At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazoleresistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-g metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-g disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-g metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.Clostridium difficile infection (CDI) is a growing problem and the most common cause of hospital-acquired infectious diarrhea (3,11,17,18). The first-line drugs for the therapy of CDI are metronidazole and vancomycin, although metronidazole is preferred to vancomycin because of its lower cost and because, purportedly, it does not increase the appearance of vancomycin-resistant enterococci. Most CDIs respond to either drug; however, both drugs have been associated with variable relapse rates ranging from 7 to 20% (1,20,23). Reports of C. difficile strains showing resistance to metronidazole are highly unusual in the literature, with only anecdotal descriptions (2,5,6,16,19,21,29). However, in a previous study, we reported that 6.3% of 415 C. difficile isolates, recovered over a period of 8 years in our institution, were resistant to metronidazole (22).Recently, we observed that initial metronidazole MICs of 16 to 64 mg/liter against primary fresh, toxigenic C. difficile isolates, as determined by the standar...

The in vitro activities of tigecycline and other antimicrobials against 51 isolates of Nocardia spp. were evaluated. MIC 90 s and MIC ranges were as follows: tigecycline, 4 and <0.06 to 8 mg/liter, respectively; minocycline, 2 and <0.06 to 2 mg/liter, respectively; linezolid, 1 and <0.06 to 2 mg/liter, respectively; moxifloxacin, 2 and <0.06 to >64 mg/liter, respectively; ertapenem, 32 and <0.06->64 mg/liter, respectively; imipenem, 2 and <0.06 to >64 mg/liter, respectively; meropenem, 8 and <0.06 to >64 mg/liter, respectively; amikacin, 1 and <0.06 to 32 mg/liter, respectively; and trimethoprim-sulfamethoxazole, 1/19 and <0.5/9.5 to >2/38 mg/liter, respectively.Nocardia species cause serious infections, especially in immunocompromised patients (4, 6, 16). Trimethoprim-sulfamethoxazole has traditionally been the agent of choice for the treatment of nocardiosis, with alternative drugs including minocycline, amikacin, and imipenem (4, 16). Resistance to the previous drugs, toxicity, intolerance, and therapeutic failures justify the search for alternative antimicrobial agents. The activity of tigecycline (15) against Nocardia spp. has never been evaluated; and there is very little information regarding the activities of linezolid, moxifloxacin, and ertapenem (1, 2, 5, 10, 20). In the study described here, we compared the in vitro activities of tigecycline, linezolid, ertapenem, and moxifloxacin with those of minocycline, imipenem, meropenem, amikacin, and trimethoprim-sulfamethoxazole against 51 nonduplicate clinical isolates of different Nocardia species.(This study was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004 [E. Cercenado, M. Marín, J. Martínez-Alarcón, and E. Bouza, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-2062, 2006].).The isolates were obtained from 1995 to 2006 in our laboratory from respiratory samples (n ϭ 41), cutaneous abscesses (n ϭ 5), brain abscesses (n ϭ 2), blood (n ϭ 2), and cerebrospinal fluid (n ϭ 1). Molecular identification was performed by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene (hsp65 PCR-RFLP) with PCR primers TB11 and TB12 and restriction analysis with BstEII, MspI, HinfI, and BsaHI, as described previously (17, 18). Identification was confirmed by sequencing the first 500 bp of the 16S rRNA gene (7,12,14). A 5Ј-end 16S rRNA gene-specific PCR was performed with universal primers E8F and E533F. The amplicons obtained were sequenced by the BigDye Terminator method and were detected with an ABI Prism 3100 automatic DNA sequencer (Applied Byosystems, Inc.). The sequences of well-characterized Nocardia isolates (14) deposited in GenBank were used as references for phylogenetic tree construction with ClustalX 1.8 software. Only identifications with 100% similarity were considered. The strains were stored at Ϫ70°C in skim milk until they were tested for their susceptibilities.The solutions of the antimicrobials and the cation-adjusted Mueller-Hinton broth us...

This multicenter, population-based study evaluated the laboratory workload produced by zygomycetes and the number of cases of zygomycosis in Spain during 2005. Less than 8% of the patients who harbored zygomycete isolates had zygomycosis. The incidence of zygomycosis (6 cases) was 0.43 cases/1,000,000 inhabitants and 0.62 cases/100,000 hospital admissions.

We evaluated the activities of amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole against zygomycetes by CLSI M38-A, Etest and Sensititre. The most active drug was posaconazole, followed by amphotericin B and itraconazole. The correlation of the Etest and Sensititre with CLSI M38-A was moderate for posaconazole but poor for the others.The incidence of zygomycosis is increasing in some institutions, purportedly due to voriconazole prophylaxis (16,19,20,22,23,25,28,31,32). The introduction of new antifungal agents and the availability of alternative antifungal susceptibility procedures necessitate studies comparing the in vitro activity of these new antifungal agents against zygomycetes.In this study, the in vitro activities of amphotericin B (AMB), itraconazole (ITC), voriconazole (VC), caspofungin (CAS), and posaconazole (POS) were assessed against 45 clinical strains of zygomycetes (Table 1). In addition, we compared the results obtained by the CLSI M38-A method with the Etest and Sensititre YeastOne.CLSI M38-A method. The antifungal drugs used were AMB (Sigma Chemical Co., St. Louis, MO), ITC (Janssen Pharmaceutical Research and Development, Madrid, Spain), VC (Pfizer Pharmaceutical Group, New York, NY), POS (ScheringPlough, Kenilworth, NJ), and CAS (Merck Research Laboratories, Rahway, NJ). The broth microdilution method was performed according to CLSI guidelines (21). The final concentration of the VC, ITC, POS, and AMB in the wells ranged from 0.03 to 16 g/ml. The final concentration of CAS in the wells ranged from 0.250 to 256 g/ml. The MIC endpoint was defined as the lowest concentration producing complete inhibition of growth (MIC-0) for all the antifungal drugs studied. The minimum effective concentration (MEC) endpoint for CAS was defined as the lowest concentration at which an abnormal growth was observed.Quality control was ensured by testing Aspergillus flavus ATCC 204304 and Aspergillus fumigatus ATCC 204305. All results were within the recommended limits of CLSI M38-A.Etest and Sensititre YeastOne procedures. Etest strips (AB Biodisk, Solna, Sweden) of ITC, VC, CAS, and AMB were supplied by Tec Laim (Madrid, Spain), and those of POS were supplied by Schering-Plough (Kenilworth, NJ). Inoculum suspensions were prepared in the same way as for the CLSI method. The MIC was defined as the lowest drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the antifungal strip.The activities of AMB, VC, ITC, and CAS were studied by means of Sensititre YeastOne (Trek Diagnostic Systems, Ltd., East Grinstead, United Kingdom). The trays containing serial twofold dilutions of the drug (0.008 to 16 g/ml) were used. Inoculum suspensions were prepared in the same way as for the CLSI method, and the adjusted suspensions were diluted at 1:100 in RPMI.The CLSI microtiter panels, Sensititre panels, and Etest plates were incubated at 35°C and read after 16,24, 36, 48, and 72 h of incubation.We compared CLSI M38-A results (incubation at 24 h) with the altern...

The Sensititre YeastOne (SYO) method is a widely used method to determine the susceptibility of Candida spp. to antifungal agents. CLSI clinical breakpoints (CBP) have been reported for antifungals, but not using this method. In the absence of CBP, epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (those without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of any susceptibility test. The ECVs for five agents, obtained using the MIC distributions determined by the SYO test, were calculated and contrasted with those for three statistical methods and the MIC 50

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions ( R e < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Influenza epidemics affect all age groups, although children, the elderly and those with underlying medical conditions are the most severely affected. Whereas co-morbidities are present in 50 % of fatal cases, 25-50 % of deaths are in apparently healthy individuals. This suggests underlying genetic determinants that govern infection severity. Although some viral factors that contribute to influenza disease are known, the role of host genetic factors remains undetermined. Data for small cohorts of influenza-infected patients are contradictory regarding the potential role of chemokine receptor 5 deficiency (CCR5-D32 mutation, a 32 bp deletion in the CCR5 gene) in the outcome of influenza virus infection. We tested 171 respiratory samples from influenza patients (2009 pandemic) for CCR5-D32 and evaluated its correlation with patient mortality. CCR5-D32 patients (17.4 %) showed a higher mortality rate than WT individuals (4.7 %; P50.021), which indicates that CCR5-D32 patients are at higher risk than the normal population of a fatal outcome in influenza infection. Influenza A viruses are a major source of acute respiratory infections and continue to be an important cause of acute illness and death worldwide. They cause annual epidemics and occasional pandemics with potentially fatal outcome. The mean global burden of seasonal influenza is more than 600 million cases, with 3 million cases of severe illness and almost 500 000 deaths per year worldwide (http://www. who.int/en/). A new H1N1 subtype influenza A virus emerged in 2009 [A(H1N1)pdm09], which was highly transmissible with relatively low virulence and caused the first pandemic of the 21st century (Neumann et al., 2009).Differences in disease severity can be due to pre-existing health conditions, predisposing host genetic factors, differences in the virulence of circulating viruses or a combination of these factors. The co-morbid conditions for A(H1N1)pdm09 include chronic metabolic disease, primarily diabetes mellitus and renal disease, chronic lung and cardiac disease, immunosuppressive conditions, neoplasms, obesity and pregnancy (Falagas et al., 2011; Louie et al., 2011;Singanayagam et al., 2011).Although co-morbidities are present in half of fatal cases, one-third of fatal cases have no co-morbid conditions (http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm), suggesting that host genetic variation accounts for the distinct disease severity of A(H1N1)pdm09 infection. Several potential genetic determinants associated with A(H1N1)pdm09 infection have been described, including TNF (Antonopoulou et al., 2012), IFN-inducible transmembrane (Everitt et al., 2012), killer-cell immunoglobulin-like receptor (Aranda-Romo et al., 2012), complement regulatory protein CD55 (Zhou et al., 2012) and Toll-like receptor 3 (Esposito et al., 2012). Data relating to the role of chemokine receptor 5 (CCR5) in severe A(H1N1)pdm09-infected patients are contradictory and have been debated (Keynan et al., 2010;Rodriguez et al., 2013;Sironi et al., 2014).CCR5 regulates various aspec...

Several reports of increases in invasive zygomycosis (IZ) at individual institutions across the USA and Europe have contributed to a generalized concept that IZ is an increasing problem and the overestimation of the clinical significance of the isolation of zygomycetes in microbiology departments. We assessed the workload and clinical significance of zygomycetes isolates recovered from clinical samples in our institution over a 19-year period (1988-2006). We retrospectively reviewed the charts of those patients from who isolates of zygomycetes were obtained and calculated the workload of its isolation, the incidence of IZ during this period and the positive predictive value (PPV) of a positive culture. Zygomycetes were recovered from 210 samples (176 patients), i.e., 0.086/1,000 clinical samples processed and 6.3/1,000 samples submitted for fungal isolation. Zygomycetes represented 0.6% of the total fungi recovered. The mean incidence of the disease was 1.2 cases/100,000 admissions (range 0-20). Only 16 of the samples which grew zygomycetes (7.6%) were from infected patients. The workload generated by zygomycetes in our institution and the PPV for IZ of their isolation in our laboratory were very low and the disease was not found to have significantly increased in recent years in our institution. Data from specific institutions cannot be generalized.