Genetic and Phenotypic Changes Related to the Development of mec-Independent Oxacillin Non-Susceptibility in ST8 Staphylococcus aureus Recovered after Antibiotic Therapy in a Patient with Bacteremia.

The mec-independent oxacillin non-susceptible S. aureus (MIONSA) strains represent a great clinical challenge, as they are not easily detected and can lead to treatment failure. However, the responsible molecular mechanisms are still very little understood. Here, we studied four clinical ST8-MSSA-t024 isolates recovered during the course of antibiotic treatment from a patient suffering successive episodes of bacteremia. The first isolates (SAMS1, SAMS2, and SAMS3) were susceptible to cefoxitin and oxacillin. The last one (SA2) was susceptible to cefoxitin, resistant to oxacillin, lacked mec genes, and had reduced susceptibility to teicoplanin. SA2 showed higher ß-lactamase activity than SAMS1. However, ß-lactamase hyperproduction could not be linked to oxacillin resistance as it was not inhibited by clavulanic acid, and no genetic changes that could account for its hyperproduction were found. Importantly, we hereby report the in vivo acquisition and coexistence of different adaptive mutations in genes associated with peptidoglycan synthesis (pbp2, rodA, stp1, yjbH, and yvqF/vraT), which is possibly related with the development of oxacillin resistance and reduced susceptibility to teicoplanin in SA2. Using three-dimensional models and PBP binding assays, we demonstrated the high contribution of the SA2 PBP2 Ala450Asp mutation to the observed oxacillin resistance phenotype. Our results should be considered as a warning for physicians and microbiologists in the region, as MIONSA detection and treatment represent an important clinical challenge.

First evaluation in Argentina of the GenoType® MTBDRplus assay for multidrug-resistant Mycobacterium tuberculosis detection from clinical isolates and specimens.

Tuberculosis (tB) and multidrug and extensively drug-resistant (dR) tB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the genotype® mtBdRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using naoH-n-acetyl-l-cystein or naoH-Clna hypertonic solution for Ziehl-neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 °C, 70 of which were selected to be included in this study according to their dR profile. thirty dR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (InH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the InH-R samples were properly identified. mutations in the katGS315t gene and inhA C-15t gene promoter region were present in 59.7 % (37/62) and 25.8 % (16/62) of the InH-R samples, respectively. the system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531l (66.7 %, 28/42), d516V (19.0 %, 8/42), H526Y and S531p/W (4.8 %, 2/42 each one), and S522l/Q (2.4 %, 1/42). a 98.8 % concordance between the genotype assay and sequencing was obtained. genotype® mtBdRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of dR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict InH-R and/or RIF-R in dR risk cases.

[Whooping cough: clinical and epidemiological characteristics of 20 confirmed cases of the Paediatric Hospital of Misiones province]. / Tos convulsa: características clinicoepidemiológicas de 20 casos confirmados atendidos en el Hospital Pediátrico de la provincia de Misiones.

Pertussis or whooping cough is a respiratory disease that has emerged in recent years in several countries including Argentina. The aim is to retrospectively describe clinical and epidemiological characteristics of 20 patients in the Pediatric Hospital of Misiones with confirmed diagnosis of whooping cough, according to criteria set by WHO and CDC, during the years 2005 and 2006. The median age was 4 months, 13 were male (65%). Fourteen patients (70%) were younger than 6 months and 9 (45%) younger than 3 months. All had cough (average duration of 7.6 days), 5 (25%) paroxysmal cough and 1 (5%) apnea and cyanosis. Two children died. Sixteen (80%) had not the three doses of vaccine quadruple, 7 children (35%) were younger than 3 months and had no dose. The disease remains a public health problem affecting not only children but also adults.

Pulsed-field gel electrophoresis, pertactin, pertussis toxin S1 subunit polymorphisms, and surfaceome analysis of vaccine and clinical Bordetella pertussis strains.

To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical isolate. The differing proteins observed have cellular roles associated with amino acid and carbohydrate transport and metabolism. Some of them have been proposed as novel vaccine candidate proteins for other pathogens. Overall, the global strategy described here is presented as a good tool for the development of next-generation acellular vaccines.

Modulation of yersinia type three secretion system by the S1 domain of polynucleotide phosphorylase.

Both low temperatures and encounters with host phagocytes are two stresses that have been relatively well studied in many species of bacteria. Previous work has shown that the exoribonuclease polynucleotide phosphorylase (PNPase) is required for Yersiniae to grow at low temperatures. Here, we show that PNPase also enhances the ability of Yersinia pseudotuberculosis and Yersinia pestis to withstand the killing activities of murine macrophages. PNPase is required for the optimal functioning of the Yersinia type three secretion system (TTSS), an organelle that injects effector proteins directly into host cells. Unexpectedly, the effect of PNPase on the TTSS is independent of its ribonuclease activity and instead requires its S1 RNA binding domain. In contrast, catalytically inactive enzyme does not enhance the low temperature growth effect of PNPase. Surprisingly, wild-type-like TTSS functioning was restored to the pnp mutant strain by expressing just the approximately 70 amino acid S1 domains from either PNPase, RNase R, RNase II, or RpsA. Our findings suggest that PNPase plays multifaceted roles in enhancing Yersinia survival in response to stressful conditions.

Tos convulsa: características clinico epidemiológicas de 20 casos confirmados atendidos en el Hospital Pediátrico de la provincia de Misiones / Whooping cough: clinical and epidemiological characteristics of 20 confirmed cases of the Paediatric Hospital of Misiones province

La tos convulsa (coqueluche o pertussis) es una enfermedad respiratoria que ha resurgido en los últimos años en varios países, incluida la Argentina. El objetivo de este trabajo es describir, en forma retrospectiva, las características clínicas y epidemiológicas de 20 pacientes del Hospital Pediátrico de Misiones con diagnóstico confirmado de coqueluche, según criterios establecidos por la OMS y el CDC, atendidos durante 2005 y 2006. La mediana de edad fue de 4 meses, 13 pacientes eran varones (65 por ciento). Catorce (70 por ciento) eran menores de 6 meses y 9 (45 por ciento) menores de 3 meses. Todos presentaron tos (duración promedio: 7,6 días), 5 (25 por ciento) tos paroxística y 1 (5 por ciento) apnea y cianosis. Dos niños fallecieron. Dieciséis (80 por ciento) no tenían las tres dosis de vacuna cuádruple, 7 niños (35 por ciento) menores de 3 meses, no tenía ninguna dosis. La enfermedad continúa siendo un problema para la salud pública y afecta no sólo a niños sino también a adultos.

Pertussis or whooping cough is a respiratory disease that has emerged in recent years in several countries including Argentina. The aim is to retrospectively describe clinical and epidemiological characteristics of 20 patients in the Pediatric Hospital of Misiones with confirmed diagnosis of whooping cough, according to criteria set by WHO and CDC, during the years 2005 and 2006. The median age was 4 months, 13 were male (65 percent). Fourteen patients (70%) were younger than 6 months and 9 (45 percent) younger than 3 months. All had cough (average duration of 7.6 days), 5 (25 percent) paroxysmal cough and 1 (5 percent) apnea and cyanosis. Two children died. Sixteen (80 percent) had not the three doses of vaccine quadruple, 7 children (35 percent) were younger than 3 months and had no dose. The disease remains a public health problem affecting not only children but also adults.