Streptococcus pneumoniae galU gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo.

Streptococcus pneumoniae, the most common cause of bacterial pneumonia, has developed a wide range of virulence factors to evade the immune system of which the polysaccharide capsule is the most important one. Formation of this capsule is dependent on the cps gene locus, but also involves other genes-like galU. The pyrophosphorylase encoded by galU plays a role in the UDP-glucose metabolism of prokaryotes and is required for the biosynthesis of capsular polysaccharides. In this paper, the effect of a galU mutation leading to a dysfunctional UDP-glucose pyrophosphorylase (UDPG:PP) on in vitro biofilm biomass, adherence to lung epithelial cells and macrophage phagocytosis is studied. Last, in vivo virulence using a Galleria mellonella model has been studied. We show that the mutation improves streptococcal adherence to epithelial cells and macrophage phagocytosis in vitro, while there is no definitive correlation on biofilm formation between parent and mutant strains. Moreover, in vivo virulence is attenuated for all mutated strains. Together, these results demonstrate that a galU mutation in S. pneumoniae influences host cell interactions in vitro and in vivo and can strongly influence the outcome of a streptococcal infection. As such, UDPG:PP is worth investigating further as a potential drug target.

First case of Streptococcus lutetiensis bacteremia involving a clindamycin-resistant isolate carrying the lnuB gene.

Here, we describe the first case of a Streptococcus lutetiensis isolate harboring the lnuB gene.

Methicillin-resistant Staphylococcus aureus ST30-SCCmec IVc clone as the major cause of community-acquired invasive infections in Argentina.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have become a major concern worldwide. We conducted a prospective multicenter study of invasive CA-MRSA to evaluate clinical features and genotype of strains causing invasive infections in Argentina. A total of 55 patients with invasive CA-MRSA infections were included. Most patients (60%) had bloodstream infections, 42% required admission to intensive care unit and 16% died. No CA-MRSA isolates were multiresistant (resistant ⩾3 classes of antibiotics). All isolates carried Panton-Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome (SCCmec) type IV. The majority CA-MRSA strains belonged to ST30 and had identical pulsed-field gel electrophoresis (PFGE) patterns, qualifying as a clonal dissemination of a highly transmissible strain. The main clone recovered from patients with CA-MRSA invasive infections was genotyped as pulsed-field gel electrophoresis type C-ST30, SCCmec type IVc-spa type 019, PVL positive. It has become predominant and replaced the previously described CA-MRSA clone (PFGE type A, ST5, SCCmec type IV, spa type 311).

Prevalence and characterization of methicillin-resistant Staphylococcus aureus among healthy children in a city of Argentina.

Community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a major global problem. Healthy carriers of S. aureus strains have an important role in the dissemination of this bacterium. The aim of this study was to estimate the prevalence of S. aureus and methicillin-resistant S. aureus (MRSA) carriage among healthy children in a city of Buenos Aires province, Argentina, and to determine the potential risk factors for its acquisition. We also described the molecular features of MRSA strains circulating in this population. S. aureus carriage was investigated in all children attending the last year of kindergarten during the 2008 school- year period. Household contacts of MRSA carriers were also screened. Of 316 healthy children, 98 (31.0%) carried S. aureus, including 14 MRSA carriers (4.4%) and 84 methicillin susceptible S. aureus (MSSA) carriers (26.6%). All MRSA isolates carried the SCCmec type IV cassette. Eight of the fourteen isolates were closely related to the clone responsible for most severe community-acquired MRSA infections caused in our country (CAA: PFGE A, SCCmec IV, spa t311, ST5). Two subtypes (A(1) and A(2)) were distinguished in this group by PFGE. Both had agr type II and presented the same virulence determinants, except for PVL coding genes and sea that were only harbored by subtype A(1). Our results, based on the analysis of MRSA isolates recovered in the screening of healthy children, provide evidence of a community reservoir of the major CA-MRSA clone described in Argentina.

[Community-acquired methicillin-resistant Staphylococcus aureus infections in a hospital for acute diseases]. / Infecciones adquiridas en la comunidad por Staphylococcus aureus resistente a meticilina en un hospital de agudos.

Community-acquired methicillin-resistant Staphylococcus aureus infections in a hospital for acute diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent pathogens associated with nosocomial infections. However, most recently, MRSA has arisen as an emerging community pathogen, causing serious infections, mainly among young patients. We herein describe 33 cases of infections caused by community-acquired MRSA (C-MRSA), diagnosed between May 2005 and June 2006, at "Eva Perón" Hospital. The isolations were retrospectively studied. Methicillin resistance was confirmed by means of the detection of the mecA gene, and the genes for two virulence factors (Panton-Valentine Leucocidin -PVL- and gamma-haemolysin) as well as the cassette mec type were screened by PCR. All the patients were previously healthy. Four patients under 12, presented bacteremia, one had serious pneumonia, and the three remaining patients had osteoarticular infections; all the patients over 12, had skin and soft tissue infections without systemic damage. The C-MRSA strains harboured cassette mec type IV, and the PVL and gamma-haemolysin genes. They were methicillin-resistant, with no other associated resistances. It is important to consider the presence of these community- acquired strains in order to develop strategies for their correct treatment.

Infecciones adquiridas en la comunidad por Staphylococcus aureus resistente a meticilina en un hospital de agudos / Community-acquired methicillin-resistant Staphylococcus aureus infections in a hospital for acute diseases

Staphylococcus aureus resistente a meticilina (SAMR) es uno de los principales agentes asociados a infecciones intrahospitalarias; sin embargo, en los últimos años ha surgido como un patógeno emergente de la comunidad, causando infecciones graves, principalmente en jóvenes. Se describen 33 casos de infecciones por SAMR de origen comunitario, diagnosticadas entre mayo de 2005 y junio de 2006 en el HIGA "Eva Perón". Se estudiaron retrospectivamente los aislamientos; se confirmó la resistencia a meticilina mediante la detección del gen mecA, se investigó la presencia de genes que codifican dos factores de virulencia (leucocidina de Panton-Valentine -LPV- y g-hemolisina) y el tipo de casete mec mediante PCR. Todos los pacientes se encontraban sanos previamente. Cuatro pacientes menores de 12 años presentaron bacteriemia, uno con neumonía grave y los 3 restantes con infección osteoarticular; todos los pacientes mayores de 12 años presentaron infecciones de piel y partes blandas sin compromiso sistémico. Se constató la presencia de casete mec tipo IV en todos los aislamientos; la resistencia a meticilina no se acompañó de resistencia a otros antimicrobianos; los aislamientos fueron portadores de genes que codifican para LPV y para g-hemolisina. Es importante considerar la presencia de estas cepas de origen comunitario a fin de elaborar estrategias para su correcto tratamiento.

Methicillin- resistant Staphylococcus aureus (MRSA) is one of the most prevalent pathogens associated with nosocomial infections. However, most recently, MRSA has arisen as an emerging community pathogen, causing serious infections, mainly among young patients. We herein describe 33 cases of infections caused by community-acquired MRSA (CMRSA), diagnosed between May 2005 and June 2006, at "Eva Perón" Hospital. The isolations were retrospectively studied. Methicillin resistance was confirmed by means of the detection of the mecA gene, and the genes for two virulence factors (Panton-Valentine Leucocidin -PVL- and g-haemolysin) as well as the cassette mec type were screened by PCR. All the patients were previously healthy. Four patients under 12, presented bacteremia, one had serious pneumonia, and the three remaining patients had osteoarticular infections; all the patients over 12, had skin and soft tissue infections without systemic damage. The C-MRSA strains harboured cassette mec type IV, and the PVL and g-haemolysin genes. They were methicillin-resistant, with no other associated resistances. It is important to consider the presence of these community- acquired strains in order to develop strategies for their correct treatment.

[Surveillance of Neisseria meningitidis in Argentina, 1993-2005: distribution of serogroups, serotypes and serosubtypes isolated from invasive disease]. / Vigilancia de Neisseria meningitidis en Argentina, 1993-2005: distribución de serogrupos, serotipos y serosubtipos causantes de enfermedad invasiva.

Neisseria meningitidis is an important cause of meningitis, bacteremia and septic shock syndrome. We herein present the distribution of serogroups, serotypes and serosubtypes of 2244 isolates of N. meningitidis from patients with meningitis or meningococcemia, received within the period 1993-2005, in the National Reference Laboratory, INEI-ANLIS "Dr. Carlos G. Malbrán", from 33 Argentine hospitals that are included in a National Network devoted to for the study of bacterial meningitis. Between 1993-1995, serogroup B was prevalent (66%) whereas in the period from 1995-2001, serogroup C prevailed (65%). However, following but after that period, the prevalence of serogroup B was recovered. In the last 5 years of the studied period, the serogroups Y and W135 represented as a whole a 15.6% as a whole whereas up to the year 2000 during the first 6 years they accounted for it was of 4.7%. Higher diversity in the distribution of serotypes and serosubtypes was observed within serogroup B. The nonsubtypable isolates throughout the period of study represented the 52.8%, this high percentage demonstrates the limited capacity of the serotyping for the determination of meningococcal/meningococcus subtypes. of meningococco.

Vigilancia de Neisseria meningitidis en Argentina, 1993-2005: distribución de serogrupos, serotipos y serosubtipos causantes de enfermedad invasiva / Surveillance of Neisseria meningitidis in Argentina, 1993-2005: Distribution of serogroups, serotypes and serosubtypes isolated from invasive disease

Neisseria meningitidis es agente causal de enfermedades severas como meningitis, bacteriemia y síndrome de shock séptico. Se presenta la distribución en serogrupos, serotipos y serosubtipos de 2244 aislamientos de N. meningitidis obtenidos de cuadros de meningitis y/o meningococcemia durante el período 1993-2005 y analizados en el Laboratorio Nacional de Referencia del INEI-ANLIS "Dr. Carlos G. Malbrán". Estos aislamientos eran provenientes de 33 hospitales de todo el país, conformados en una red nacional de laboratorios para el estudio de meningitis bacteriana. Durante el período 1993-1995 prevaleció el serogrupo B (66%), mientras que entre los años 1995 y 2001 prevaleció el serogrupo C (65%); a partir de esta fecha se restableció la prevalencia de B. En los últimos 5 años los serogrupos Y y W135 representaron en su conjunto el 15,6%, mientras que hasta el año 2000 no superaron el 4,7%. Se registró mayor diversidad en la distribución de serotipos y serosubtipos dentro del serogrupo B que dentro del serogrupo C. Los aislamientos no subtipables durante todo el período de estudio representaron el 52,8%; este elevado porcentaje evidencia la limitada capacidad de la serología para la determinación de subtipos de meningococo.

Neisseria meningitidis is an important cause of meningitis, bacteremia and septic shock syndrome. We herein present the distribution of serogroups, serotypes and serosubtypes of 2244 isolates of N. meningitidis from patients with meningitis or meningococcemia, received within the period 1993-2005, in the National Reference Laboratory, INEI-ANLIS "Dr. Carlos G. Malbrán", from 33 Argentine hospitals that are included in a National Network devoted to for the study of bacterial meningitis. Between 1993-1995, serogroup B was prevalent (66%) whereas in the period from 1995-2001, serogroup C prevailed (65%). However, following but after that period, the prevalence of serogroup B was recovered. In the last 5 years of the studied period, the serogroups Y and W135 represented as a whole a 15.6% as a whole whereas up to the year 2000 during the first 6 years they accounted for it was of 4.7%. Higher diversity in the distribution of serotypes and serosubtypes was observed within serogroup B. The nonsubtypable isolates throughout the period of study represented the 52.8%, this high percentage demonstrates the limited capacity of the serotyping for the determination of meningococcal/meningococcus subtypes. of meningococco.

Methicillin-resistant Staphylococcus aureus strains in Buenos Aires teaching hospitals: replacement of the multidrug resistant South American clone by another susceptible to rifampin, minocycline and trimethoprim-sulfamethoxazole.

The aim of this study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different infectious sites of hospitalized patients at two university hospitals. Fourteen isolates were analyzed by repetitive sequence based PCR (Rep-PCR), randomly amplified polymorphic DNA assay (RAPD-PCR), and pulsed-field gel electrophoresis (PFGE). We found that a prevalent clone of MRSA, susceptible to rifampin, minocycline, and trimethoprim/sulfamethoxazole (RIF(s), MIN(s), TMS(s)) was present in both hospitals in replacement of the multiresistant MRSA South American clone, previously described in these hospitals. The staphylococcal chromosomal cassette (SCCmec) type I element was detected in this new clone.

Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación / PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination

Haemophilus influenzae es reconocido como un agente patógeno responsable de infecciones localizadas y sistémicas. Se han descrito 6 tipos de polisacáridos capsulares antigénicamente distintos (a, b, c, d, e, y f ) que se pueden identificar por aglutinación en lámina con antisueros específicos. También existen cepas no capsuladas (NC) fenotípicamente no tipificables (NT). La introducción de la vacuna conjugada produjo una marcada disminución de las enfermedades invasivas causadas por H. influenzae tipo b. En este contexto, la tipificación capsular mediante PCR es el método más apropiado para distinguir las cepas no capsuladas de las mutantes b deficientes en cápsula (b-) y detectar la presencia de cepas pertenecientes a otros serotipos que no puedan ser tipificables por aglutinación. Se determinó el genotipo capsular a 38 aislamientos de Haemophilus influenzae no tipificables por aglutinación, derivados al servicio de Bacteriología Clínica del INEI-ANLIS "Dr. Carlos G. Malbrán" en el período 2002-2004. El 78,9% de los aislamientos provenían de hemocultivos y la mayor parte de ellos estaban asociados a foco respiratorio. El 100% de los aislamientos fueron identificados como H. influenzae no capsulados mediante la técnica de PCR.

Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f ) which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b-) and for detecting strains of other serotypes that cannot be detected by slide agglutination. Capsular genotype was studied in 38 isolates of non-typeable Haemophilus influenzae received at INEIANLIS "Dr. Carlos G. Malbrán" between 2002-2004. Of the isolates included in this study 78.9% of them were recovered from blood cultures and most of them were associated with a respiratory focus. By PCR technique 100% of the isolates were identified as non-capsulate H. influenzae and genotype b-was not detected.

Methicillin-resistant Staphylococcus aureus strains in Buenos Aires Teaching Hospitals: replacement of the multidrug resistant South American clone by another susceptible to rifampin, minocycline and trimethoprim-sulfamethoxazole / Staphylococcus aureus meticilina-resistente en hospitales universitarios de Buenos Aires: reemplazo del clon Sudamericano multi-resistente por otro sensible a rifampicina, minociclina y trimetoprima-sulfametoxazol

The aim of this study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different infectious sites of hospitalized patients at two university hospitals. Fourteen isolates were analyzed by repetitive sequence based PCR (Rep-PCR), randomly amplified polymorphic DNA assay (RAPD-PCR), and pulsed-field gel electrophoresis (PFGE). We found that a prevalent clone of MRSA, susceptible to rifampin, minocycline, and trimethoprim/sulfamethoxazole (RIF S, MIN S, TMS S) was present in both hospitals in replacement of the multiresistant MRSA South American clone, previously described in these hospitals. The staphylococcal chromosomal cassette (SCCmec) type I element was detected in this new clone.

El objetivo de este trabajo fue la caracterización de aislamientos de Staphylococcus aureus meticilina-resistentes (SAMR), provenientes de diferentes procesos infecciosos de pacientes internados en dos hospitales universitarios. Catorce aislamientos fueron analizados mediante la PCR de secuencias repetitivas (Rep-PCR), la amplificación al azar de ADN polimórfico (RAPD-PCR) y la electroforesis de campo pulsado (PFGE). Encontramos que un clon prevalente de SAMR, sensible a rifampicina, minociclina y trimetoprima-sulfametoxazol (RIF S, MIN S, TMS S) estaba presente en ambos hospitales, reemplazando al clon SAMR y multi-resistente previamente descrito en estos mismos hospitales. En este nuevo clon se detectó el cassette cromosómico estafilocócico SCCmec tipo I.

Phenotypic and genotypic characterization of macrolide resistant Streptococcus pneumoniae recovered from adult patients with community-acquired pneumonia in an Argentinian teaching hospital.

Streptococcus pneumoniae isolates (n = 262) were recovered from adult patients with community-acquired pneumonia. Erythromycin-resistance levels increased from 9% (1997-1998) to 16% (2000-2002). Sampling for resistance mechanisms prevalent within 19 erythromycin-resistant S. pneumoniae showed mef(E) in 13/19 isolates while 4/19 carried the erm(B) gene (3/19 cMLS(B) and 1/19 iMLS(B) phenotype). MIC ranges for erythromycin and clindamycin were 0.5-16 mg/l and <0.008-0.063 mg/l for the M phenotype, 128-512 mg/l and 128-256 mg/l for the cMLS(B) phenotype, and 4 and <0.008 mg/l for the iMLS(B) phenotype. This is the first report studying the prevalence of macrolide resistance determinants in S. pneumoniae in our country.

[PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination]. / Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación.

Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f ) which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b-) and for detecting strains of other serotypes that cannot be detected by slide agglutination. Capsular genotype was studied in 38 isolates of non-typeable Haemophilus influenzae received at INE-ANLIS "Dr. Carlos G. Malbrán" between 2002-2004. Of the isolates included in this study 78.9% of them were recovered from blood cultures and most of them were associated with a respiratory focus. By PCR technique 100% of the isolates were identified as non-capsulate H. influenzae and genotype b-was not detected.

Methicillin-resistant Staphylococcus aureus strains in Buenos Aires teaching hospitals: replacement of the multidrug resistant South American clone by another susceptible to rifampin, minocycline and trimethoprim-sulfamethoxazole.

The aim of this study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different infectious sites of hospitalized patients at two university hospitals. Fourteen isolates were analyzed by repetitive sequence based PCR (Rep-PCR), randomly amplified polymorphic DNA assay (RAPD-PCR), and pulsed-field gel electrophoresis (PFGE). We found that a prevalent clone of MRSA, susceptible to rifampin, minocycline, and trimethoprim/sulfamethoxazole (RIF(s), MIN(s), TMS(s)) was present in both hospitals in replacement of the multiresistant MRSA South American clone, previously described in these hospitals. The staphylococcal chromosomal cassette (SCCmec) type I element was detected in this new clone.

Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación. / [PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination]

Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f ) which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b-) and for detecting strains of other serotypes that cannot be detected by slide agglutination. Capsular genotype was studied in 38 isolates of non-typeable Haemophilus influenzae received at INE-ANLIS [quot ]Dr. Carlos G. Malbrán[quot ] between 2002-2004. Of the isolates included in this study 78.9

of them were recovered from blood cultures and most of them were associated with a respiratory focus. By PCR technique 100

of the isolates were identified as non-capsulate H. influenzae and genotype b-was not detected.

Invasive Streptococcus pneumoniae isolates from Argentinian children: serotypes, families of pneumococcal surface protein A (PspA) and genetic diversity.

PspA is an antigenically variable virulence factor of Streptococcus pneumoniae that inhibits complement deposition and is a potential candidate for human vaccines. Of 64 published strains 96% are in PspA families 1 and 2; optimal protection is family-specific. Effective development of a PspA-containing vaccine requires more information about the PspA family of strains in parts of the world where the vaccine is most needed. In these studies we observed that of 149 isolates (of 19 capsular types) from Argentina, 54.4% were family 1, 41.6% were family 2 and 4.0% expressed both family 1 and family 2 PspAs. Box typing revealed the Argentinian strains to be from at least 10 clonally related groups.

All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level beta-lactam-resistant clinical isolate of Streptococcus mitis.

All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the beta-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 microg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a pbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.