Different evolution of S. aureus methicillin-resistant and methicillin-susceptible infections, Argentina.

Staphylococcus aureus-(SA) is widespread among healthcare-associated-(HA) and the community-associated-(CA) infections. However, the contributions of MRSA and MSSA to the SA overall burden remain unclear. In a nationally-representative-survey conducted in Argentina, 668 SA clinical isolates from 61 hospitals were examined in a prospective, cross-sectional, multicenter study in April 2015. The study aimed to analyze MRSA molecular epidemiology, estimate overall SA infection incidence (MSSA, MRSA, and genotypes) in community-onset (CO: HACO, Healthcare-Associated-CO and CACO, Community-Associated-CO) and healthcare-onset (HO: HAHO, Healthcare-associated-HO) infections, stratified by age groups. Additionally temporal evolution was estimated by comparing this study's (2015) incidence values with a previous study (2009) in the same region. Erythromycin-resistant-MSSA and all MRSA strains were genetically typed. The SA total-infections (TI) overall-incidence was 49.1/100,000 monthly-visits, 25.1 and 24.0 for MRSA and MSSA respectively (P = 0.5889), in April 2015. In adults with invasive-infections (INVI), MSSA was 15.7 and MRSA was 11.8 (P = 0.0288), 1.3-fold higher. HA SA infections, both MSSA and MRSA, surpassed CA infections by over threefold. During 2009-2015, there was a significant 23.4 % increase in the SA infections overall-incidence, mainly driven by MSSA, notably a 54.2 % increase in INVI among adults, while MRSA infection rates remained stable. The MSSA rise was accompanied by increased antimicrobial resistance, particularly to erythromycin, linked to MSSA-CC398-t1451-ermT + -IEC+-pvl- emergence. The SA-infections rise was primarily attributed to community-onset-infections (37.3 % and 62.4 % increase for TI and INVI, respectively), particularly HACO-MSSA and HACO-MRSA in adults, as well as CACO-MSSA. The main CA-MRSA-PFGE-typeN-ST30-SCCmecIVc-PVL+/- clone along with other clones (USA300-ST8-IV-LV-PVL+/-, PFGE-typeDD-ST97-IV- PVL-) added to rather than replaced CA-MRSA-PFGE-typeI-ST5-SCCmecIVa-PVL+/- clone in HA invasive-infections. They also displaced clone HA-MRSA-PFGE-typeA-ST5-SCCmecI, mainly in HAHO infections. The overall-burden of SA infections is rising in Argentina, driven primarily by community-onset MSSA, particularly in adults, linked to increased erythromycin-resistance and MSSA-CC398-t1451-ermT + -IEC+-pvl- emergence. Novel knowledge and transmission-control strategies are required for MSSA.

Differential expression patterns of purinergic ectoenzymes and the antioxidative role of IL-6 in hospitalized COVID-19 patient recovery.

Introduction: We have acquired significant knowledge regarding the pathogenesis of severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). However, the underlying mechanisms responsible for disease recovery still need to be fully understood. Methods: To gain insights into critical immune markers involved in COVID-19 etiopathogenesis, we studied the evolution of the immune profile of peripheral blood samples from patients who had recovered from COVID-19 and compared them to subjects with severe acute respiratory illness but negative for SARS-CoV-2 detection (controls). In addition, linear and clustered correlations between different parameters were determined. Results: The data obtained revealed a significant reduction in the frequency of inflammatory monocytes (CD14+CD16+) at hospital discharge vs. admission. Remarkably, nitric oxide (NO) production by the monocyte compartment was significantly reduced at discharge. Furthermore, interleukin (IL)-6 plasma levels were negatively correlated with the frequency of NO+CD14+CD16+ monocytes at hospital admission. However, at the time of hospital release, circulating IL-6 directly correlated with the NO production rate by monocytes. In line with these observations, we found that concomitant with NO diminution, the level of nitrotyrosine (NT) on CD8 T-cells significantly diminished at the time of hospital release. Considering that purinergic signaling constitutes another regulatory system, we analyzed the kinetics of CD39 and CD73 ectoenzyme expression in CD8 T-cells. We found that the frequency of CD39+CD8+ T-cells significantly diminished while the percentage of CD73+ cells increased at hospital discharge. In vitro, IL-6 stimulation of PBMCs from COVID-19 patients diminished the NT levels on CD8 T-cells. A clear differential expression pattern of CD39 and CD73 was observed in the NT+ vs. NT-CD8+ T-cell populations. Discussion: The results suggest that early after infection, IL-6 controls the production of NO, which regulates the levels of NT on CD8 T-cells modifying their effector functions. Intriguingly, in this cytotoxic cell population, the expression of purinergic ectoenzymes is tightly associated with the presence of nitrated surface molecules. Overall, the data obtained contribute to a better understanding of pathogenic mechanisms associated with COVID-19 outcomes.

Health-Related Quality of Life in Patients Living with Wilson Disease in Spain: A Cross-Sectional Observational Study.

Wilson disease (WD) is a rare copper metabolism disorder caused by mutations in the ATP7B gene. It usually affects young individuals and can produce hepatic and/or neurological involvement, potentially affecting health-related quality of life (HRQoL). We assessed HRQoL in a cohort of Spanish patients with WD and evaluated disease impact on several domains of patients' lives, treatment adherence, drug preference and satisfaction, and healthcare resource utilisation in a cross-sectional, retrospective, multicentric, observational study. A total of 102 patients were included: 81.4% presented isolated liver involvement (group H) and 18.6% presented neurological or mixed involvement (group EH). Up to 30% of patients reported a deteriorated emotional status with anxiety and depression, which was greater in the EH subgroup; the use of neuropsychiatric drugs was high. Over 70% of the patients were satisfied with their current treatment but complained about taking too many pills, stating they would consider switching to another more patient-friendly treatment if available. The Simplified Medication Adherence Questionnaire revealed only 22.5% of patients were fully adherent to therapy, suggesting that alternative therapies are needed. This real-world study, even though is highly enriched with hepatic patients and mild disease, shows that WD impacts patients' HRQoL, especially in the emotional domain.

Oxidative stress response in reference and clinical Staphylococcus aureus strains under Linezolid exposure.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) strains are some of the most widespread pathogens with multi-resistant to antimicrobial agents (AA). AA provoke several changes inside bacteria, which cannot be solely explained by the main mechanisms of action reported. OBJECTIVE: The role of oxidative stress in bacteria exposed to bacteriostatic AA has not been widely studied; hence, the aim of our work was to investigate the effect of linezolid (LZD) on S. aureus strains. METHODS: Oxidative stress markers, such as superoxide dismutase (SOD) enzyme activity, the global antioxidant response, advanced oxidation protein products (AOPP) and basal levels of glutathione in 28 clinical and 2 reference strains were measured. RESULTS AND CONCLUSIONS: We identified 10 of 30 strains showing a slight increase in reactive species under LZD treatment with respect to the untreated control (between 22% and 56%). Higher generation was detected in clinical strains compared with the reference strains; however, the impact on the antioxidant response was not significant, and the oxidized protein levels were almost undetectable. The strains exposed to this oxazolidinone did not suffer acute oxidative stress. This is the first work reporting the behaviour of clinical and reference strains of S. aureus exposed to LZD, showing negligible oxidative stress.

MRSA dynamic circulation between the community and the hospital setting: New insights from a cohort study.

Dissemination of methicillin-resistant-Staphylococcus aureus/(MRSA) is a worldwide concern both in hospitals [healthcare-associated-(HA)-MRSA] and communities [community-associated-(CA)-MRSA]. Knowledge on when and where MRSA colonization is acquired and what clones are involved is necessary, to focus efforts for prevention of hospital-acquired MRSA-infections. METHODS: A prospective/longitudinal cohort study was performed in eight Argentina hospitals (Cordoba/ October-December/2014). Surveillance cultures for MRSA (nose-throat-inguinal) were obtained on admission and at discharge. MRSA strains were genetically typed as CA-MRSAG and HA-MRSAG genotypes. RESULTS: Overall, 1419 patients were screened and 534 stayed at hospital for ≥3 days. S. aureus admission prevalence was 30.9% and 4.2% for MRSA. Overall MRSA acquisition rate was 2.3/1000 patient-days-at-risk with a MRSA acquisition prevalence of 1.96% (95%CI: 1.0%-3.4%); 3.2% of patients were discharged back to community with MRSA. CA-MRSAG accounted for 84.6% of imported, 100.0% of hospital-acquired and 94% of discharged MRSA strains. Most imported and acquired MRSA strains belonged to two major epidemic CA-MRSA clones spread in Argentina: PFGEtypeI-ST5-IVa-t311-PVL+ and PFGEtypeN/ST30-IVc-t019-PVL+. CONCLUSIONS: CA-MRSA clones, particularly ST5-IV-PVL+ and ST30-IV-PVL+, with main reservoir in the community, not only enter but also are truly acquired within hospital, causing healthcare-associated-hospital-onset infections, having a transmission capacity greater or similar than HA-MRSAG. This information is essential to develop appropriate MRSA infection prevention-control programs, considering hospital and community.

Synthesis and structure-activity relationships of novel abietane diterpenoids with activity against Staphylococcus aureus.

Aim: To find alternative compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), novel derivatives from dehydroabietic acid were synthesized. Methods & results: Compound 12 was the most effective against 15 MRSA and 11 MSSA with minimum inhibitory concentration values ranging from 3.9 to 15.6 µg/ml. Although less active than 12, compound 11, followed by 25 and 13, also exhibited anti-staphylococcal activity. Additional studies showed that compound 12 is devoid of toxic effect on non-target cells. A structure-activity relationship study revealed that an oxime at C-13 together with a hydroxyl at C-12 could play a key role in the activity. Conclusion: These structures, in particular compound 12, could arise as templates for the development of agents against MRSA and MSSA.

Antibacterial effects of extracts obtained from plants of Argentina: Bioguided isolation of compounds from the anti-infectious medicinal plant Lepechinia meyenii.

ETHNOPHARMACOLOGICAL RELEVANCE: The mostly native species from Argentina are used in traditional medicine generally for the treatment of pain and inflammation, respiratory, gastro-intestinal and urinary disorders and as antiseptics. AIM OF THE STUDY: Since these ailments may be associated with bacterial infections and that it is necessary to discover alternative compounds with antibacterial activity, 69 extracts from these plants were screened for their activity against pathogenic bacteria. The most effective extract was then submitted to bioguided isolation to obtain the compounds responsible for this activity. MATERIALS AND METHODS: Extracts and fractions were screened using agar dilution, and compounds using microbroth dilution methods. A large panel of pathogenic bacteria was used, especially methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Bioguided fractionation was performed using successive chromatographic techniques, while the chemical structures of the isolated compounds were determined by nuclear magnetic resonance (NMR). Additionally, a series of derivatives of the most active compound were prepared in order to study the chemical features required for achieving the antibacterial effect. RESULTS: Lepechinia meyenii (Walp.) Epling (Lamiaceae) extract showed itself the most effective, with minimum inhibitory concentration (MIC) against Gram positive and negative bacteria ranging from 62.5 to 500 µg/mL, and showing better activity on MRSA than on MSSA. Activity-guided fractionation yielded the abietanes carnosol (1), rosmanol (2) and carnosic acid (3) as active principles, with MICs ranging from 15.6-31.2, 15.6-62.5 and 7.8-15.6 µg/mL, respectively against 15 MRSA strains, and 15.6-31.2, 31.2-62.5 and 7.8-15.6 µg/mL, respectively against 11 MSSA strains, maintaining higher activity against the resistant bacteria, as does the extract. In addition, Enterococcus faecalis was sensitive to 1-3 with MICs of 15.6-62.5 µg/mL. The structure activity analysis showed that 12-OH is necessary for remarkable activity, but methylation in C-20 significantly increased this, as observed with 20-methyl carnosate (5) displaying the greatest effect, even more so than 3, with MICs of 3.9 µg/mL against all the tested MRSA and 3.9-7.8 µg/mL against the MSSA. CONCLUSIONS: The results of this study contribute to validate the traditional antibacterial use of species native to Argentina, particularly of L. meyenii. The chemical structures of the compounds obtained may aid the design of antibacterial agents, especially those effective against MRSA.

c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin.

c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase) signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase) pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant component of the complex early machinery mounted to deal with staphylococcal infections.

New patterns of methicillin-resistant Staphylococcus aureus (MRSA) clones, community-associated MRSA genotypes behave like healthcare-associated MRSA genotypes within hospitals, Argentina.

Methicillin-resistant Staphylococcus aureus (MRSA) burden is increasing worldwide in hospitals [healthcare-associated (HA)-MRSA] and in communities [community-associated (CA)-MRSA]. However, the impact of CA-MRSA within hospitals remains limited, particularly in Latin America. A countrywide representative survey of S. aureus infections was performed in Argentina by analyzing 591 clinical isolates from 66 hospitals in a prospective cross-sectional, multicenter study (Nov-2009). This work involved healthcare-onset infections-(HAHO, >48 hospitalization hours) and community-onset (CO) infections [including both, infections (HACO) in patients with healthcare-associated risk-factors (HRFs) and infections (CACO) in those without HRFs]. MRSA strains were genetically typed as CA-MRSA and HA-MRSA genotypes (CA-MRSAG and HA-MRSAG) by SCCmec- and spa-typing, PFGE, MLST and virulence genes profile by PCR. Considering all isolates, 63% were from CO-infections and 55% were MRSA [39% CA-MRSAG and 16% HA-MRSAG]. A significantly higher MRSA proportion among CO- than HAHO-S. aureus infections was detected (58% vs 49%); mainly in children (62% vs 43%). The CA-MRSAG/HA-MRSAG have accounted for 16%/33% of HAHO-, 39%/13% of HACO- and 60.5%/0% of CACO-infections. Regarding the epidemiological associations identified in multivariate models for patients with healthcare-onset CA-MRSAG infections, CA-MRSAG behave like HA-MRSAG within hospitals but children were the highest risk group for healthcare-onset CA-MRSAG infections. Most CA-MRSAG belonged to two major clones: PFGE-type N-ST30-SCCmecIVc-t019-PVL(+) and PFGE-type I-ST5-IV-SCCmecIVa-t311-PVL(+) (45% each). The ST5-IV-PVL(+)/ST30-IV-PVL(+) clones have caused 31%/33% of all infections, 20%/4% of HAHO-, 43%/23% of HACO- and 35%/60% of CACO- infections, with significant differences by age groups (children/adults) and geographical regions. Importantly, an isolate belonging to USA300-0114-(ST8-SCCmecIVa-spat008-PVL(+)-ACME(+)) was detected for the first time in Argentina. Most of HA-MRSAG (66%) were related to the Cordobes/Chilean clone-(PFGE-type A-ST5-SCCmecI-t149) causing 18% of all infections (47% of HAHO- and 13% of HACO-infections). Results strongly suggest that the CA-MRSA clone ST5-IV-PVL(+) has begun to spread within hospitals, replacing the traditional Cordobes/Chilean-HA-MRSA clone ST5-I-PVL(-), mainly in children. Importantly, a growing MRSA reservoir in the community was associated with spreading of two CA-MRSA clones: ST5-IV-PVL(+), mainly in children with HRFs, and ST30-IV-PVL(+) in adults without HRFs. This is the first nationwide study in Argentina providing information about the molecular and clinical epidemiology of CA-MRSA, particularly within hospitals, which is essential for designing effective control measures in this country and worldwide.

Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007-2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6 fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an important increase of invasive CA-MRSA infections-(8.5 fold). In all regions analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%), which showed pulsed-field-gel electrophoresis-(PFGE)-type-"I", sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL(+), accounted for 11.5% of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina belonging to South American-USA300 clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL(+)/ACME(-)) were detected. We also demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec type closely related to SCCmecIV(2B&5). CONCLUSIONS/SIGNIFICANCE: The dissemination of epidemic MRSA clone, ST5-IV-PVL(+) was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003-2008), conversely to other countries. The predominance of this clone, which has capacity to express the h-VISA phenotype, in healthcare-associated community-onset cases suggests that it has infiltrated into hospital-settings.

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina.

BACKGROUND: Community-Associated Methicillin Resistant Staphylococcus aureus (CA-MRSA) has traditionally been related to skin and soft tissue infections in healthy young patients. However, it has now emerged as responsible for severe infections worldwide, for which vancomycin is one of the mainstays of treatment. Infective endocarditis (IE) due to CA-MRSA with heterogeneous vancomycin-intermediate susceptibility-(h-VISA) has been recently reported, associated to an epidemic USA 300 CA-MRSA clone. CASE PRESENTATION: We describe the occurrence of h-VISA phenotype in a case of IE caused by a strain belonging to an epidemic CA-MRSA clone, distinct from USA300, for the first time in Argentina. The isolate h-VISA (SaB2) was recovered from a patient with persistent bacteraemia after a 7-day therapy with vancomycin, which evolved to fatal case of IE complicated with brain abscesses. The initial isolate-(SaB1) was fully vancomycin susceptible (VSSA). Although MRSA SaB2 was vancomycin susceptible (≤ 2 µg/ml) by MIC (agar and broth dilution, E-test and VITEK 2), a slight increase of MIC values between SaB1 and SaB2 isolates was detected by the four MIC methods, particularly for teicoplanin. Moreover, Sab2 was classified as h-VISA by three different screening methods [MHA5T-screening agar, Macromethod-E-test-(MET) and by GRD E-test] and confirmed by population analysis profile-(PAP). In addition, a significant increase in cell-wall thickness was revealed for SaB2 by electron microscopy. Molecular typing showed that both strains, SaB1 and SaB2, belonged to ST5 lineage, carried SCCmecIV, lacked Panton-Valentine leukocidin-(PVL) genes and had indistinguishable PFGE patterns (subtype I2), thereby confirming their isogenic nature. In addition, they were clonally related to the epidemic CA-MRSA clone (pulsotype I) detected in our country. CONCLUSIONS: This report demonstrates the ability of this epidemic CA-MRSA clone, disseminated in some regions of Argentina, to produce severe and rapidly fatal infections such as IE, in addition to its ability to acquire low-level vancomycin resistance; for these reasons, it constitutes a new challenge for the Healthcare System of this country.

Mucoidy, quorum sensing, mismatch repair and antibiotic resistance in Pseudomonas aeruginosa from cystic fibrosis chronic airways infections.

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.

Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells.

Staphylococcus aureus is a pathogen that causes serious infectious diseases eventually leading to septic and toxic shock. Classically S. aureus has been considered an extracellular pathogen, but cumulative evidence indicates that it invades cells and replicates intracellularly leading to staphylococcal persistence and chronic disease. It has been previously shown that this pathogen localizes to LC3-labeled compartments and subverts the autophagy pathway. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. In the present report we present evidence that the pore-forming toxin alpha-hemolysin (Hla) is a S. aureus secreted factor which participates in the activation of the autophagic pathway. In addition, our results indicate that although the toxin elicits an autophagic response this pathway is dysfunctional as indicated by the accumulation of the LC3-II form in cell lysates obtained from intoxicated cells. In addition, not only the purified Hla toxin but also the toxin-secreting pathogen prevented the maturation of autophagosomes. Interestingly, in cells infected with the wild-type strain of S. aureus the bacteria-containing compartments which recruited LC3 onto the limiting membrane did not accumulate the acidotropic probe LysoTracker. In contrast, those phagosomes containing the Hla(-) mutant (unable to produce the toxin) localized in an acidic compartment unlabeled by LC3. These results suggest that the LC3 protein is recruited only to those damaged vacuoles (i.e., perforated by the toxin), perhaps as an attempt to protect the cells. Furthermore, we have demonstrated that the toxin-dependent activation of autophagy (although it is regulated by calcium and requires Atg5) is independent of both PI3Kinase activity and Beclin 1 suggesting the involvement of a non-canonical autophagy pathway.

Emergence and dissemination of a community-associated methicillin-resistant Panton-Valentine leucocidin-positive Staphylococcus aureus clone sharing the sequence type 5 lineage with the most prevalent nosocomial clone in the same region of Argentina.

Epidemiological surveillance for community-associated methicillin-resistant Staphylococcus aureus revealed prevalences of 33% and 13% in pediatric and adult patients, respectively, in Cordoba, Argentina, in 2005. This study describes for the first time the emergence and dissemination of the sequence type 5 (ST5) lineage as the most prevalent clone (89%) (pulsed-field gel electrophoresis type I-ST5-staphylococcal cassette chromosome type IVa-spa type 311) harboring the Panton-Valentine leukocidin and enterotoxin A genes.

Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

Evolution and molecular characterization of methicillin-resistant Staphylococcus aureus epidemic and sporadic clones in Cordoba, Argentina.

Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the "Cordobes clone," has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.

Identification of a novel methicillin-resistant Staphylococcus aureus epidemic clone in Córdoba, Argentina, involved in nosocomial infections.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.