María José Revillo

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

BackgroundMolecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).ResultsClinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised.Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively.Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.ConclusionThis study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.

Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis.

The virulence factor gliotoxin (GT) and its inactive derivative, bis(methylthio)gliotoxin (bmGT), are produced by pathogens of the genus Aspergillus. Here we report the detection of GT and bmGT in serum of humans at risk of invasive aspergillosis (IA) as well as in cultures of fungal isolates derived from patients with proven infection with A. fumigatus. Although both compounds are readily recoverable from spiked human serum or plasma, only bmGT is retained in whole blood, indicating that bmGT may be the better marker for in vivo detection. Accordingly, bmGT was found more frequently than GT in samples from patients at risk of IA and incultures of clinical isolates of A. fumigatus. In some cases, bmGT was detected before mycologic evidence ofinfection was gained. Importantly, neither GT nor bmGT was found in serum from healthy donors or from neutropenic patients without any sign of infection. Thus, bmGT presence might provide a more reliable indicator of A. fumigatus infections than GT. Due to its simplicity and sensitivity, a diagnostic technology based on this test could be easily adopted in clinical laboratories to help in the diagnosis of this often fatal fungal infection.

Identification of novel vga(A)-carrying plasmids and a Tn5406-like transposon in meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis of human and animal origin.

Nine staphylococcal strains of human and animal origin with a lincomycin-resistant/erythromycin-susceptible phenotype and carrying vga genes were characterised to determine the genetic elements involved in the dissemination of these uncommon resistance genes. These strains were typed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and/or spa typing. Antimicrobial susceptibility was studied by disk diffusion and agar dilution methods. Presence of the genes lnu(A), lnu(B), vga(A), vga(A)v, vga(B), vga(C), vga(E), lsa(B) and cfr was studied by PCR. Transformation experiments were carried out in all strains, and the plasmid or chromosomal gene location was determined by Southern blot analysis. Genetic environments of the vga genes were analysed by PCR mapping or inverse PCR and sequencing. Five meticillin-resistant Staphylococcus aureus (MRSA) ST398 strains and three Staphylococcus epidermidis strains harboured the gene vga(A), and one MRSA-ST8 strain contained the gene vga(A)v. One MRSA-ST398 strain, which also contained the gene lnu(A), showed the highest minimum inhibitory concentration (MIC) to lincomycin. The vga(A)v-positive strain presented lower MIC values than the vga(A)-positive strains. Presence of the pVGA plasmid was confirmed in two MRSA-ST398 strains. Four novel vga(A)-carrying plasmids were detected: pUR2355 (in two MRSA and one meticillin-susceptible S. epidermidis); pUR4128 (one MRSA); pUR3036 [one meticillin-resistant S. epidermidis (MRSE)]; and pUR3937 (one MRSE). The plasmid pUR4128 was very similar to pUR2355. Plasmids pUR3036 and pUR3937 were related and were very similar to plasmid pSE-12228-06. The gene vga(A)v was located in a transposon analogous to Tn5406. Therefore, four novel vga(A)-carrying plasmids and a variant of Tn5406 were identified in this study.

A photobleaching resistant polymer supported hexanuclear molybdenum iodide cluster for photocatalytic oxygenations and photodynamic inactivation of Staphylococcus aureus

The ability of a hexanuclear molybdenum cluster, [Mo6I8(CH3COO)6]2-, to carry out, upon irradiation, singlet oxygen mediated photocatalytic oxygenation reactions has been tested. This complex has been also supported on a solid polymeric matrix, yielding an immobilized photosensitizer with remarkable photostability and recyclability. Preliminary studies of antibacterial photoinactivation of Staphylococcus aureus have been conducted, with positive results.

Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole-resistant Candida albicans strains.

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole‐resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9‐dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole‐resistant and the azole‐susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light‐emitting diode light fluences (18 and 37 J cm−2). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log10 CFU reduction (0.5 McFarland) was 0.62 μmol l−1 for most strains, whereas for DMMB it ranged between 1.25 and 2.5 μmol l−1. Increasing the fluence to 37 J cm−2 allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log10 reduction (4 McFarland). H2O2 was the main phototoxic species involved in the fungicidal effect of HYP‐aPDT whereas 1O2 was more important for DMMB‐based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: is livestock-contact a risk factor in infections caused by MRSA CC398?

UNLABELLED Tetracycline-resistance (Tet(R)) has been postulated as a marker of the livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage CC398. OBJECTIVES OF THE STUDY to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-Tet(R) strains recovered during 2011-2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n=25); (B) no-contact with livestock-animals (n=42); (C) no information about animal contact (n=31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p<0.05), being the percentage in group C of 61.3%. Most of MRSA-Tet(R)-CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolide-susceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasion-cluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission.

Detection and characterization of a ST6 clone of vanB2-Enterococcus faecalis from three different hospitals in Spain.

Thirteen vancomycin-resistant and teicoplanin-susceptible Enterococcus faecalis isolates were recovered from unrelated patients in three Spanish hospitals from November 2009 to December 2010. All isolates carried the vanB2 gene, showed indistinguishable or closely-related PFGE patterns and were ascribed to the sequence type ST6 (included into the high-risk clonal-complex CC2). They showed a multiresistance phenotype (erythromycin, tetracycline, ciprofloxacin and high-level-resistance to streptomycin, gentamicin and kanamycin) and harboured the aac(6’)-aph(2”), ant(6)-Ia, and tet(M)+/−tet(L) genes. All isolates produced gelatinase and harboured the gelE gene, but not the esp or hyl genes. The inclusion of the vanB2 gene into the Tn5382 transposon was demonstrated in one isolate. Clonal dissemination of vanB2-containing the E. faecalis strain is demonstrated.

Resistencia de Mycobacterium tuberculosis en Zaragoza (1993-1997) y factores asociados

Fundamento Conocer la frecuencia de resistencias de Mycobacterium tuberculosis Zaragozay estudiar los factores asociados. Pacientes y metodo Estudio transversal de la sensibilidad de Mycobacterium tuberculosis el Hospital Universitario Miguel Servet (Zaragoza) entre 1993 y 1997; el metodo utilizado fue el de las proporciones. Se realizo un analisis estadistico con pruebas convencionales utilizando un nivel de significacion de p Resultados Se estudiaron 428 pacientes con cultivo positivo frente a Mycobacterium tuberculosis : 136 (31,8%) VIH positivos, 121 (28,3%) VIH negativos y en 171 (39,9%) no constaba la serologia. Las resistencias al menos a un farmaco se observaron en 47 pacientes (10,9%). En 22 fueron primarias (5,9%) y en 25 pacientes fueron adquiridas (42,4%). La resistencia primaria observada en los VIH positivos fue del 9,2%, mientras que en los VIH negativos fue del 7,5%; la resistencia adquirida tambien fue mayor en los pacientes infectados por el VIH, 51,8% frente al 42,8% de los seronegativos. Cuando se comparo la aparicion de resistencias entre ambos colectivos, no se observaron diferencias estadisticamente significativas. En 20 pacientes aparecieron resistencias multiples (4,7%), y 10 (2,3%) lo eran al menos a isoniacida y rifampicina. Los factores asociados con la aparicion de resistencia adquirida fueron: habito enolico ( odds ratio [OR] = 2,65; intervalo de confianza [IC] del 95%, 1,24–5,65), usuarios de drogas por via parenteral (OR = 2,33; IC del 95%, 1,05–5,17), episodios previos de tuberculosis (OR = 109,40; IC del 95%, 15,02–796,43) y ser transeunte (OR = 3,75; IC del 95%, 1,26–11,17); cuando estudiamos la resistencia primaria, no encontramos diferencias significativas entre los diferentes factores de riesgo. Conclusiones El porcentaje de resistencias a M. tuberculosis observado en Zaragoza es similar a otros descritos en diferentes regiones de Espana. No se han observado diferencias significativas entre seropositivos y seronegativos para el VIH. Los factores asociados significativamente con la aparicion de resistencia adquirida fueron: habito enolico, adiccion a drogas por via parenteral, episodios previos de tuberculosis y ser transeunte.

Long-term molecular surveillance of multidrug-resistant tuberculosis in Spain.

The data presented here span 11 years (1998-2008) of monitoring of multidrug-resistant tuberculosis (MDR-TB) clustering through molecular typing techniques in Spain. The molecular and epidemiological data of 480 multidrug-resistant Mycobacterium tuberculosis complex isolates were analyzed. Thirty-one clusters involving 157 (32.7%) patients were identified. The proportion of immigrants increased substantially over the study period reaching 65% in 2008; however, the clustering rate remained stable indicating that local transmission was little influenced by imported MDR-TB. The three major clusters respond to the persistence of two autochthonous strains throughout the study period and an extensively drug-resistant (XDR) Mycobacterium bovis outbreak with only two cases was reported since 2002. Molecular and epidemiological evidence for the importation of new strains and their spread within the community was found. Immigrant-only clusters most often grouped patients infected abroad with strains belonging to rare spoligotypes. Conversely, widespread spoligotypes of the Latin-American and Mediterranean (LAM) and Haarlem families were responsible for the majority of the MDR-TB local transmission. The demonstration of clusters spanning several Spanish regions that have been ongoing throughout the study period makes it advisable to maintain a continuous molecular surveillance in order to monitor the spread of MDR-TB.

First Spanish Case of Nocardiosis Caused by Nocardia takedensis

ABSTRACT Nocardia takedensis is a recently described species isolated from soil. The first clinical isolate in Japan has recently been reported. This report describes the first clinical isolate of N. takedensis in Spain from a respiratory specimen.