Genotyping Reveals High Clonal Diversity and Widespread Genotypes of Candida Causing Candidemia at Distant Geographical Areas.

The objectives of this study were to gain further insight on Candida genotype distribution and percentage of clustered isolates between hospitals and to identify potential clusters involving different hospitals and cities. We aim to genotype Candida spp. isolates causing candidemia in patients admitted to 16 hospitals in Spain, Italy, Denmark, and Brazil. Eight hundred and eighty-four isolates (Candida albicans, n = 534; C. parapsilosis, n = 282; and C. tropicalis, n = 68) were genotyped using species-specific microsatellite markers. CDC3, EF3, HIS3, CAI, CAIII, and CAVI were used for C. albicans, Ctrm1, Ctrm10, Ctrm12, Ctrm21, Ctrm24, and Ctrm28 for C. tropicalis, and CP1, CP4a, CP6, and B for C. parapsilosis. Genotypes were classified as singletons (genotype only found once) or clusters (same genotype infecting two or more patients). Clusters were defined as intra-hospital (involving patients admitted to a single hospital), intra-ward (involving patients admitted to the same hospital ward) or widespread (involving patients admitted to different hospitals). The percentage of clusters and the proportion of patients involved in clusters among species, genotypic diversity and distribution of genetic diversity were assessed. Seven hundred and twenty-three genotypes were detected, 78 (11%) being clusters, most of which (57.7%; n = 45/78) were intra-hospital clusters including intra-ward ones (42.2%; n = 19/45). The proportion of clusters was not statistically different between species, but the percentage of patients in clusters varied among hospitals. A number of genotypes (7.2%; 52/723) were widespread (found at different hospitals), comprising 66.7% (52/78) of clusters, and involved patients at hospitals in the same city (n = 21) or in different cities (n = 31). Only one C. parapsilosis cluster was a widespread genotype found in all four countries. Around 11% of C. albicans and C. parapsilosis isolates causing candidemia are clusters that may result from patient-to-patient transmission, widespread genotypes commonly found in unrelated patients, or insufficient microsatellite typing genetic discrimination.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Epidemiological and clinical characteristics of Streptococcus tigurinus endocarditis.

BACKGROUND: Streptococcus tigurinus was recently described as a new streptococcal species within the viridans group streptococci (VGS). The objectives of the present work were to analyse the clinical and microbiological characteristics of S. tigurinus isolated from patients with bacteraemias, to determine the prevalence of S. tigurinus among VGS endocarditis in Spain, and to compare the clinical characteristics and outcomes of endocarditis caused by S. tigurinus and other VGS. METHODS: Retrospective nationwide study, performed between 2008 and 2016 in 9 Spanish hospitals from 7 different provinces comprising 237 cases of infective endocarditis. Streptococcal isolates were identified by sequencing fragments of their 16S rRNA, sodA and groEL genes. Clinical data of patients with streptococcal endocarditis were prospectively collected according to a pre-established protocol. RESULTS: Patients with endocarditis represented 7/9 (77.8%) and 26/86 (30.2%) of the bacteraemias caused by S. tigurinus and other VGS, respectively (p < 0.001), in two of the hospital participants. Among patients with streptococcal endocarditis, 12 different Streptococcus species were recognized being S. oralis, S. tigurinus and S. mitis the three more common. No relevant statistical differences were observed in the clinical characteristics and outcomes of endocarditis caused by the different VGS species. CONCLUSIONS: In this multicenter study performed in Spain, S. tigurinus showed a higher predilection for the endocardial endothelium as compared to other VGS. However, clinical characteristics and outcomes of endocarditis caused by S. tigurinus did not significantly differ from endocarditis caused by other oral streptococci.

Emergence of linezolid-resistant coagulase-negative staphylococci in an intensive care unit.

BACKGROUND: The aim of this study was to report the emergence of linezolid-resistant coagulase-negative staphylococci (CoNS) in an intensive care unit. METHODS: An observational study was conducted in critically ill patients with colonization or infection by linezolid-resistant CoNS between January 2010 and December 2014. We analyzed the epidemiological and clinical features, and the mechanism of resistance to linezolid. We also evaluated the association between the incidence of linezolid-resistant CoNS strains and the consumption of linezolid in the study period. RESULTS: During the study period 49 patients had a linezolid-resistant CoNS strain isolated from clinical samples (blood in 42 cases, urine in 6, peritoneal fluid in 1). Molecular study showed a combination of mechanisms of resistance. Most patients were critically ill (APACHE II score = 21.9 ± 8.3) and nearly all had undergone surgery and invasive procedures, and had prior exposure to antibiotics. Linezolid-resistant CoNS were considered to be contaminants in 42 patients and associated with infection in 7 patients, comprising bacteremia and septic shock in most of them. They were successfully treated with glycopeptides or daptomycin. A modest significant correlation was observed between the decrease in linezolid consumption and the lower incidence of resistant isolates. CONCLUSIONS: Linezolid-resistant CoNS had emerged in critically ill patients with severe underlying diseases and prior antibiotic exposure. Most isolates represented colonization; however, linezolid-resistant CoNS can produce serious infections in critically ill patients. Glycopeptides and daptomycin seem to provide useful alternatives for therapy of these infections. A relationship was found between linezolid consumption and the incidence of linezolid-resistant CoNS strains.

Antifungal stewardship in a tertiary hospital.

BACKGROUND: The inappropriate use of antifungals is an important health problem related to increasing adverse effects, unnecessary cost and promotion of resistant and emerging fungal infections. Despite its relevance, many health institutions assign few resources to improve prescribing practices. AIMS: To evaluate the efficiency of an antifungal stewardship programme (ASP) centered on restricted antifungal agents. METHODS: The main activity during the eight-month study was to perform a programmed review of restricted antifungals (lipid formulations of amphotericin B, echinocandins and voriconazole) prescribed in hospitalized patients. In the case of amendable antifungal treatment, a recommendation was included in the electronic medical record. RESULTS: A total of 280 antifungal prescriptions for 262 patients were revised during the study period. The indications were prophylactic in 85 cases (30.4%), pre-emptive in 10 cases (3.5%), empiric in 122 cases (43.6%), and directed in 63 cases (22.5%). A total of 70 prescriptions (25%) in 61 patients were considered to be amendable. In most of these cases, treatment could have been reduced considering the patient's clinical improvement and microbiological results. The most common advice was antifungals change (70%), antifungal withdrawal (21%), removal of one antifungal drug in cases of combined therapy (7%), and switching to oral route (1%). Proposed recommendations were addressed in 28 cases (40%). There was no significant difference in adherence with respect to the type of recommendation (p=0.554). There was a 42% lower use of antifungals during the period of the study compared to that observed during a similar previous period. Mortality among patients who were treated according to the recommendations of the ASP was 17% and in whom treatment was not modified it was 30% (p=0.393). CONCLUSIONS: ASPs centered on hospitalized patients may be an efficient strategy to ameliorate antifungal use in hospitals.

Gonococcal endocarditis: a case report and review of the literature.

A 59-year-old Caucasian male presented with progressive dyspnea, arthralgias and fever for three days. A diastolic regurgitation murmur was detected in the aortic area. A transesophageal echocardiograph showed several vegetations and severe aortic regurgitation. Blood cultures yielded Neisseria gonorrhoeae beta-lactamase negative. The patient had not noticed any urogenital discomfort or urethral discharge. The patient successfully underwent surgery for septal abscess debridement. The patient received ceftriaxone 2 g bid for eight weeks and the clinical follow-up was uneventful. The review of the literature revealed a total of the 38 additional cases reported between 1980 and the present. The majority of the patients were young, male and with native valve involvement. There has been a clear tendency for left-sided valve involvement (especially in the aortic valve). All valve cultures were reported negative despite, in most cases, the marked tissue destruction. Polymerase chain reaction was performed in two patients and positive results were shown in both. Cultures of exudates from other locations were negative in most cases. One striking fact is the high proportion of patients who underwent surgery (72 %). Information regarding antibiotic sensitivity was available in 28 cases, with penicillin resistance reported in six patients (21 %) and intermediate sensitivity in four patients (14 %). Resistance to ciprofloxacin was reported in two cases (7 %). A rapid increase and distribution of isolates resistant to third generation cephalosporins have been recently detected. The mortality is high, particularly taking into account that most were young patients who had not presented previous heart disease.

AmpC beta-lactamases in Escherichia coli: emergence of CMY-2-producing virulent phylogroup D isolates belonging mainly to STs 57, 115, 354, 393, and 420, and phylogroup B2 isolates belonging to the international clone O25b-ST131.

Cephalosporins resistance is increasing in Escherichia coli in Spain. We characterize infections by E. coli with reduced susceptibility to third-generation cephalosporins (3GCs) with the AmpC phenotype. Between January 2004 and March 2007, 121 E. coli isolates with the AmpC phenotype were collected (4.8% of all the 2538 E. coli isolates with reduced susceptibility to 3GCs). These isolates were further characterized by clinical and molecular analysis. Plasmid-encoded ampC genes were detected in 46 (38%) isolates (43 CMY-2); 75 isolates (62%) had modifications in the chromosomal ampC promoter region (c-AmpC). CMY-2 producers belonged primarily to the more virulent phylogroup D (48.4%), whereas most isolates of c-AmpC belonged to phylogroup A (56.4%). Bacteremia and infections in children were more frequently produced by CMY-2 producers. CMY-2-producing phylogroup D E. coli belonged to 8 multilocus sequence typing types. Three CMY-2 producers belonged to O25b/ST131/B2 clone. Infections caused by E. coli with the AmpC phenotype may be spreading primarily because of CMY-2-producing phylogroup D isolates, although this enzyme was also detected in the O25b/ST131/B2 clone.

Prevalence and clonal characterization of Streptococcus pyogenes clinical isolates with reduced fluoroquinolone susceptibility in Spain.

The aim of this study was to determine the prevalence and characteristics of non-fluoroquinolone (FQ)-susceptible Streptococcus pyogenes isolates and to study their mechanisms of resistance. We performed a prospective prevalence study with 468 isolates collected from 2005 to 2007 and a retrospective study that was based on the examination of existing data collected from 1999 to 2008. The retrospective study included data for isolates with high-level resistance (HR) to ciprofloxacin (MIC >or= 32 microg/ml) (HR isolates) and isolates with the same emm types as those reported in the literature with low-level resistance (LR) to ciprofloxacin (MICs, 2 to 8 microg/ml) (LR isolates, n = 205). Genetic characterization of the isolates was performed by means of emm typing and multilocus sequence typing. The prevalence of LR ranged from 1.9% in 2005 to 30.8% in 2007. This increase was mainly due to the circulation of an emm6 subtype (emm6.4) that represented 77.1% of the LR isolates in 2007. Notably, another emm6 subtype, also detected in 2007 (emm6.37), showed coresistance to 14- and 15-membered macrolides mediated by the mefA gene. Only three HR isolates were detected (isolates emm68.1/ST247/T3,13,B3264, emm77/ST399/T28, and emm28/ST52/T28), and all were identified in the retrospective study. Overall, the 673 isolates represented 25 emm types. All LR isolates were clustered into two emm types: emm6 (six emm6 subtypes) and emm75. All the 156 emm6 isolates had LR, harbored the Ser79/Ala mutation in the parC gene product, and had the same sequence type (ST), ST382. Most (21/33) of the emm75 isolates had LR, showed the Ser79/Phe plus Asp91/Asn double mutation in the parC gene product, and were ST150. The Asp91/Asn mutation by itself did not confer resistance to FQs.

CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin.

OBJECTIVES: To describe trends in fosfomycin resistance in urinary isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBLs) in relation to fosfomycin consumption and to characterize representative fosfomycin-resistant isolates. METHODS: In 2007-08, an unexpected increase in fosfomycin resistance in ESBL-producing urinary E. coli was observed. Laboratory records were reviewed and a prospective surveillance study was initiated on all urinary tract infections caused by ESBL-producing, fosfomycin-resistant E. coli. bla(ESBL) types, phylogroups, genetic environment and afa/dra operon were determined by PCR and sequencing. Molecular epidemiology was analysed by PFGE and multilocus sequence typing. To elucidate possible mechanisms of fosfomycin resistance, uhpT, glpT, uhpA, ptsI, cyaA and murA genes were analysed. Fosfomycin consumption was determined as recommended by WHO. RESULTS: From 2004 to 2008, fosfomycin consumption increased by 50%, while fosfomycin resistance in ESBL producers increased from 2.2% to 21.7%. Of 26 isolates studied, 24 produced CTX-M-15 and belonged to the O25b-ST131-phylogroup B2 clonal strain. PFGE revealed two clusters. Cluster I included 18 isolates, 16 of them indistinguishable from strains producing CTX-M-15 previously described in Madrid. The five isolates of Cluster II had the IS26 linked to bla(CTX-M-15) and the afa/dra operon. In Cluster I isolates, no mutations in glpT, uhpT, uhpA, ptsI, cyaA and murA were detected. Cluster II isolates showed a 15 bp deletion (A(169)-C(183)) in uhpA. CONCLUSIONS: Fosfomycin resistance in urinary E. coli has increased due to the acquisition of this resistance by a previously circulating CTX-M-15-producing E. coli O25b-ST131-phylogroup B2 strain. This happened during a period when the use of fosfomycin increased by 50%.

Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005.

The aim of this study was to describe the genetic characteristics of Streptococcus pyogenes showing the MLSB phenotype of macrolide resistance from 1999 to 2005 in Spain and to highlight the substantial increase in these isolates in the last few years. The antimicrobial susceptibilities of 17,232 group A streptococci isolated from Madrid and Gipuzkoa from 1999 to 2005 were studied. The presence of the resistance genes ermA, ermB, mef, tetM, and tetO and the presence of the intTn and xis genes of the Tn916-Tn1545 transposon family were studied in a sample of 739 MLSB-resistant isolates. The epidemiological relationships among these isolates were analyzed by emm typing, T typing, and multilocus sequence typing. Erythromycin resistance was found in 21.3% of the isolates analyzed (annual variation of 14.3% to 28.9%). Until 2003, most erythromycin-resistant isolates showed the M phenotype, but in 2004 and 2005, about 50% of isolates showed the MLSB phenotype. Among the MLSB-resistant isolates studied, 16 clones were identified. The most prevalent clone was a strange emm11/T11/ST403 clone with a null yqiL allele. All but one of the 463 emm11/T11/ST403 isolates carried the ermB, tetM, intTn, and xis genes. The second most prevalent MLSB-resistant clone was emm28/T28/ST52, which comprised two subclones: one bacitracin-resistant, tetracycline-susceptible subclone carrying the ermB gene (n=115) and another bacitracin-susceptible, tetracycline-resistant subclone carrying the ermB and tetM genes (n=33). The rapid diffusion of these two clones, and especially of emm11/T11/ST403, caused the large increase in MLSB-resistant S. pyogenes isolates in Spain, suggesting a potential ability for international dissemination.

Characterisation of the main clones of Streptococcus pyogenes carrying the ermA (subclass TR) gene in Spain.

Seventy-four Streptococcus pyogenes isolates showing the macrolide-lincosamide-streptogramin B (MLS(B)) resistance phenotype carrying the ermA gene (72 of which showed the inducible resistance phenotype) were obtained between 1999 and 2004. Seven different sequence types (STs) and emm types were detected: emm22/ST46 (n=33); emm77/ST63 (n=22); emm73/ST331 (n=10); emm94/ST89 (n=6); and one isolate each of emm28/ST52, emm11/ST403 and emm4/ST38. All ST46 isolates were susceptible to tetracycline and almost all reacted against the T12 type (all agglutinated into the T-pattern 3/12/13/B3264). Resistance to tetracycline was observed in all ST63 (tetO+) and ST89 (tetM+) isolates. Most of the ST63 isolates reacted against the T28 type (all agglutinated into the T-pattern 9/13/28). The 74 isolates were grouped into eight pulsed-field gel electrophoresis pulsotypes (one cluster for each emm/ST type, except for emm77/ST63).

Spread of Escherichia coli strains with high-level cefotaxime and ceftazidime resistance between the community, long-term care facilities, and hospital institutions.

A total of 151 Escherichia coli strains resistant to cefotaxime and ceftazidime were isolated during a prospective surveillance study. These strains were characterized by clinical, microbiological, and molecular analyses and were distributed into four clusters of 103, 11, 6, and 5 isolates, along with 25 unrelated strains. The principal cluster was isolated from urine, wound, blood, and other samples in three hospitals, eight nursing homes, and a community healthcare center. This cluster was associated with both nosocomial (65%) and community-acquired (35%) infections. Most strains were resistant to ciprofloxacin, gentamicin, tobramycin, cefepime, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole but were susceptible to imipenem. All isolates from the four clusters expressed the extended-spectrum beta-lactamase (ESBL) CTX-M-15. This enzyme was also present in 8 (30.8%) of the 26 unrelated isolates. The other ESBLs, CTX-M-14 and CTX-M-32, were detected in five and seven cases, respectively, but they were detected in individual E. coli isolates only. In three clusters, blaCTX-M-15 alleles were linked to an ISEcp1-like element, while in eight strains of cluster II an IS26 element preceded the blaCTX-M-15 allele. An additional pool of resistance genes included tetA, drfA14 or dfrA17, sul1 or sul2, aac(6')Ib, and aac(3)IIb. All except one of the 27 isolates tested for genetic virulence markers harbored the same three virulence genes: iutA and fyuA (siderophores), and traT (serum survival factor). Epidemic or occasional isolates of cefotaxime- and ceftazidime-resistant E. coli can spread between distinct health facilities including hospitals, community health centers, and long-term care centers.

[Clinical significance of Streptococcus agalactiae isolation from urine samples of outpatients from health care centers]. / Significado clínico del aislamiento de Streptococcus agalactiae de orina de pacientes de centros de salud.

INTRODUCTION: Streptococcus agalactiae is a well-recognized pathogen in the obstetric population and is a cause of invasive infection in adults with underlying diseases. Nevertheless, the role of S. agalactiae in urinary tract infections in the adult non-pregnant population treated in outpatient health care centers has been less extensively studied. METHODS: The clinical significance of S. agalactiae isolation in urine samples from health care center patients was studied in five areas of Spain over a period of six months. The study protocol included the collection of personal, epidemiological and clinical data. A total of 85 patients were studied; 79 were women (17 pregnant) and 6 men. RESULTS: Pyuria was found in 72.9% of the cases. A total of 26.4% (n = 18) of non-pregnant women and all the men had some type of chronic underlying disease and 35.3% (n = 24) had urinary disease. Among the pregnant women, 53% had asymptomatic bacteriuria and 35.3% had vaginal and/or urethral colonization. Among the group of men and non-pregnant women, the most frequent entity was non-complicated cystitis, seen in 66.1% (45 of 68) of cases, followed by complicated infection in 19.1% and asymptomatic bacteriuria in 11.8%. Of the 45 patients with non-complicated cystitis, 35 (77.7%) were over 40 years old and 19 (42.2%) were over 60 years old. CONCLUSIONS: Among the population of non-pregnant adults treated in outpatient health care centers, S. agalactiae isolation from urine was found mainly in women over 40 years old and was the cause of non-complicated urinary tract infection in more than half of cases.

The aetiology of paediatric inflammatory vulvovaginitis.

UNLABELLED: Vulvovaginitis is the most common gynaecological problem in prepubertal girls and clear-cut data on the microbial aetiology of moderate to severe infections are lacking. Many microorganisms have been reported in several studies, but frequently the paediatrician does not know the pathogenic significance of an isolate reported in vaginal specimens of girls with vulvovaginitis. A multicentre study was performed, selecting 74 girls aged 2 to 12 years old with a clinical picture of vulvovaginitis and inflammatory cells on Gram stain. All the specimens were cultured following standard microbiological techniques and the paediatricians completed a questionnaire to highlight risk factors after interviewing the parents or tutors. The data were compared with those obtained in a control group of 11 girls without vulvovaginitis attending a clinic. Streptococcus pyogenesand Haemophilus spp.were isolated in 47 and 12 cases, respectively. Upper respiratory infection in the previous month ( P<0.001) and vulvovaginitis in the previous year ( P<0.05) were identified as significant risk factors. Foreign bodies, sexual abuse, poor hygiene and bad socioeconomic situation were not identified as risk factors for the infection. CONCLUSION: Paediatric inflammatory vulvovaginitis is mainly caused by pathogens of the upper respiratory tract and the most common risk factor for this infection is to have suffered an upper respiratory tract infection in the previous month.

Clonal dissemination of Yersinia enterocolitica strains with various susceptibilities to nalidixic acid.

Ten epidemiologically related Yersinia enterocolitica clinical isolates were studied. Six isolates were nalidixic acid resistant (MIC > 512 microg/ml), with mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene, suggesting clonal dissemination of a nalidixic acid-susceptible Y. enterocolitica strain which has acquired different mutations generating resistance to nalidixic acid.