, but there are many flaws linked to resistance, tolerance or persistence. This study aimed to verify changes in resistance, virulence and clonal profiles of S. aureus isolated from persistent mastitis goat milk before and after enrofloxacin treatment. RESULTS: MIC increased to at least one antimicrobial in S. aureus isolates after enrofloxacin treatment compared to before. The most detected resistance genes before and after treatment were tetK, tetM, and blaZ, with more resistance genes detected after enrofloxacin treatment (p < 0.05). Occasional variations in efflux system gene detection were observed before and after treatment. Nine virulence genes (hla, fnbA, fnbB, eta, etb, sea, sec, seh, and sej) were detected at both times, and between these, the hla and eta genes were detected more in isolates after treatment. All isolates of S. aureus belonged to the same sequence type (ST) 133, except for two S. aureus isolates prior to enrofloxacin treatment which were classified as ST5 and the other as a new one, ST4966. Isolates of S. aureus 4, 8, and 100 from before and after treatment had identical pulse types, while others obtained from other animals before and after treatment were classified into distinct pulse types. CONCLUSION: There were occasional changes in the studied profiles of S. aureus isolated before and after treatment of animals with enrofloxacin, which may have contributed to the permanence of bacteria in the mammary gland, even when using traditional treatment, resulting in persistent mastitis.

Milk microbial composition of Brazilian dairy cows entering the dry period and genomic comparison between Staphylococcus aureus strains susceptible to the bacteriophage vB_SauM-UFV_DC4.

Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.

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

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.

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.

Foodborne disease outbreaks in Barbados (1998-2009): a 12-year review.

INTRODUCTION: Microbes such as Salmonella, Campylobacter and S. aureus have been implicated in Foodborne disease outbreaks (FBDOs) worldwide, yet information on their occurrence in Barbados is scanty. The purpose of this study was to determine the aetiological agents, food vehicles, locations and peak seasons of FBDOs in Barbados; assess the quality of epidemiological investigations; and identify deficiencies in food production practices and laboratory detection. METHODOLOGY: A search of FBDOs occurring in Barbados between 1998-2009 was conducted among published and unpublished literature sources and reports. The search terms included the keywords "foodborne disease," "outbreaks" and "Barbados". RESULTS: During the period 1998 to 2009, there were 24 foodborne outbreaks, 215 cases of illness, one hospitalisation and no deaths. Overall, 37.5% of outbreaks were associated with hotels/resorts. Salmonella Enteritidis phage type 8 was most commonly implicated with eggs and poultry being the primary vehicles. Three outbreak reports were available for assessment and revealed that there were deficiencies in the outbreak investigations. These reports also recorded high levels of food contamination with indicator organisms, suggesting that improvements in food hygiene and production practices were required. CONCLUSIONS: The number of FBDOs is low in comparison to developed countries. However, the data was likely affected by under-reporting and inadequacies in the outbreak investigations and laboratory detection. Improvements in these areas would lead to not only better detection and characterisation of FBDOs in Barbados but improved food safety control measures.

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 epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) isolated from milk of cows with subclinical mastitis.

Bovine mastitis has been a concern for dairy herd for decades. The adaptation capacity of one of the main species responsible for this disease, Staphylococcus aureus (S. aureus), plays a pivotal role in this issue. The aim of this study was to establish a molecular and phenotypic profile of 285 S. aureus strains isolated from milk of subclinical mastitis cows from 18 different farms in São Paulo State using spa typing, multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), agr cluster (I, II, III and IV) typing, PCR for genes including enterotoxins (sea, seb, sec, sed, see, seg, seh, sei), toxic shock syndrome toxin (tsst-1), and Panton-Valentine leucocidin (pvl), as well as in vitro resistance assays for 12 antibiotics. The results showed a wide variety of strains with a high toxigenic potential; concomitantly, sec, seg and seh were prevalent. In addition, we observed a predominance of the spa types t605 (ST 126, CC126) and t127 (ST1, CC1) and the unusual presence of t321 causing bovine mastitis, which has been previously reported only in swine. The most frequent ST were ST126 (70.5%) and ST1 (10.5%). Regarding PFGE, we observed four major groups and six profile patterns. The highest resistance was observed for streptomycin (9.5%), followed by tetracycline (3.5%), clindamycin (9.3%), and erythromycin (2.8%). The tsst-1 gene was detected in 36.8% of isolates and pvl was not observed. One hundred and thirty-six (47.7%) isolates possessed agr type II, followed by types III (20%) and I (8.1%), with type IV not being detected. We observed that the same spa type could result in different PFGE profiles, so the exclusive use of spa type sequences can lead to incorrect interpretations regarding the spread of clones in an epidemiological context.

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)

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)