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Objectives: Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptibleStaphylococcus aureus (MSSA). This study investigated the clinical and molecular epidemiology of MSSA causing bacteraemia, with emphasis on the reduced susceptibility to vancomycin (RSV) phenotype (MIC ≥1.5 mg/L) and its relationship with agr dysfunction. Methods: All MSSA bloodstream isolates obtained at our hospital during 2010 were analysed. Antimicrobial susceptibility was determined and time –kill experiments were performed for oxacillin. Multilocus sequence type andagr genotype were determined and DNA microarray analysis of virulence factors was performed.agr dysfunction was assessed phenotypically and by RT–PCR quantification of RNAIII. Results: Of 84 MSSA, 55 (65.5%) exhibited the RSV phenotype, comprising 13 clonal complexes.agr II polymorphism was more prevalent in RSV than non-RSV isolates (41.8% versus 17.2%,P ¼ 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (DCt 4.05+3.29 versus 1.5+2.11, P ¼ 0.005), implying greater agr dysfunction in RSV MSSA. Conclusions: We demonstrated a correlation between RSV phenotype in MSSA and reducedagr expression, particularly in association with theagr II genotype. These results may help to understand the role ofagr dysfunction in the increased mortality in MSSA infections.

Multilocus sequence typing and nrdA sequence analysis identified 6 different species or genogroups and 13 sequence types (STs) among 15 Achromobacter isolates from cystic fibrosis (CF) patients and 7 species or genogroups and 11 STs among 11 isolates from non-CF patients. Achromobacter xylosoxidans was the most frequently isolated species among CF patients.

A total of 183 patients were colonized or infected with multidrug-resistant Pseudomonas aeruginosa isolates at a hospital in Spain during 2007-2010; prevalence increased over this period from 2.8% to 15.3%. To characterize these isolates, we performed molecular epidemiologic and drug resistance analysis. Genotyping showed that 104 (56.8%) isolates belonged to a single major clone (clone B), which was identified by multilocus sequence typing as sequence type (ST) 175. This clone was initially isolated from 5 patients in 2008, and then isolated from 23 patients in 2009 and 76 patients in 2010. PCR analysis of clone B isolates identified the b/a(VIM-2) gene in all but 1 isolate, which harbored b/a(IMP-22). ST175 isolates were susceptible to only amikacin (75%) and colistin (100%). Emergence of the ST175 clone represents a major health problem because it compromises therapy for treatment of P aeruginosa nosocomial infections., This study was supported by Spanish Network for the Research in Infectious Diseases (RD06/0008) from the Instituto de Salud Carlos III, Spain.

In February 2006, a patient colonized with a multidrug-resistant sequence type 56 Acinetobacter baumannii strain was admitted to a hospital in Madrid, Spain. This strain spread rapidly and caused a large outbreak in the hospital. Clinicians should be alert for this strain because its spread would have serious health consequences.

Introduction Diagnosis of HSV-1 keratitis (HK) is frequently based on clinical findings. Invasive specimens (corneal scrapings, biopsies) are required for microbiological diagnosis. Methods Corneal scrapings and conjunctival swabs were collected on patients with/without clinical suspicion of HK from 2007 to 2012. Results The sensitivity, specificity, positive and negative predictive values for conjunctival swabs by PCR was 77.8, 92.1, 84.4 and 88.3, respectively. Discussion Conjunctival swabs by PCR may help in the diagnosis of HK, despite the limited sensitivity.

Background The influence of acute graft pyelonephritis (AGPN) on graft outcome in renal transplant recipients still remains controversial. Methods We retrospectively analysed 189 patients (113 males; mean age: 49.7 ± 13.1 years) undergoing renal transplantation at the University Hospital 12 de Octubre (Madrid, Spain) from January 2002 to December 2004, with a minimum follow-up of 36 months. Factors associated with AGPN were assessed by logistic regression analysis. Long-term graft function was compared according to the occurrence of this complication during follow-up. 'Decline in renal graft function' was defined as the increase in serum creatinine (SC) levels > 0.33 mg/dL between Month 3 and Year 1 after transplantation. Results Nineteen patients (10.0%) were diagnosed with 25 episodes of AGPN (incidence rate: 4.4 episodes per 100 patient-years). The presence of glomerulonephritis as the underlying disease [odds ratio (OR) 4.2; 95% confidence interval (95%CI): 1.3-14.1] and the previous occurrence of two to five (OR 9.4; 95%CI: 1.5-56.8) or more than five episodes of asymptomatic bacteriuria after transplantation (OR 19.8; 95%CI: 2.4-160.2) emerged as independent predictors for AGPN. A near-significant association was found for cytomegalovirus infection (OR 4.2; 95%CI: 0.9-18.4), whereas receiving a single-kidney transplant (vs. double-kidney) showed a protective effect (OR 0.2; 95%CI: 0.0-0.8). During the 36-month follow-up, levels of SC, creatinine clearance and 24-h proteinuria did not differ significantly between patients with or without AGPN, and this complication did not exert any effect on the risk for decline in renal graft function. Conclusions AGPN does not impair long-term graft function in renal transplant recipients.

Background. Approximately 25% of haemodialysis (HD) patients use catheters as vascular access. Catheter-related bloodstream infections (CRBSI) are a major risk in this population. The objective of our study was to determine whether endoluminal catheter colonization (ECC) predicts CRBSI. Methods. We followed up a cohort of HD patients in our institution who underwent HD with tunnelled cuffed central venous catheters (TCC) between December 2006 and June 2008. Colonization of the inner catheter lumen was assessed every 15 days immediately before HD by culture of blood―heparin mixture and the time to positivity (TTP) was recorded by the BacT/Alert automated system. CRBSI was confirmed by differential TTP (>2 h) between TCC and peripheral blood cultures. Results. We studied 51 patients who required 64 TCC. The incidence of CRBSI was 1.65 episodes per 1000 catheter-days, with Staphylococcus epidermidis being the most common cause of infection (76.2%). ECC was more frequent in the CRSBI group than in the non-CRBSI group (100 vs 5.4%, P < 0.001). For S. epidermidis CRBSIs, the median time from ECC to CRBSI was 31.5 days (interquartile range, 27.0―79.0). The sensitivity, specificity and negative and positive predictive values of arterial lumen cultures for S. epidermidis CRBSIs were 100, 96.3, 92.3 and 100%, respectively, while for venous culture, these values were 92.3, 96.3, 92.3 and 96.3%, respectively. For predicting S. epidermidis CRBSI, endoluminal cultures with a TTP of ≤ 14 h had sensitivity and specificity of 52.1 and 97.7%, respectively. Conclusions. This study shows that ECC may predict the risk of developing CRSBI. Surveillance cultures could, therefore, be used to triage individual HD patients who might benefit from specific intervention measures.

Most Staphylococcus aureus small-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO 2 . We conducted a prospective investigation of all clinical cases of CO 2 -dependent S. aureus during a 3-year period. We found 14 CO 2 -dependent isolates of S. aureus from 14 patients that fulfilled all requirements to be considered SCVs, 9 of which were methicillin resistant. The clinical presentations included four cases of catheter-related bacteremia, one complicated by endocarditis; two deep infections (mediastinitis and spondylodiscitis); four wound infections; two respiratory infections; and two cases of nasal colonization. Pulsed-field gel electrophoresis typing showed that the 14 isolates were distributed into 4 types corresponding to sequence types ST125- agr group II ( agr II), ST30- agr III, ST34- agr III, and ST45- agr I. An array hybridization technique performed on the 14 CO 2 -dependent isolates and 20 S. aureus isolates with normal phenotype and representing the same sequence types showed that all possessed the enterotoxin gene cluster egc , as well as the genes for α-hemolysin and δ-hemolysin; biofilm genes icaA , icaC , and icaD ; several microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes ( clfA , clfB , ebh , eno , fib , ebpS , sdrC , and vw ); and the isaB gene. Our study confirms the importance of CO 2 -dependent SCVs of S. aureus as significant pathogens. Clinical microbiologists should be aware of this kind of auxotrophy because recovery and identification are challenging and not routine. Further studies are necessary to determine the incidence of CO 2 auxotrophs of S. aureus , the factors that select these strains in the host, and the genetic basis of this type of auxotrophy.

Objectives:To describe the relative proportions of nosocomial and community-onsetStaphylococcus aureusbacteremia at our institution and the epidemiologic characteristics and clonal diversity ofS. aureusisolates, as determined by pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance patterns.Design:Retrospective cohort study of all cases ofS. aureusbacteremia between October 2001 and October 2002.Setting:A 1,300-bed, tertiary-care hospital.Results:One hundred sixty-two unique episodes ofS. aureusbacteremia were identified. Forty-three cases (26.5%) were caused by methicillin-resistantS. aureus(MRSA). Most cases ofS. aureusbacteremia, whether MRSA or methicillin susceptible (MSSA), were nosocomial in origin (77.2%) or were otherwise associated with the healthcare system (16%). Only 11 (6.8%) of the cases (all MSSA) were strictly community acquired. Thirty-five unique macrorestriction patterns were identified among the 154 isolates that were typed by PFGE. Four major genotypes were defined among the isolates of MRSA, with 36 (85.7%) represented by a single PFGE type. Of the isolates within this major clone, all (100%) were ciprofloxacin resistant and 77.8% were erythromycin resistant. In contrast, the 112 isolates of MSSA comprised 31 different PFGE types, 3 of which represented 42.9% of all MSSA isolates and were associated with both nosocomial and community-onset bacteremia.Conclusions:Most cases ofS. aureusbacteremia in our healthcare region are nosocomial in origin or are acquired through contact with the healthcare system and are thus potentially preventable. To preclude dissemination of pathogenic clones, it is therefore necessary to redouble preventive measures in both the hospital and the community.

The annual incidence of Serratia marcescens bacteremia in a neonatal intensive care unit increased significantly between 2002 and 2010. Molecular epidemiology studies revealed that 8 clones were responsible for 85.2% of cases. Given that these infections are potentially preventable, even the appearance of 1 case of bacteremia should be an indicator for outbreak management.

Over a 6-year period (2007-2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients' clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007-2012. PCR amplification revealed the presence of bla(VIM-1) gene in 37 isolates, bla(VIM-2) in 1 isolate and bla(KPC) in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla(VIM-1). The genetic environment of bla(VIM-1) was investigated and two integron structures were found: intI-bla(VIM-1)-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1 (In624); and intI-bla(VIM-1)-aacA4-aadA1-qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300 kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla(VIM-1) may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital.

We report recurrent bacteremia due to mixed infection with two clonally derived isolates of Staphylococcus aureus in a patient with Sezary syndrome. The two isolates, one gentamicin resistant and methicillin susceptible and the other gentamicin susceptible and methicillin resistant, developed by the deletion of the mecA, ant(4 ′ )Ia , and aacA-aphD genes from a common gentamicin-resistant and methicillin-susceptible ancestor.

From 2002 to 2003, 32 isolates of Staphylococcus hominis subsp. novobiosepticus (SHN) were recovered from 21 patients, 18 of whom were neonates, with 13 considered to have late-onset SHN sepsis. All isolates from neonates had an indistinguishable pulsed-field gel electrophoresis pattern. Our data support SHN as an important nosocomial pathogen in neonates.

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.

New commercial techniques for determination of the viral load (VL) in plasma are able to detect as few as 20 copies of HIV-1 RNA/ml. The relevance of this new technical threshold is uncertain. Upon multivariate analysis, factors associated with detection of VLs between 20 and 49 copies/ml by the Cobas TaqMan HIV-1 v2.0 assay in an HIV clinic were the basal VL and time on antiretroviral therapy.

This report documents the nosocomial spread for an 18-month period of a single clone of linezolid- and teicoplanin-resistant Staphylococcus haemolyticus associated primarily with catheter-related bacteremia in intensive care unit patients. All linezolid-resistant isolates had the same G2576T mutation in at least 1 copy of the 23S rRNA gene.

Molecular epidemiology of Staphylococcus aureus strains causing bacteremia in neonates during 2002 to 2005 revealed seven clones, with four MSSA clones responsible for 80% of the cases. Some clones persisted or reappeared throughout the study. Three bacteremic clones were found colonizing health care workers (HCWs), particularly clone C, which was harbored by at least 15% of HCWs.

Most studies focusing on respiratory infections in immunocompromised children have been addressed to bacterial etiology. However, respiratory virus infections in this population can also lead to severe disease. The objective of this study is to evaluate the clinical significance of respiratory virus infections in children with cancer or human immunodeficiency virus (HIV) infection. Retrospective study conducted in a teaching hospital in Madrid. Medical records from children

During 2003, an increase in the number of cases of listeriosis was observed in a tertiary hospital in Madrid. The objectives of this study were to review the clinical characteristics of the cases diagnosed from 2001 to 2003 and to investigate clonal relationships among the clinical isolates of Listeria monocytogenes.A retrospective analysis was performed of all cases of listeriosis diagnosed in Hospital 12 de Octubre (Madrid) during 2001-2003. Clinical records for each patient were reviewed and all clinical isolates were compared using serotyping and pulsed-field gel electrophoresis (PFGE) with the AscI and SmaI restriction enzymes.A total of 18 patients were diagnosed: 4 in 2001, 2 in 2002, and 12 in 2003. The estimated incidence rates were 7.3 cases per 1,000,000 inhabitants in 2001, 3.6 in 2002 and 21.8 in 2003. The most frequent serotype during the study period was 4b (66.7%), and this serotype represented 83.3% of the cases diagnosed in 2003. PFGE yielded 12 different genotypes; one of them (PFGE type G) was common to 5 cases diagnosed in 2003.The expansion of a single clone of L. monocytogenes during 2003 partially contributed to increasing the incidence of listeriosis that year. Molecular epidemiology techniques are useful for detecting outbreaks of a possible foodborne origin and their results should promote epidemiological studies to investigate the food products involved.

In liver transplant recipients the most frequent infection is that produced by cytomegalovirus (CMV). One of the methods to reduce the incidence of that infection is the CMV pp65 antigenemia-guided preemptive therapy with intravenous ganciclovir.Liver transplant recipients were tested for CMV antigenemia at days 14, 28, 45, 60, 90 and 180 postransplantation and when clinically indicated. Patients showing50/200.000 leukocytes received ganciclovir 5 mg/kg/12 h for 14 days. Risk factors for active CMV disease where studied.182 CMV seropositive patients where included in the study. 16 patients with50/200.000 leukocytes received ganciclovir as preemptive therapy. CMV disease appeared in 9/182 patients (4.9%): 2/16 who received PT and 7/166 among those who did not receive preemptive therapy. The only factor associated with increased incidence of CMV disease was to have missing samples for CMV antigenemia during the follow up (p0.0042; OR = 8,17; 95% CI, 1.94-34.36).Ganciclovir antigenemia-guided preemptive therapy is associated with a low incidence of CMV disease. Bad adherence to the protocol of antigenemia samples increases the risk for CMV disease.

Streptococcus pneumoniae is the major cause of bacterial meningitis. We report a case of meningitis due to a mixed infection with two distinct strains of S. pneumoniae : one penicillin-resistant strain of serotype 9V and one penicillin-susceptible strain of serotype 7. The two strains exhibited different pulsed-field gel electrophoresis profiles.

We report the case of a teenager with chronic lymphocytic meningitis for whom Tropheryma whipplei 16S rRNA PCR results were positive in two cerebrospinal fluid samples and one duodenal biopsy specimen. PCR targeting another specific sequence of Tropheryma whipplei and sequencing of the initially amplified 16S rRNA fragment did not confirm the results.

We compared the efficacy of ganciclovir versus that of cytomegalovirus (CMV) immunoglobulin for the prevention of CMV disease in 31 CMV-seropositive heart transplant recipients who had received early immunoprophylaxis with OKT3 monoclonal antibodies. The incidence of CMV disease and visceral involvement was much higher in the CMV immunoglobulin group than in the ganciclovir group (40 versus 6%, respectively; P = 0.03). No adverse effects were found in the CMV immunoglobulin group, but 19% of the patients in the ganciclovir group developed mild leukopenia or a mild increase in their serum creatinine levels.

The efficacy of ganciclovir, given prophylactically, to prevent cytomegalovirus-related disease was evaluated in liver transplant recipients, mostly seropositive, under treatment with OKT3 monoclonal antibodies. The incidence of cytomegalovirus disease and visceral involvement was reduced, respectively, from 52 and 36% in the control group to 12 and 8% in the ganciclovir-treated patients. Leukopenia was a frequent (32%) side effect of ganciclovir administration.

Determining the acquisition routes of infection is crucial to designing specific preventive approaches against Staphylococcus aureus poststernotomy mediastinitis.From 2002 to 2004, a nasal sample was obtained from patients before cardiac surgery. We collected clinical and microbiologic data of all episodes of S aureus poststernotomy mediastinitis. A case-control study (3:1) was performed to confirm the role of previous preoperative nasal colonization by S aureus as a risk factor for S aureus poststernotomy mediastinitis. Pulsed field gel electrophoresis molecular analysis of nasal and surgical site S aureus isolates was performed to analyze their relatedness in each patient with poststernotomy mediastinitis and with other patients of the study cohort.S aureus nasal cultures were positive in 228 (15.9%) of 1432 patients: methicillin-susceptible S aureus in 222 (15.5%) and meticillin-resistant S aureus in 6 (0.4%). S aureus poststernotomy mediastinitis was diagnosed in 17 (1.2%) of 1432 patients: 9 (3.95%) of 228 in colonized patients versus 8 (0.66%) of 1204 in noncolonized patients (P.0001). Seven of 9 patients (1.2%) with methicillin-susceptible S aureus had an identical isolate by pulsed field gel electrophoresis in preoperative nasal and surgical-site cultures, but no clonal relatedness was shown among the isolates from these 9 patients. None of the 8 patients with methicillin-resistant S aureus poststernotomy mediastinitis had an identical isolate by pulsed field gel electrophoresis in preoperative nasal and surgical-site cultures, and the same clone of methicillin-resistant S aureus was responsible for all these cases.Endogenous [corrected] nasal colonization often precedes methicillin-resistant S aureus poststernotomy mediastinitis, which suggests that preoperative [corrected] decontamination is adequate for preventing methicillin-susceptible [corrected] S aureus poststernotomy mediastinitis, whereas hospital infection control measures seem to be the major factor for preventing methicillin-resistant S aureus poststernotomy mediastinitis.

We sought to examine the impact of asymptomatic bacteriuria on renal transplant outcome by retrospectively analyzing 189 renal transplant recipients for whom systematic screening uncovered 298 episodes of asymptomatic bacteriuria in 96 recipients. These patients were treated and all were followed for 36 months. Significant risk factors included female gender, glomerulonephritis as the disease that led to transplantation, and double renal transplant. There were no differences in serum creatinine, creatinine clearance, or proteinuria between patients with and without bacteriuria. The incidence of pyelonephritis in these patients was 7.6 episodes per 100 patient-years compared with 1.07 in those without asymptomatic bacteriuria. Between two to five and more than five bacteriuria episodes were significant independent factors associated with pyelonephritis whereas more than five episodes was a significant independent factor associated with rejection. Thus, we found no differences in renal function prognosis between patients who do not develop asymptomatic bacteriuria and those uncovered by systematic screening and who received treatment following kidney transplantation. Despite this treatment, the incidence of pyelonephritis was much higher in the group of patients with detected asymptomatic bacteriuria.