Carmen Aspiroz

Detection, molecular characterization, and clonal diversity of methicillin-resistant Staphylococcus aureus CC398 and CC97 in Spanish slaughter pigs of different age groups.

The objective of this study was to determine the frequency of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs, to characterize the recovered isolates, and to investigate their genomic relatedness. Nasal swabs were collected from 53 finishing-pigs (F-pigs) and 53 suckling-piglets (S-piglets) at two different abattoirs in La Rioja (Northern Spain) coming from six production holdings. MRSA isolates were characterized by spa−, agr−, SCCmec−, and multilocus sequence typing, pulsed-field gel electrophoresis (PFGE)-ApaI, toxin gene profiling, antimicrobial susceptibility, and determination of antimicrobial resistance genes. MRSA isolates were recovered from 11 F-pigs (14 isolates) and 26 S-piglets (30 isolates). Forty of the 44MRSA presented the spa-types t011, t108, t1197, and t2346, which corresponded to the sequence type ST398 and to the clonal complex CC398. Interestingly, the remaining four isolates from F-pigs presented the spa-type t3992, and they were ascribed to a new sequence type named ST1379 (a single-locus variant of ST97), which was included in clonal complex CC97. Five PFGE-ApaI clusters with up to nine individual patterns detected among our MRSA and low genomic relatedness was observed between F-pig and S-piglet isolates. All MRSA were positive for hla, hld, and hlg hemolysin genes. ST1379 isolates harbored eta, lukE/D, and hlg-2 toxin genes, whereas ST398 isolates were positive for hlb. A great variety of distinct resistance gene patterns were observed, most of them coming from F-pig isolates. MRSA virulence properties seem to be dependent of the isolate clonal lineage. This study showed that slaughter pigs are frequently colonized by MRSA CC398; moreover, the detection of strains belonging to CC97 underlines that other lineages are also able to spread in livestock. Further studies should assess the risk of CC398 and non-CC398 MRSA to enter the food chain as well as the human health implications.

Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains.

S. aureus nasal carriage was investigated in 278 healthy humans, determining the antibiotic resistance mechanisms, virulence traits, and genetic lineages of recovered isolates. Nasal samples were cultured in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was detected in 53 of 278 nasal samples (19.1%): MRSA was found in one sample (0.4%) and methicillin-susceptible S. aureus (MSSA) in the remaining 52 samples. The MRSA isolate was typed as ST1649-t701-agrI-SCCmec-IVc and only exhibited resistance to beta-lactams. A high diversity of spa types (n=37) was identified among the 52 MSSA, identifying 5 new spa-types. Multi-locus sequence typing (MLST) typing was performed in 30 selected MSSA, detecting 16 different sequence types, 2 of them being new. MSSA strains presented agr types I (30.2%), II (30.2%), III (34%), and IV (5.6%). Eleven strains showed erythromycin resistance and harbored different combinations of erm(A), erm(B), erm(C), erm(T), and msr(A) genes. Two strains exhibited ciprofloxacin resistance, and one of them presented amino acid changes in GyrA and GrlA proteins. The presence of 28 genes encoding staphylococcal toxins was investigated by PCR in all 53 S. aureus isolates. The toxic shock syndrome toxin 1 (TSST-1) gene was detected in 15 MSSA isolates (11 of them typed within the clonal complex CC30) and the gene of exfoliative toxin A in 2 strains. Different combinations of enterotoxin genes were identified among S. aureus strains. None of the S. aureus isolates harbored the Panton-Valentine leukocidin gene. Two MSSA presented the sequence-type ST398 [harboring erm(T) gene], and 2 additional isolates were typed as ST97. Interestingly, MSSA CC398 and CC97 isolates were detected. These clonal complexes are associated with food-producing animals.

Isolation of Malassezia globosa and M. sympodialis from patients with pityriasis versicolor in Spain

Pityriasis versicolor is a superficial infection of the stratum corneum by several yeast species formerly collectively named Malassezia furfur. The genus Malassezia has been recently enlarged with new species. With the exception of M. pachydermatis, the remaining six species have an absolute requirement in vitro for supplementation of long-chain fatty acids in media. These lipophilic yeasts comprise six species: M. furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae and M. sympodialis. The aim of this study was to establish whether there was any association between the various species of Malassezia and pityriasis versicolor lesions. Thus, we studied the isolates from 79 patients with pityriasis versicolor, both from lesions and from apparently healthy skin close to the visible lesions. In pityriasis versicolor lesions, the species most frequently isolated was M. globosa (90%), followed by M. sympodialis (41%). Almost all isolates (99%) belonged to one of these two species. The most frequent pattern was M. globosa as the sole species (58% of cases), although the association with M. sympodialis was also frequent (30%). These results confirmed M. globosa as the main agent of pityriasis versicolor and M. sympodialis as the second agent in importance. Malassezia globosa was found to be a species with high levels of esterase and lipase enzymes of probable importance in their pathogenicity.

Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin

OBJECTIVES To detect the presence of lnu genes in staphylococcal strains with the unusual phenotype lincosamide resistance/macrolide susceptibility (L(R)/M(S)), and to determine their locations and genetic environments. METHODS Six staphylococcal strains of human and animal origin with the phenotype L(R)/M(S) were studied. The presence of 15 resistance genes was tested by PCR. SCCmec typing was performed for all methicillin-resistant strains. agr typing, spa typing and multilocus sequence typing were carried out for Staphylococcus aureus strains. Transformation experiments were carried out by electrotransformation. Plasmid or chromosomal gene location was determined by Southern blot analysis and the genetic environments of the lnu genes were studied in all strains. RESULTS Three methicillin-resistant staphylococcal strains contained the lnu(A) gene. The presence of the pLNU1 plasmid carrying lnu(A) was confirmed in one methicillin-resistant S. aureus (MRSA) ST398-t108 and one methicillin-resistant Staphylococcus sciuri. A novel lnu(A)-carrying plasmid (pUR5425) was identified in one MRSA ST125-t067 strain. Transformants of the three lnu(A)-positive strains presented increased lincomycin MIC values. The remaining three studied staphylococcal strains harboured the lnu(B) gene: two methicillin-susceptible S. aureus (MSSA) ST9-t337 and one MRSA ST398-t011. The lnu(B) gene was embedded in the chromosome in the two MSSA strains and in a large-sized plasmid in the MRSA strain. The same lnu(B) genetic environment was detected in these three strains. CONCLUSIONS The resistance phenotype L(R)/M(S) seems to be related to S. aureus animal-associated clonal lineages (ST398 and ST9). A novel lnu(A)-carrying plasmid was identified and this is the first detection of the lnu(B) gene in MRSA ST398.

Differentiation of three biotypes of Malassezia species on human normal skin. Correspondence with M. globosa, M. sympodialis and M. restricta

One hundred and twenty lipid dependent Malassezia spp. isolates were obtained from the clinically normal skin of 38 healthy adult volunteers by swabbing three different body sites (back, chest and scalp). Ninety-six percent of these strains could be grouped into three biotypes on the basis of microscopic, cultural, metabolic and biochemical (catalase, esculin and lipase (C-14)) characteristics. The differential features were simple to determine and easily reproduced. Moreover, the three biotypes were referable to the species M. globosa (biotype 1), M. sympodialis (biotype 2) and M. restricta (biotype 3). Based on their microscopic features, cultural properties and body site locations, we suggest that biotype 1/ M. globosa corresponds to the description of Pityrosporum orbiculare (round yeast cells with a narrow base, very frequently found on the upper trunk), and biotype 3/ M. restricta corresponds to the concept of P. ovale (oval yeast cells with a broad budding base, located mainly on the scalp). Pleomorphic biotype 2/ M. sympodialis, most frequently found in the back, does not clearly fit into any of the Pityrosporum species.

Treatment of refractory fingernail onychomycosis caused by nondermatophyte molds with methylaminolevulinate photodynamic therapy

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In Vitro Fungicidal Photodynamic Effect of Hypericin on Candida Species

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm−2 (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μm, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm−2). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm−2. C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.

Genome Sequence of Lactococcus garvieae 21881, Isolated in a Case of Human Septicemia

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.

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.

Significant ecological impact on the progression of fluoroquinolone resistance in Escherichia coli with increased community use of moxifloxacin, levofloxacin and amoxicillin/clavulanic acid

OBJECTIVES To determine trends in ciprofloxacin resistance and co-resistance to other antibiotic classes in blood isolates of Escherichia coli, and to investigate if there is an ecological relationship to the community use of fluoroquinolones and other antibiotics. METHODS Forty-two Spanish hospitals of the European Antimicrobial Resistance Surveillance Network collected ciprofloxacin and other antibiotic susceptibility data for non-duplicate consecutive E. coli isolates from patients with bacteraemia between 2001 and 2009. The nationwide ambulatory use of antibiotics between 1997 and 2008 was determined by WHO methods, and the co-evolution of both parameters was further analysed. RESULTS Of the 28 307 E. coli blood isolates, 27.9% were ciprofloxacin non-susceptible (CIPNS), increasing from 17.6% in 2001 to 32.7% in 2009. A continuous increase was observed between CIPNS and other resistances, including cephalosporin resistance due to the production of extended-spectrum β-lactamases (ESBLs) and non-susceptibility to both amoxicillin/clavulanic acid and tobramycin. Although the total use of antibiotics did not increase, community use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid increased by 307.2%, 62.6% and 70.1%, respectively. Yearly rates of CIPNS E. coli strongly correlated with the use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid (r(2 )> 0.80; P < 0.005 in all cases). CONCLUSIONS The rapid increase in CIPNS E. coli causing bacteraemia was closely related to the increase in resistance to amoxicillin/clavulanic acid, production of ESBLs and resistance to aminoglycosides. Community use of fluoroquinolones (mainly moxifloxacin and levofloxacin) and of amoxicillin/clavulanic acid represents a significant driver in the progression of fluoroquinolone resistance in bacteraemic E. coli.