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.

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants.

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

Mycobacteriophage CRB2 defines a new subcluster in mycobacteriophage classification.

Mycobacteriophages are viruses -mostly temperates- that infect Mycobacterium smegmatis and sometimes Mycobacterium tuberculosis. Mycobacteriophages are grouped in clusters on the basis of the overall nucleotide sequence homology, being further divided in subclusters as more mycobacteriophage genomes are sequenced and annotated. As part of our on-going screening for novel isolates, we herein report the bioinformatics analysis of CRB2, a mycobacteriophage belonging into the Siphoviridae family that propagates at 30°C. CRB2 has a 72,217 bp genome with a 69.78% GC content that belongs to Cluster B; nucleotide comparison with other B cluster members positions CRB2 as the sole member of a new subcluster, B9, being mycobacteriophage Saguaro (belonging into subcluster B7) its closest relative. Sequencing and annotation of 14 mycobacteriophages isolated by our group has yielded six cluster A members, a singleton, four of the five members of subcluster B6, one of the three reported members of subcluster G4, and CRB2 which defines subcluster B9. Considering the massive mycobacteriophage search performed in USA and the relatively rarity of our phages, we propose that factors other than size of the sampling determine the variability of mycobacteriophage distribution, and thus a world-wide concerted mining would most likely bring extremely rare and yet undiscovered mycobacteriophages.

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.

Complete genome sequences of nine mycobacteriophages.

Genome analyses of a large number of mycobacteriophages, bacterial viruses that infect members of the genus Mycobacterium, yielded novel enzymes and tools for the genetic manipulation of mycobacteria. We report here the complete genome sequences of nine mycobacteriophages, including a new singleton, isolated using Mycobacterium smegmatis mc(2)155 as a host strain.