M. Ángeles Orellana

VIM-2-producing multidrug-resistant Pseudomonas aeruginosa ST175 clone, Spain.

This clone is a major public health problem because it limits antimicrobial drug therapy.

Molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae in a hospital in Madrid: Successful establishment of an OXA-48 ST11 clone

Here we report a retrospective clinical and molecular study conducted in a tertiary care facility in southern Madrid, Spain, from January 2009 to February 2014 to investigate the epidemiology of carbapenemase-producing Klebsiella pneumoniae (CPKp). Carbapenemase genes were identified in 97 non-duplicate K. pneumoniae isolates, including 59 harbouring blaOXA-48, 37 harbouring blaVIM-1 and 1 harbouring blaKPC-2. Pulsed-field gel electrophoresis (PFGE) analysis verified the presence of 20 different clonal types, whilst multilocus sequence typing (MLST) assigned the isolates to eight sequence types (STs). A gradual increase was noted in the number of CPKp isolated, ranging from 0.8% in 2009 to 4.3% in 2013. A large outbreak was also identified, initiated in 2013 owing to a blaOXA-48 and blaCTX-M-15 co-producing ST11 clone and involving a total of 44 patients. Whole-genome sequencing was used to characterise the resistome of a representative isolate from this outbreak. Bioinformatics analysis revealed the presence of 121 genes related to antibiotic and antiseptic resistance, mutations in the ompk35 and ompk36 genes, and the presence of the blaOXA-48 gene on a 62 811bp IncL/M-type plasmid as part of a Tn1999.2 composite transposon. These results portray the increasing trend in carbapenemase-producing isolates in this hospital and highlight the successful establishment of a blaOXA-48 and blaCTX-M-15 co-producing ST11 clone that has led to the displacement of previous circulating clones.

Clinical and Molecular Characteristics of Infections with CO2-Dependent Small-Colony Variants of Staphylococcus aureus

ABSTRACT Most Staphylococcus aureus small-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO2. We conducted a prospective investigation of all clinical cases of CO2-dependent S. aureus during a 3-year period. We found 14 CO2-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 (agrII), ST30-agrIII, ST34-agrIII, and ST45-agrI. An array hybridization technique performed on the 14 CO2-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 CO2-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 CO2 auxotrophs of S. aureus, the factors that select these strains in the host, and the genetic basis of this type of auxotrophy.

Molecular Characterization of Achromobacter Isolates from Cystic Fibrosis and Non-Cystic Fibrosis Patients in Madrid, Spain

ABSTRACT 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.

Relationship between agr dysfunction and reduced vancomycin susceptibility in methicillin-susceptible Staphylococcus aureus causing bacteraemia

OBJECTIVES Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptible Staphylococcus 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 and agr 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 = 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (ΔCt 4.05 ± 3.29 versus 1.5 ± 2.11, P = 0.005), implying greater agr dysfunction in RSV MSSA. CONCLUSIONS We demonstrated a correlation between RSV phenotype in MSSA and reduced agr expression, particularly in association with the agr II genotype. These results may help to understand the role of agr dysfunction in the increased mortality in MSSA infections.

Clonal diversity among Burkholderia cepacia complex isolates from cystic fibrosis patients in a reference unit

INTRODUCTION The epidemiology of Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) is not widely known. METHODS All CF patients with Bcc between 2002 and 2011 were reviewed, and a molecular analysis of isolates was performed. RESULTS The prevalence of Bcc infection was 7.2% (18/250). Molecular analysis of 16 Bcc isolates showed 5 species (7 B. contaminans, 6 B. cepacia, 1 B. cenocepacia, 1 B. multivorans, and 1 B. stabilis) and 13 sequence types. There were no cases of cross-transmission. CONCLUSION A high diversity of Bcc species was found in infected CF patients.

Usefulness of endoluminal catheter colonization surveillance cultures to reduce catheter-related bloodstream infections in hemodialysis

BACKGROUND To evaluate the use of surveillance cultures (SCs) to prevent catheter-related bloodstream infections (CRBSIs) in asymptomatic hemodialysis (HD) patients. METHODS In 2011-2012, we conducted a prospective study of HD patients with tunneled cuffed central venous catheters (TCCs). Colonization of the catheter lumen was assessed every 15 days by inoculating ~5 mL endoluminal blood into aerobic culture bottles. Individual patients were triaged based on SC results: group 1 (negative); group 2 (coagulase-negative Staphylococcus [CoNS] with time-to-positivity (TTP) >14 hours); group 3 (CoNS with TTP ≤14 hours); and group 4 (any microorganism other than CoNS and any TTP). RESULTS We studied 104 patients (129 TCCs). Median follow-up was 262.5 days (interquartile range [IR], 135.0-365.0). A total of 1,734 SCs were collected (median, 18 per patient; IR, 10.0-24.0), of which 1,634 (94.2%) were negative (group 1) and 100 (5.8%) were positive (group 2: 79; group 3: 12, group 4: 9). In groups 2 and 3, 19 TCCs required antibiotic lock therapy (ALT). In group 4, all patients received intravenous therapy and ALT. Under this protocol, there were 0.27 episodes of CRBSI per 1,000 catheter days compared with 1.65 (P < .001) prior to its implementation. CONCLUSION SCs based on easily accessible samples proved useful in triaging HD patients at a high risk of infection.

Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples

Rapid and accurate detection of the pathogens that cause gastrointestinal infection is important for appropriate therapy and proper infection control. This study assesses the performance of a new molecular assay for simultaneous detection of 13 different gastrointestinal bacteria in stool specimens. Using the Allplex GI-Bacteria (AGI-BI/AGI-BII) assay, a total of 394 stool samples were tested and the results were compared with culturing on selective differential followed by identification by mass spectroscopy. Discordant results were analyzed by a different multiplex PCR method, the Fast-Track Diagnostics Bacterial gastroenteritis (FTD-BG). The routine method (RM) detected 109 (27.7%) positive samples and the Allplex-GI assay, 261 (66.2%). Analysis of discordant results revealed that the molecular assay detected 44 pathogens that were not detected by the RM, including 23 Campylobacter spp., 11 Salmonella spp, 3 Y. enterocolitica, 2 EIEC/Shigella spp, 2 E. coli 0157, 2 C. difficile and 1 Aeromonas spp. Five cases not detected by the molecular method were detected by the RM (3 Aeromonas spp, 1 Salmonella spp and 1 Y. enterocolitica). For all targets, the percentages of sensitivity and specificity were >95%, except for Aeromonas spp., which were 81% and 99% respectively. This study suggests that Allplex-GI multiplex PCR is a sensitive and specific assay that enables a rapid and accurate diagnosis of bacterial gastrointestinal infections.