Comparación de las características y curso clínico de la endocarditis infecciosa por Staphylococcus aureus meticilino sensible versus meticilino resistente / Characteristics and clinical course of endocarditis due to Methicillin-sensitive versus methicillin-resistant Staphylococcus aureus

Resumen Introducción: Staphylococcus aureus es uno de los agentes causales más comunes de la endocarditis infecciosa. Se reportan pocos estudios en Latinoamérica acerca de las diferencias entre los perfiles de resistencia a la meticilina. Objetivo: Describir las características y el curso clínico de los pacientes con S. aureus sensible a meticilina frente al resistente. Métodos: Estudio observacional retrospectivo, cohorte histórica de pacientes adultos con diagnóstico confirmado de endocarditis entre los años 2011 y 2015. Se seleccionaron pacientes positivos para S. aureus comparando las características y el curso clínico entre los casos S. aureus sensible a meticilina frente al resistente. Resultados: Se estudiaron 86 pacientes con endocarditis. 28 (33%) tenían infección por S. aureus. 21 (75%) tenían endocarditis por S. aureus sensible a meticilina y 7 (25%) por S. aureus resistente a meticilina. En el grupo S. aureus sensible a meticilina, 11 (52,3%) fueron infecciones asociadas a atención en salud. La mayoría de casos de S. aureus resistente a meticilina fueron (85,7%) adquiridos en comunidad. La mortalidad de endocarditis por S. aureus sensible a meticilina fue superior a la causada por el resistente (33,3% vs. 14%). Conclusiones: S. aureus sigue siendo el agente más frecuente en endocarditis, más comúnmente el sensible a la meticilina. Los eventos embólicos y la gravedad fueron mayores en S. aureus sensible a meticilina. La mayor proporción de endocarditis debido a S. aureus resistente a meticilina se adquirió en la comunidad, por lo que se sugiere iniciar cobertura empírica contra S. aureus resistente a meticilina en todo caso de endocarditis adquirida en la comunidad.

Abstract Introduction: Staphylococcus aureus is one of the most common sources of infectious endocarditis. There are few studies in Latin America that report on the differences between the methicillin resistance profiles. Objective: To describe the characteristics and clinical course of patients with methicillin-sensitive S. aureus (MSSA) compared to methicillin-resistance S. aureus (MRSA) Methods: An observational, retrospective study was conducted on a historical cohort of adult patients with a confirmed diagnosis of endocarditis between the years 2011 and 2015. Patients positive for S. aureus were selected and the characteristics and clinical course and the cases of MSSA were compared with those of MRSA. Results: A total of 86 patients with endocarditis were included, of whom 28 (33%) had an infection due to S. aureus, and 21 (75%) had endocarditis due to methicillin-sensitive S. aureus, and 7 (25%) due to MRSA. In the MSSA group, 11 (52.3%) were infections associated with health care. The majority (85.7%) of cases of MRSA were community acquired. The endocarditis mortality due to MSSA was higher than that caused by MRSA (33.3% vs. 14%). Conclusions: S. aureus continues to be the most common agent in endocarditis, with MSSA being more common. The embolic events and the severity were greater in MSSA. The majority of endocarditis due to MRSA is acquired in the community, and for this reason it is suggested starting empirical cover against MRSA in all cases of community acquired endocarditis.

Comparison of effects of staphylococcal nuclease A fused with different exogenous DNA fragments / 生物工程学报

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.

Nasal carriage of Panton Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in healthy preschool children / Colonización nasal de Staphylococcus aureus Meticilino Resistente portador de la Leucocidina Panton-Valentine en preescolares

Objective Determining the prevalence of nasal carriage of S. aureus, both sensitive to methicillin and resistant to it, in preschool children and evaluating the presence of Panton-Valentine leukocidin genes in the isolates. Methods This was a cross-sectional study in which cultures from anterior nares were obtained from healthy preschool children. Isolates were identified as S. aureus based on morphological and biochemical tests. Antibiotic susceptibility profiles were determined by the disk diffusion method. All the isolates were further analyzed by multiplex PCR to determine the presence of mecA and PVL genes; methicillin-resistant isolates were also SCCmec typed by multiplex PCR. Results Overall S. aureus nasal colonization prevalence was 38.5 % and 4.8 % for methicillin-resistant strains. All the methicillin-resistant isolates carried the genes for PVL; two isolates possessed the SCCmec type IV, two were SCCmec type I and one was SCCmec type II. Conclusion This study revealed high PVL-positive, methicillin-resistant S. aureus colonization prevalence in healthy preschool children from Cartagena, which may play a key role in the epidemiology of community-associated infection by methicillin-resistant S. aureus in healthy children from this particular geographical area.

Objetivo Determinar la prevalencia de colonización nasal de S. aureus, tanto sensible como resistente a meticilina, en niños preescolares y evaluar la presencia de los genes de la leucocidina Panton-Valentine en estos aislamientos. Métodos Estudio de corte transversal en el que se realizaron cultivos de flora nasal de niños preescolares. Los aislamientos fueron identificados como S. aureus con base en su morfología y pruebas bioquímicas. La susceptibilidad a antibióticos se determinó por el método de difusión en disco. Todos los aislamientos fueron analizados por PCR múltiple para determinar la presencia de los genes mecA y PVL, y para la tipificación del casete cromosómico SCCmec de los aislamientos resistentes a meticilina. Resultados La colonización nasal por S. aureus fue 38,5 %, y la de cepas meticilino-resistentes fue 4,8 %. Todos los aislamientos SARM portaban los genes para PVL, dos portaban el elemento SCCmec tipo IV, dos fueron tipo I y uno fue tipo II. Conclusión Encontramos una alta prevalencia de colonización por cepas meticilino-resistentes, PVL-positivos en la población estudiada, lo que podría jugar un papel clave en la epidemiología de las infecciones por S.aureus meticilino-resistente en esta área geográfica.

Establishment and identification of classical swine fever virus (CSFV) capsid targeted nuclease expression system / 病毒学报

One pair of primers was designed based on the sequence encoding capsid protein C of classical swine fever virus (CSFV). The C gene fragment was amplified by RT-PCR and PCR products were inserted into eukaryotic expression vector pcDNA-SN containing staphylococcal nuclease (SN) gene resulting in recombinant plasmid pcDNA-C-SN. 48h after transfection of the recombinant into porcine kidney (PK)-15 cells using liposome, the expression of fusion protein was identified through RT-PCR, Western blot and indirect immunofluorescence, and nuclease activity was detected by in vitro DNA digestion assay. The results showed that fusion protein of C-SN was expressed stably in PK-15 cells, and could be identified by rabbit polyclonal antibody against CSFV capsid protein and had good nuclease activity to cleave DNA. Meanwhile, the expressed fusion protein of C-SN in the transfected cells could effectively inhibit the proliferation of CSFV, reducing the infection rate by 10(2)-10(3) times. Our findings laid a foundation for further application of capsid-targeted antiviral strategies for CSFV.

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Evaluation of CHROMagar Staph aureus, a New Chromogenic Medium, for the Detection of Nosocomial Methicillin-Resistant Staphylococcus aureus / 감염

BACKGROUND: Staphylococcus aureus remains one of the most frequently encountered bacterial pathogens and is responsible for a variety of mild to life- threatening infections. There is a substantial body of evidence that individuals who are asymptomatic nasal carriers of S. aureus are at increased risk of developing serious staphylococcal infections. Approximately 20% to 30% of health care workers at any given time are also nasal carriers of S. aureus. A subset of these may spread the organism to patients by direct contact transmission. CHROMagar Staph aureus (CSA) is a new chromogenic medium for identification of S. aureus on the basis of colony pigmentation. METHODS: The abilities of CSA, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates (n=70) and discriminate between S. aureus and coagluase-negative staphylococci (CoNS; n=8) were compared. RESULTS: CSA proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study could be easily differentiated from S. aureus on the medium. The supplementation with 4 microgram/mL of oxacillin allowed simple identification of methicillin resistance in hospital acquired S. aureus strains which show multiple drug resistance profiles. CONCLUSION: CSA proved to be simple and reliable method for the identification of nasal carriers of S. aureus of health care workers.

Asp 280 residue is important in the activity of the Escherichia coli leader peptidase

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.

Conformational features of a peptide model Ac-DTVKLMYKGQPMTFR-NH2, corresponding to an early folding beta hairpin region of staphylococcal nuclease.

Recent H-D exchange 1H NMR studies of the refolding of Staphylococcal nuclease (P117G) variant suggest that, a region of the protein corresponding to a beta hairpin in the native structure folded early in the refolding process. In order to investigate whether the formation of beta hairpin is an early folding event, we investigated the conformational features of the beta hairpin peptide model Ac-DTVKLMYKGQPMTFR-NH2 from Staphylococcal nuclease with 1H NMR techniques. It appears that the peptide aggregates even at a low concentration. However, based on the observation of weak dnn(i, i + 1) NOEs between K8-G9, G9-Q10, an upfield shift of Gly9 NH and a low temperature coefficient (-d delta/dT) for Gly9 NH, we suggest that the sequence YKGQP as part of the beta hairpin peptide model samples conformational forms with reduced conformational entropy and turn potential. The presence of aggregation could be restricting the population of folded conformational forms and formation of beta hairpin at detectable concentrations. We suggest that, formation of beta hairpin could be an early event in the folding of Staphylococcal nuclease and this observation correlates with H-D exchange 1H NMR results and also with the prediction of a protein folding model proposed in literature.

Staphylococcus Aureus, Actividad Termonucleasa y Enterotoxinas en Alimentos / Staphylococcus aureus, thermonuclease activity and enterotoxins in food

Se inocularon 3 tipos de alimentos (embutidos cárnicos, pescados y leche) con diferentes concentraciones de cepas de Staphyloccoccus aureus (FRI-790 y FRI-S6), productoras de enterotoxinas estafilocócica y terminucleasa. Estas sustancias fueron extraídas y detectadas de los alimentos por métodos bioquímicos e inmunológicos. Todas las muestras dieron resultados positivos cuando el conteo de los microorganismos fuemayor menor que 10(6) ufc/g de alimento

Enterotoxigenicidad, actividad termonucleasa y fagotipia en cepas de staphylococcus aureus aislada / Enterotoxigenicity thermonuclease activity, and phago type staphylococus aureus strains isolated in food

Se estudiaron 51 cepas de Staphylococcus aureus involucradas en brotes de intoxicación alimentaria ocurridos en 13 provincias del país. La detección de las toxinas A y C se realizó por el método de Ouchterlony. Se utilizó la prueba la lachica para la determinación de la termonucleasa y el fagotipaje se empleó el método normalizado por el Ministerio de Salud Pública. La enterotoxina del serotipo A fue predominante con el 31,1 %de positividad. El 96 %de las cepas enterotoxigénicas fueron positivas a la prueba de producción de termonucleasa. Los grupos fagos indicaron la procedencia humana de las cepas