Molecular and phylogenetic characterization of the monkeypox outbreak in the South of Spain.

Background and Aim: Until the May 2022 Monkeypox (MPXV) outbreak, which spread rapidly to many non-endemic countries, the virus was considered a viral zoonosis limited to some African countries. The Andalusian circuit of genomic surveillance was rapidly applied to characterize the MPXV outbreak in the South of Spain. Methods: Whole genome sequencing was used to obtain the genomic profiles of samples collected across the south of Spain, representative of all the provinces of Andalusia. Phylogenetic analysis was used to study the relationship of the isolates and the available sequences of the 2022 outbreak. Results: Whole genome sequencing of a total of 160 MPXV viruses from the different provinces that reported cases were obtained. Interestingly, we report the sequences of MPXV viruses obtained from two patients who died. While one of the isolates bore no noteworthy mutations that explain a potential heightened virulence, in another patient the second consecutive genome sequence, performed after the administration of tecovirimat, uncovered a mutation within the A0A7H0DN30 gene, known to be a prime target for tecovirimat in its Vaccinia counterpart. In general, a low number of mutations were observed in the sequences reported, which were very similar to the reference of the 2022 outbreak (OX044336), as expected from a DNA virus. The samples likely correspond to several introductions of the circulating MPXV viruses from the last outbreak. The virus sequenced from one of the two patients that died presented a mutation in a gene that bears potential connections to drug resistance. This mutation was absent in the initial sequencing before treatment.

A case of fatal monkeypox infection: necropsy and molecular findings, with some considerations related to clinical management.

OBJECTIVE: Human monkeypox (mpox) is usually self-limited infection; however, rising data show a worse outcome in patients with impaired immune status, particularly those co-infected with HIV [Mitjà O, Alemany A, Marks M, Lezama Mora JI, Rodríguez-Aldama JC, Torres Silva MS et al. Mpox in people with advanced HIV infection: A global case series. Lancet. 2023; 401:939-49. DOI:https://doi.org/10.1016/S0140-6736(23)00273-8] [Govind A, Lazarte SM, Kitchell E, Chow JY, Estelle CD, Fixsen E et al. Severe mpox infections in people with uncontrolled human immunodeficiency virus (HIV). Clin Infect Dis. 2023; 76:1843-6. DOI:https://doi.org/10.1093/cid/ciad052]. METHODS: We report the clinical, pathological, and molecular study of a patient with mpox infection and a late HIV diagnosis, with fatal outcome. RESULTS: Necropsy revealed visceral spread of mpox. Mpox virus was sequenced twice during the admission, uncovering an emerging mutation near a genomic region where mutations associated with tecovirimat resistance have been documented. DISCUSSION: Monkeypox can manifest as an opportunistic infection in individuals with advanced HIV-associated immunosuppression.

Usefulness of UF-5000 automatic screening system in UTI diagnosis.

We have evaluated the Sysmex UF-5000 cytometer use in microbiology for the screening of negative urines, looking for cut-off points to detect bacteria and leukocytes. The number of processed urines was 3569, the highest to date in these studies. The best general cut-off point has been 100 bact/µl, giving an area under the ROC curve of 0.868, a sensitivity of 96%, a specificity of 50%, 1.17% of false negatives, and saving 40% of cultures. The PPV and NPV have been 35.5 and 95.4 respectively. The leukocyte count has not been useful. Finally, we have evaluated urine screening usefulness, concluding that in laboratories such as ours (284 urines/working day) or smaller, it is not cost-effective.

Detection of High Level of Co-Infection and the Emergence of Novel SARS CoV-2 Delta-Omicron and Omicron-Omicron Recombinants in the Epidemiological Surveillance of Andalusia.

Recombination is an evolutionary strategy to quickly acquire new viral properties inherited from the parental lineages. The systematic survey of the SARS-CoV-2 genome sequences of the Andalusian genomic surveillance strategy has allowed the detection of an unexpectedly high number of co-infections, which constitute the ideal scenario for the emergence of new recombinants. Whole genome sequence of SARS-CoV-2 has been carried out as part of the genomic surveillance programme. Sample sources included the main hospitals in the Andalusia region. In addition to the increase of co-infections and known recombinants, three novel SARS-CoV-2 delta-omicron and omicron-omicron recombinant variants with two break points have been detected. Our observations document an epidemiological scenario in which co-infection and recombination are detected more frequently. Finally, we describe a family case in which co-infection is followed by the detection of a recombinant made from the two co-infecting variants. This increased number of recombinants raises the risk of emergence of recombinant variants with increased transmissibility and pathogenicity.

In Vitro and In Vivo Virulence Study of Listeria monocytogenes Isolated from the Andalusian Outbreak in 2019.

In 2019, the biggest listeriosis outbreak by Listeria monocytogenes (Lm) in the South of Spain was reported, resulting in the death of three patients from 207 confirmed cases. One strain, belonging to clonal complex 388 (Lm CC388), has been isolated. We aimed to determine the Lm CC388 virulence in comparison with other highly virulent clones such as Lm CC1 and Lm CC4, in vitro and in vivo. Four L. monocytogenes strains (Lm CC388, Lm CC1, Lm CC4 and ATCC 19115) were used. Attachment to human lung epithelial cells (A549 cells) by these strains was characterized by adherence and invasion assays. Their cytotoxicities to A549 cells were evaluated by determining the cells viability. Their hemolysis activity was determined also. A murine intravenous infection model using these was performed to determine the concentration of bacteria in tissues and blood. Lm CC388 interaction with A549 cells is non-significantly higher than that of ATCC 19115 and Lm CC1, and lower than that of Lm CC4. Lm CC388 cytotoxicity is higher than that of ATCC 19115 and Lm CC1, and lower than that of Lm CC4. Moreover, Lm CC388 hemolysis activity is lower than that of the Lm CC4 strain, and higher than that of Lm CC1. Finally, in the murine intravenous infection model by Lm CC388, higher bacterial loads in tissues and at similar levels of Lm CC4 were observed. Although a lower rate of mortality of patients during the listeriosis outbreak in Spain in 2019 has been reported, the Lm CC388 strain has shown a greater or similar pathogenicity level in vitro and in an animal model, like Lm CC1 and Lm CC4.

Assessing the Impact of SARS-CoV-2 Lineages and Mutations on Patient Survival.

OBJECTIVES: More than two years into the COVID-19 pandemic, SARS-CoV-2 still remains a global public health problem. Successive waves of infection have produced new SARS-CoV-2 variants with new mutations for which the impact on COVID-19 severity and patient survival is uncertain. METHODS: A total of 764 SARS-CoV-2 genomes, sequenced from COVID-19 patients, hospitalized from 19th February 2020 to 30 April 2021, along with their clinical data, were used for survival analysis. RESULTS: A significant association of B.1.1.7, the alpha lineage, with patient mortality (log hazard ratio (LHR) = 0.51, C.I. = [0.14,0.88]) was found upon adjustment by all the covariates known to affect COVID-19 prognosis. Moreover, survival analysis of mutations in the SARS-CoV-2 genome revealed 27 of them were significantly associated with higher mortality of patients. Most of these mutations were located in the genes coding for the S, ORF8, and N proteins. CONCLUSIONS: This study illustrates how a combination of genomic and clinical data can provide solid evidence for the impact of viral lineage on patient survival.

Highly accurate whole-genome imputation of SARS-CoV-2 from partial or low-quality sequences.

BACKGROUND: The current SARS-CoV-2 pandemic has emphasized the utility of viral whole-genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and, therefore, useless sequences. Viral sequences evolve in the context of a complex phylogeny and different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data. RESULTS: We have developed the impuSARS application, which takes advantage of the enormous number of SARS-CoV-2 genomes available, using a reference panel containing 239,301 sequences, to produce missing data imputation in viral genomes. ImpuSARS was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing), showing great fidelity when reconstructing the original sequences, recovering the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (<20%). CONCLUSIONS: Imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. ImpuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole-genome sequencing.

Efficacy and safety of a comprehensive educational antimicrobial stewardship program focused on antifungal use.

OBJECTIVE: Few data exist regarding the impact of antimicrobial stewardship programs on antifungal use. We evaluated the efficacy and safety of a comprehensive long-term antimicrobial stewardship program (ASP) focused on antifungal use. METHODS: During a 9-year period, we quarterly assessed antifungal consumption, incidence density of hospital-acquired candidemia, Candida spp. distribution, antifungal resistance, and crude death rate per 1000 occupied bed days (OBDs) of hospital-acquired candidemia. We performed segmented regression analysis of interrupted time series. RESULTS: A significant change in trend was observed for antifungal consumption, with a sustained reduction of -0.87% per quarter (95% confidence interval [CI], -1.36 -0.38, p < 0.001), accounting for a final reduction of -38.4%. The main reduction was produced in fluconazole, with a sustained reduction of -1.37% per quarter (95%CI, -1.96 -0.68, p<0.001). The incidence density of hospital-acquired candidemia decreased, with a change in slope of -5.06% cases per 1000 OBDs per year (95%CI, -8.23 -1.77, p = 0.009). The 14-day crude death rate per 1000 OBDs dropped from 0.044 to 0.017 (-6.36% deaths per 1000 OBDs per year; 95%CI, -13.45 -1.31, p = 0.09). CONCLUSIONS: This ASP has succeeded in optimizing the use of antifungal with a long-lasting reduction without increasing the incidence, neither the mortality, of hospital-acquired candidemia.

Time to positivity of blood cultures in patients with bloodstream infections: A useful prognostic tool / El tiempo de positividad de los hemocultivos: una herramienta pronóstica útil en pacientes con bacteriemia

Objective: The time to positivity (TTP) of blood cultures in patients with bloodstream infections (BSIs) has been considered to be a possible prognostic tool for some bacterial species. However, notable differences have been found between sampling designs and statistical methods in published studies to date, which makes it difficult to compare results or to derive reliable conclusions. Our objective was to evaluate the clinical and microbiological implications of TTP among patients with BSI caused by the most common pathogens. Methods: A total of 361 episodes of BSI were reported for 332 patients. The survival of the entire cohort was measured from the time of blood culture sampling. In order to compare our results with those of previous studies, TTP was divided in three different groups based on log rank (short TTP <12h; medium TTP ≥12h to ≤27h, and long TTP >27h). Cox proportional hazard models were used to calculate crude and adjusted hazard ratios (HR). Results: The Cox proportional hazard model revealed that TTP is an independent predictor of mortality (HR=1.00, p=0.031) in patients with BSIs. A higher mortality was found in the group of patients with the shortest TTP (<12h) (HR=2.100, p=0.047), as well as those with longest TTP (>27h) (HR=3.277, p=0.031). Conclusions: It seems that TTP may provide a useful prognostic tool associated with a higher risk of mortality, not only in patients with shorter TTP, but also in those with longer TTP (AU)

Objetivo: El tiempo de positividad (TP) de los hemocultivos en pacientes con bacteriemia ha sido considerado como una posible herramienta pronóstica. Sin embargo, en los estudios publicados hasta la fecha, hemos observado importantes diferencias tanto en el diseño experimental como en la metodología utilizada. Esto dificulta el poder comparar los resultados obtenidos u obtener conclusiones consistentes. El objetivo de este estudio ha sido evaluar las implicaciones clínicas y microbiológicas del TP en pacientes con bacteriemia causada por los microorganismos más frecuentes, revisando la metodología utilizada en estudios anteriores. Métodos: Se estudiaron un total de 361 episodios de bacteriemia de 332 pacientes. La supervivencia de nuestra cohorte se midió desde que se tomó la muestra de hemocultivo. El TP fue dividido en tres grupos en base al log rank (TP cortos <12h; TP medios ≥12h y ≤27h; TP largos >27h), con el objetivo de comparar nuestros resultados con los obtenidos en estudios previos. Se utilizó el modelo de riesgos proporcionales (Cox) para calcular los hazard ratios (HR) tanto crudos como ajustados. Resultados: El modelo Cox mostró que el TP es un factor independiente relacionado con la mortalidad en pacientes con bacteriemia (HR = 1,00, p = 0,031). Concretamente, encontramos una mayor mortalidad en aquellos pacientes con TP cortos (<12 horas) (HR=2.100, p=0,047), así como en pacientes con TP largos (>27h) (HR=3.277, p=0,031). Conclusiones: En el presente estudio demostramos que el TP puede ser utilizado como una herramienta pronóstica útil de mortalidad no solo en pacientes con TP cortos, sino también en aquellos con TP largos (AU)

Long-Term Impact of an Educational Antimicrobial Stewardship Program on Hospital-Acquired Candidemia and Multidrug-Resistant Bloodstream Infections: A Quasi-Experimental Study of Interrupted Time-Series Analysis.

BACKGROUND: The global crisis of bacterial resistance urges the scientific community to implement intervention programs in healthcare facilities to promote an appropriate use of antibiotics. However, the clinical benefits or the impact on resistance of these interventions has not been definitively proved. METHODS: We designed a quasi-experimental intervention study with an interrupted time-series analysis. A multidisciplinary team conducted a multifaceted educational intervention in our tertiary-care hospital over a 5-year period. The main activity of the program consisted of peer-to-peer educational interviews between counselors and prescribers from all departments to reinforce the principles of the proper use of antibiotics. We assessed antibiotic consumption, incidence density of Candida and multidrug-resistant (MDR) bacteria bloodstream infections (BSIs) and their crude death rate per 1000 occupied bed days (OBDs). RESULTS: A quick and intense reduction in antibiotic consumption occurred 6 months after the implementation of the intervention (change in level, -216.8 defined daily doses per 1000 OBDs; 95% confidence interval, -347.5 to -86.1), and was sustained during subsequent years (average reduction, -19,9%). In addition, the increasing trend observed in the preintervention period for the incidence density of candidemia and MDR BSI (+0.018 cases per 1000 OBDs per quarter; 95% confidence interval, -.003 to .039) reverted toward a decreasing trend of -0.130 per quarter (change in slope, -0.029; -.051 to -.008), and so did the mortality rate (change in slope, -0.015; -.021 to -.008). CONCLUSIONS: This education-based antimicrobial stewardship program was effective in decreasing the incidence and mortality rate of hospital-acquired candidemia and MDR BSI through sustained reduction in antibiotic use.

Comparison of the in vitro activity of ampicillin and moxifloxacin against Listeria monocytogenes at achievable concentrations in the central nervous system.

PURPOSE: The aim of this study was to compare the in vitro activity of ampicillin and moxifloxacin against six isolates selected from 154 invasive clinical isolates of Listeria monocytogenes and evaluate their intra- and extracellular activities with achievable central nervous system concentrations obtained using Monte Carlo simulations with conventional and unconventional dosages. METHODOLOGY: The MICs and minimal bactericidal concentrations (MBCs) of ampicillin and moxifloxacin were determined by using the broth microdilution method. The intra- and extracellular activities were compared using time-kill curves and inhibition of intracellular growth assays. RESULTS: The MICs50/90 of ampicillin were 0.125/0.5 mg l-1 and the MBC50/90 was ≥16 mg l-1, while the moxifloxacin MICs50/90 were 0.25/0.5 mg l-1 and the MBC50/90 was 0.5 mg l-1. Ampicillin did not show any extracellular bactericidal activity at 24 h, although bactericidal activity was detected at 48 h. For moxifloxacin, the bactericidal effect was evident after 6 h of incubation. Both antibiotics achieved significant reductions in intracellular inoculum after 1-24 h of incubation; however, moxifloxacin becomes bactericidal more rapidly, producing a much greater reduction in the inoculum in the first hour than ampicillin. There were no differences among the MIC and MBC values of moxifloxacin and ampicillin among the strains belonging to different serotypes and/or epidemic clones. This fact was also found in the intra- and extracellular studies. CONCLUSION: The results of this study demonstrated the faster bactericidal activity of moxifloxacin at achievable central nervous system concentrations against intra- and extracellular forms of L. monocytogenes in comparison with ampicillin.

Time to positivity of blood cultures in patients with bloodstream infections: A useful prognostic tool.

OBJECTIVE: The time to positivity (TTP) of blood cultures in patients with bloodstream infections (BSIs) has been considered to be a possible prognostic tool for some bacterial species. However, notable differences have been found between sampling designs and statistical methods in published studies to date, which makes it difficult to compare results or to derive reliable conclusions. Our objective was to evaluate the clinical and microbiological implications of TTP among patients with BSI caused by the most common pathogens. METHODS: A total of 361 episodes of BSI were reported for 332 patients. The survival of the entire cohort was measured from the time of blood culture sampling. In order to compare our results with those of previous studies, TTP was divided in three different groups based on log rank (short TTP <12h; medium TTP ≥12h to ≤27h, and long TTP >27h). Cox proportional hazard models were used to calculate crude and adjusted hazard ratios (HR). RESULTS: The Cox proportional hazard model revealed that TTP is an independent predictor of mortality (HR=1.00, p=0.031) in patients with BSIs. A higher mortality was found in the group of patients with the shortest TTP (<12h) (HR=2.100, p=0.047), as well as those with longest TTP (>27h) (HR=3.277, p=0.031). CONCLUSIONS: It seems that TTP may provide a useful prognostic tool associated with a higher risk of mortality, not only in patients with shorter TTP, but also in those with longer TTP.

Clinical characteristics, treatment and outcomes of MRSA bacteraemia in the elderly.

OBJECTIVES: To compare clinical and microbiological characteristics, treatment and outcomes of MRSA bacteraemia among elderly and younger patients. MATERIAL AND METHODS: Prospective study conducted at 21 Spanish hospitals including patients with MRSA bacteraemia diagnosed between June/2008 and December/2009. Episodes diagnosed in patients aged 75 or more years old (≥75) were compared with the rest of them (<75). RESULTS: Out of 579 episodes of MRSA bacteraemia, 231 (39.9%) occurred in patients ≥75. Comorbidity was significantly higher in older patients (Charlson score ≥4: 52.8 vs. 44%; p = .037) as was the severity of the underlying disease (McCabe ≥1: 61.9 vs. 43.4%; p < .001). In this group the acquisition was more frequently health-care related (43.3 vs. 33.9%, p = .023), mostly from long-term care centers (12.1 vs. 3.7%, p < .001). An unknown focus was more frequent among ≥75 (19.9 vs. 13.8%; p = .050) while severity at presentation was similar between groups (Pitt score ≥3: 31.2 vs. 27.6%; p = .352). The prevalence of vancomycin resistant isolates was similar between groups, as was the appropriateness of empirical antibiotic therapy. Early (EM) and overall mortality (OM) were significantly more frequent in the ≥75 group (EM: 12.1 vs. 6%; p = .010 OM: 42.9 vs. 23%; p < .001). In multivariate analysis age ≥75 was an independent risk factor for overall mortality (aOR: 2.47, CI: 1.63-3.74; p < .001). CONCLUSION: MRSA bacteraemia was frequent in patients aged ≥75 of our cohort. This group had higher comorbidity rates and the source of infection was more likely to be unknown. Although no differences were seen in severity or adequacy of empiric therapy, elderly patients showed a higher overall mortality.

Wood impregnation of yeast lees for winemaking.

This study develops a new method to produce more complex wines by means of an indirect diffusion of wood aromas from yeast cell-walls. An exogenous lyophilized biomass was macerated with an ethanol wood extract solution and subsequently dried. Different times were used for the adsorption of polyphenols and volatile compounds to the yeast cell-walls. The analysis of polyphenols and volatile compounds (by HPLC/DAD and GC-MS, respectively) demonstrate that the adsorption/diffusion of these compounds from the wood to the yeast takes place. Red wines were also aged with Saccharomyces cerevisiae lees that had been impregnated with wood aromas and subsequently dried. Four different types of wood were used: chestnut, cherry, acacia and oak. Large differences were observed between the woods studied with regards to their volatile and polyphenolic profiles. Sensory evaluations confirmed large differences even with short-term contact between the wines and the lees, showing that the method could be of interest for red wine making. In addition, the results demonstrate the potential of using woods other than oak in cooperage.

Determining accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus by the microdilution method.

OBJECTIVES: To model the standard broth microdilution method, based on a modified Gompertz function, to obtain accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus (MRSA). The effect of these MIC values on the vancomycin therapeutic target of AUC(0-24)/MIC ≥ 400 was evaluated. METHODS: Three clinical isolates of MRSA with different vancomycin MIC values were used in this model. The optical densities (OD) of each MIC determination were modelled by a non-linear regression method using an F-test. The OD data were adjusted to the Gompertz equation to obtain the MIC values. The mean vancomycin AUC(0-24) obtained with a 30 mg/kg/day dosing schedule was calculated using a Monte Carlo simulation over 5000 subjects, using the pharmacokinetic data obtained in vancomycin-treated patients in our hospital. RESULTS: Although the MIC values obtained with this model were lower than those of the diffusion method (Etest) in all three cases, this did not affect the AUC(0-24)/MIC ratio for the strains with MICs of 1 mg/L by Etest. However, in those strains with MIC values >1 mg/L, the confidence intervals obtained for this ratio included values <400. CONCLUSIONS: The inherent variability of the broth microdilution method could explain the differences in the clinical outcome in MRSA-infected patients treated with vancomycin, mainly in those due to strains with MIC values of 1.5-2 mg/L by Etest, because the corresponding MIC values would range from 0.84 to 1.52 mg/L by the microdilution method, which could affect the therapeutic target.

In vitro and intracellular activities of fosfomycin against clinical strains of Listeria monocytogenes.

This study was designed to evaluate the potential role of fosfomycin as a therapeutic agent in human listeriosis. The in vitro activity of fosfomycin against 154 Listeria monocytogenes clinical isolates under conditions that mimic the induction of prfA expression was determined and was correlated with fosfomycin intracellular antimicrobial activity. In vitro, partial induction of prfA expression is achieved through bacterial growth in brain-heart infusion agar supplemented with activated charcoal (BHIC). A fosfomycin pharmacokinetic/pharmacodynamic breakpoint of ≤64 mg/L was estimated using a Monte Carlo simulation to assess the success of an intravenous fosfomycin dose of 300 mg/kg/day over 5000 individuals. Eighty strains (51.9%) were susceptible to fosfomycin in BHIC, with minimum inhibitory concentrations (MICs) of ≤64 mg/L; 13 strains (8.4%) had the epidemic clone (EC) marker. In addition, 27 strains (17.5%) had a three doubling dilutions reduction in the MIC from ≥1024 mg/L to 128 mg/L (96-128 mg/L by Etest). The fosfomycin modal MIC is lower under prfA expression. However, this effect is smaller in terms of clinical categorisation of isolates and can be influenced by the serotype and clonal type. In A549 cells, the reductions in bacterial inocula of the two susceptible isolates studied after 1h and 24h of incubation with fosfomycin at 0.5× the human maximum serum concentration (Cmax) were 45.8% and 46.6%, and 93.8% and 99.1%, respectively. Slightly higher reductions were found with fosfomycin at 1× Cmax. The resistant strain tested showed significantly lower reductions in all assays.

Antimicrobial susceptibility among E. faecalis and E. faecium from France, Germany, Italy, Spain and the UK (T.E.S.T. Surveillance Study, 2004-2009).

We report on antimicrobial activity against E. faecalis and E. faecium collected in France, Germany, Italy, Spain, and the UK between 2004 and 2009 as part of the Tigecycline Evaluation and Surveillance Trial (UK in vitro data not included due to low isolate numbers). Overall, 1.1% (n=23/2068) of E. faecalis and 11.5% (n=103/893) of E. faecium were vancomycin-resistant. High levels of minocycline-resistant E. faecalis were reported in Germany, Spain, France, and Italy (40.2-44.2%); levofloxacin resistance was high in Germany, Italy, and Spain (31.1-41.6%). Minocycline non-susceptibility increased significantly among E. faecalis in Spain and Italy (P<0.001). No tigecycline-resistant E. faecalis were reported. Among E. faecium, resistance ranged from 72.9% (France) to 93.3% (Germany) for ampicillin, from 56.1% (France) to 90.2% (Germany) for levofloxacin, and from 75.3% (Italy) to 94.7% (Germany) for penicillin. Levofloxacin non-susceptibility increased significantly among E. faecium in France and Spain (P<0.001). The lowest rates of antimicrobial resistance among E. faecium were reported for tigecycline (2/893; 0.2%) and linezolid (3/893; 0.3%).

[Microbiological characterisation of Listeria monocytogenes isolates from human cases in Andalusia]. / Caracterización microbiológica de los aislados de Listeria monocytogenes procedentes de casos humanos en Andalucía.

OBJECTIVE: The aim of this study was to perform a retrospective study by genotyping 154 isolates from human listeriosis cases occurred in the region of Andalusia (southern Spain) in the period 2005-2009. MATERIAL AND METHODS: Serotyping was performed for 1 and 4 somatic antigens using commercial Listeria antisera, and by multiplex-PCR serogrouping according to the method described by Doumith et al. (2004). The antimicrobial susceptibility was performed by Epsilon test and interpreted by CLSI criteria. PFGE was performed according to the PulseNet protocol with the ApaI enzyme. The similarity of PFGE profiles was evaluated using the Bionumerics software. The multiplex PCR protocol described by Chen and Knabel (2007) was used for the identification of isolates belonging to L. monocytogenes ECI, ECII, and ECIII epidemic clones. RESULTS: The 154 isolates were grouped into four serotypes: 4b [94 (61%)] strains, 1/2b [30 (19%)] strains, 1/2a [27 (18%)] strains, and 1/2c [3 (2%)] strains, with 100% of susceptibility to ampicillin and cotrimoxazole. A further sixty-two ApaI distinct pulsotypes were recognized. Thirty-seven isolates (24%) showed unique ApaI pulsotypes, and the remaining 117 strains (76%) were assigned to 25 ApaI clusters (60% in clusters of more than two isolates). The EC markers were found in 62 (40.3%) of the L. monocytogenes isolates tested. The ECI marker was present in 43 (46.2%) 4b serotype isolates, ECII in 10 (10.7%) 4b serotype isolates, and ECIII in 9 (33,3%) 1/2a serotype isolates. DISCUSSION: A large proportion of the human listeriosis cases under investigation could be grouped into molecular subtype clusters, and our cases could be related to international food-borne outbreaks.