Epidemiologia molecular e estudo dos fatores de virulência de Staphylococcus aureus resistentes à oxacilina isolados de feridas em pacientes atendidos em unidades básicas de saúde da cidade de Botucatu

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epidemiologia da colonização e infecção microbiana em Unidade de Terapia Intensiva Neonatal: abordagem clínica e molecular

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Capacidade de adesão, invasão e produção de enterotoxinas de Staphylococcus aureus isolados de leite de vacas com mastite subclínica e leite humano de um banco de leite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Capacidade de adesão, invasão e produção de enterotoxinas de Staphylococcus aureus isolados de leite de vacas com mastite subclínica e leite humano de um banco de leite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studio di cavità Fabry-Perot per laser ultrastabili

Lo scopo di questo lavoro di tesi è stato quello di studiare il comportamento di un fascio laser interagente con un risonatore ottico, grazie al quale il laser può essere stabilizzato agganciando la sua frequenza di missione ad uno dei modi della cavità. In sintesi la lunghezza d’onda del fascio è vincolata ad assumere valori multipli della lunghezza della cavità, dato che in questo modo si possono decisamente migliorare le caratteristiche spettrali di un laser tipico. La stabilizzazione, e il restringimento di riga del laser, vengono effettuati agganciando la sua frequenza sul modo trasverso fondamentale tramite un sistema di feedback. La cavità è però soggetta a sua volta a fluttuazioni di tipo termico e meccanico. Una variazione in lunghezza del risonatore comporta una variazione in frequenza dei modi. Le derive di frequenza dovute agli effetti termici si possono limitare utilizzando materiali con bassa dilatazione termica posti in ambienti la cui temperatura viene stabilizzata tramite un sistema di feedback. Per le vibrazioni, invece, il lavoro è più complicato: non essendo sufficiente mettere il sistema in ambienti isolati per attenuare le fluttuazioni, è stato recentemente proposto di studiare la posizione migliore dei sostegni affinché le fluttuazioni, e quindi le conseguenti variazioni in lunghezza della cavità, risultino minime. Per analizzare questo problema è stato utilizzato un software open-source per l’analisi agli elementi finiti, Salome-Meca, tramite il quale è stata riprodotta la geometria del un risonatore ottico a nostra disposizione, per simularne il comportamento sotto l’effetto del campo gravitazionale. Da qui si sono ottenuti i dati riguardo lo spostamento degli specchi della cavità in funzione della posizione del sostegno, dai quali si è riuscito a trovare il punto di posizionamento del supporto capace di ridurre lo spostamento di un ordine di grandezza.

Low rate of methicillin-resistant coagulase-positive staphylococcal colonization of veterinary personnel in Hong Kong

Elevated rates of methicillin-resistant Staphylococcus aureus (MRSA) carriage have been reported in veterinary personnel, suggesting an occupational colonization risk. Hong Kong veterinary personnel (n = 150) were sampled for coagulase-positive staphylococci (CPS) nasal colonization. Risk factors for colonization were assessed by questionnaire. Isolates were identified and antibiotic susceptibility determined. All CPS isolates were investigated for mecA carriage, SCCmec type and PVL genes. Two subjects were colonized with methicillin-resistant CPS: one with MRSA (spa type t002 (CC5), SCCmec type II) and one with methicillin-resistant Staphylococcus pseudintermedius (MRSP) (MLST type ST71, SCCmec type II-III). MLST type ST71 S. pseudintermedius strain is the predominant MRSP clone circulating in dogs in Europe and in Hong Kong. The low MR-CPS colonization rate may be associated with low levels of large animal exposure or low rates of MRSA colonization of companion animals in Hong Kong. Colonization with non-aureus CPS, which may cause human infection, must also be considered in veterinary personnel.

The increase of methicillin-resistant Staphylococcus aureus (MRSA) and the presence of an unusual sequence type ST49 in slaughter pigs in Switzerland

Background: In years past, methicillin-resistant S. aureus (MRSA) has been frequently detected in pigs in Europe, North America and Asia. Recent, yet sporadic studies have revealed a low occurrence of MRSA in Switzerland. In 2009, a monitoring survey of the prevalence and genetic diversity of methicillin-resistant S. aureus (MRSA) in slaughter pigs in Switzerland was conducted using methods recommended by the EU guidelines, and using a sampling strategy evenly distributed throughout the year and representative of the Swiss slaughter pig population. Monitoring should determine if the overall prevalence of MRSA in the entire country is increasing over the years and if specific multi-resistant MRSA clones are spreading over the country.;Results: In 2009, the nasal cavities of eight out of 405 randomly selected pigs were positive for MRSA, representing a prevalence of 2.0% (95% CI 0.9-3.9). The following year, 23 out of 392 pigs were positive for MRSA [5.9% prevalence (95% CI 3.8-8.7)]. Three multilocus sequence types (ST), four spa types and two types of staphylococcal cassette chromosome mec (SCCmec) elements were detected. The most frequent genotypes were ST398 (MLST)-(spa)t034-V (SCCmec) (n = 18) and ST49-t208-V (n = 7), followed by ST398-t011-V (n = 4), ST398-t1451-V (n = 1), and ST1-t2279-IVc (n = 1). The isolates displayed resistance to beta-lactams [mecA, (31/31); blaZ, (19/31)]; tetracycline [tet(M), (31/31); tet(K), (30/31)] (n = 31); macrolides and lincosamides [erm(C) (4/31) or erm(A) (18/31)] (n = 22); tiamulin [vga(A)v (9/31) or unknown mechanism (18/31)] (n = 27); trimethoprim [dfr(G) (18/31); spectinomycin [ant(9)-Ia (19/31) or unknown mechanism (3/31)] (n = 22); streptomycin [str(19/31)]; sulphamethoxazole (7/31) and ciprofloxacin (n = 1) (mechanisms not determined).;Conclusions: This study is the first to describe the presence of MRSA ST49 in slaughter pigs, and to demonstrate a significant and nearly three-fold increase of MRSA prevalence in pigs within two years. The presence of a specific clonal lineage of MRSA from Switzerland suggests that it has been selected in Swiss pig husbandry. Effective hygiene measures should be enhanced within the entire pig production chain to suppress the spread of these pathogens into the community.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Prevalence of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) in children a multicenter cross-sectional study

In this cross-sectional multicenter study, we determined the rate of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in children admitted to 9 training hospitals in Switzerland during 1 month. From 1337 patients, 1363 nasal swabs were obtained (mean age 6.1 years, median 4.7 years, interquartile range 1.3-10.4 years) and 562 (41.3%) grew S. aureus. Only one isolate was MRSA (0.18%) which encoded mecA and femA genes as well as SCCmec type IV, whereas Panton-Valentine leukocidin (PVL) was absent.

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1˜5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

A Staphylococcus xylosus isolate with a new mecC allotype

Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses

OBJECTIVES To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. METHODS Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. RESULTS MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. CONCLUSIONS MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.