Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli.

BACKGROUND: Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts. RESULTS: We report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30-40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl2, mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content. CONCLUSION: This is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His)6-tag location and lysis buffer additives (e.g. N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.

Staphylococcus aureus nasal carriage in health care workers: First report from a major public hospital in Argentina / Portación nasal de Staphylococcus aureus en trabajadores de la salud: primer reporte en un hospital público en Argentina

Staphylococcus aureus causes numerous mild to severe infections in humans, both in health facilities and in the community. Patients and health care workers (HCWs) may disseminate strains during regular medical examinations or hospitalization. The aim of this study was to determine the nasal carriage rate of methicillin-susceptible and methicillin-resistant S. aureus among health care workers at Hospital Provincial del Centenario, a public general hospital in Rosario, Argentina. A transversal study was conducted on 320 health care workers. Nasal swabs were taken and presumptive S. aureus colonies were isolated. Bacterial identity and methicillin resistance status were confirmed by amplification of the nuc and mec genes. Chi square test and Fisher exact test were used for statistical analysis. Of 320 HCWs, 96 (30%) were nasal carriers of S. aureus, 20 of whom (6.3%) carried methicillin-resistant S. aureus (MRSA) and 76 (23.7%) methicillin-susceptible S. aureus (MSSA). Carriage was within thepublished values for physicians (30%) and higher for technicians (57%). Accompanying resistance (62/96, 64.6%) was detected, including resistance to fluoroquinolones (23/96, 24%), aminoglucosides (13/96, 13.5%) or to macrolides (33/96, 34.4%). All the strains were susceptible to vancomycin whereas only 3.1% (3/96), all of them on MSSA strains, were resistant to mupirocin. This study is the first one of its kind in Argentina and one of the few performed in South America, to highlight the relevance of nasal carriage of MRSA and MSSA in health care personnel and brings to light the need for consensus recommendations for regular S. aureus carriage screening as well as for decolonization strategies.

Staphylococcus aureus es agente causal de numerosas infecciones en humanos, que pueden ser desde leves hasta graves, y circula tanto en la comunidad como en las instalaciones de los centros de salud. Los pacientes y los trabajadores de la salud pueden diseminar cepas durante los exámenes médicos de rutina o durante la hospitalización. El foco de este estudio fue determinar la tasa de portación nasal de S. aureus sensible o resistente a meticilina en trabajadores de la salud del Hospital Provincial del Centenario, un hospital público de atención primaria en Argentina. Se llevó a cabo un estudio transversal en 320 trabajadores de la salud (TS). Se tomaron hisopados nasales y se aislaron colonias presuntivas de S. aureus. La identidad de las bacterias y su resistencia a meticilina fueron confirmadas por amplificación de los genes nuc y mec. El análisis estadístico comprendió el test de la chi al cuadrado y el test de exactitud de Fisher. De 320 TS, 96 (30%) fueron portadores nasales de S. aureus, de los cuales 20 (6,3% del total) llevaban cepas de S. aureus resistentes a meticilina (SARM) y 76 (23,7% del total) eran portadores de cepas sensibles a meticilina (SASM). La portación entre los médicos fue del 30% y estuvo dentro de los niveles publicados; dentro del subgrupo del personal técnico la portación fue superior: 57%. Se detectaron resistencias acompañantes (64,6%; 62/96) a fluoroquinolonas (24%; 23/96), aminoglucósidos (13,5%; 13/96) o macrólidos (34,4%; 33/96). Todas las cepas fueron sensibles a vancomicina y solo el 3,1% (3/96), las 3 SASM, fueron resistentes a mupirocina. Este estudio, el primero en su tipo en Argentina y uno de los pocos hechos en América del Sur, remarca la relevancia de la portación nasal de SARM y SASM en el personal de atención de la salud y evidencia la necesidad de contar con recomendaciones consensuadas para el tamizaje regular de S. aureus, así como de estrategias de descolonización.

Staphylococcus aureus nasal carriage in health care workers: First report from a major public hospital in Argentina.

Staphylococcus aureus causes numerous mild to severe infections in humans, both in health facilities and in the community. Patients and health care workers (HCWs) may disseminate strains during regular medical examinations or hospitalization. The aim of this study was to determine the nasal carriage rate of methicillin-susceptible and methicillin-resistant S. aureus among health care workers at Hospital Provincial del Centenario, a public general hospital in Rosario, Argentina. A transversal study was conducted on 320 health care workers. Nasal swabs were taken and presumptive S. aureus colonies were isolated. Bacterial identity and methicillin resistance status were confirmed by amplification of the nuc and mec genes. Chi square test and Fisher exact test were used for statistical analysis. Of 320 HCWs, 96 (30%) were nasal carriers of S. aureus, 20 of whom (6.3%) carried methicillin-resistant S. aureus (MRSA) and 76 (23.7%) methicillin-susceptible S. aureus (MSSA). Carriage was within thepublished values for physicians (30%) and higher for technicians (57%). Accompanying resistance (62/96, 64.6%) was detected, including resistance to fluoroquinolones (23/96, 24%), aminoglucosides (13/96, 13.5%) or to macrolides (33/96, 34.4%). All the strains were susceptible to vancomycin whereas only 3.1% (3/96), all of them on MSSA strains, were resistant to mupirocin. This study is the first one of its kind in Argentina and one of the few performed in South America, to highlight the relevance of nasal carriage of MRSA and MSSA in health care personnel and brings to light the need for consensus recommendations for regular S. aureus carriage screening as well as for decolonization strategies.