Jennifer Villa

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.

Draft Whole-Genome Sequence of OXA-48-Producing Multidrug-Resistant Klebsiella pneumoniae KP_ST11_OXA-48.

ABSTRACT We present the draft genome sequence of a blood culture isolate of OXA-48-producing Klebsiella pneumoniae (sequence type 11 [ST11]) obtained in the course of a hospital outbreak in Spain. Sequence analysis showed 121 genes related to antibiotic and antiseptic resistance, including blaOXA-48, which was located in an IncL/M plasmid.

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.

Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes

BackgroundThe ability of Staphylococcus aureus to invade tissues and cause an infectious disease is the result of a multi-factorial process supported by the huge number of virulence factors inherent to this microorganism tightly regulated by the accessory gene regulator (agr). During antimicrobial therapy bacteria may be exposed to sub-inhibitory concentrations (subMICs) of antibiotics that may trigger transcriptional changes that may have an impact on the pathogenesis of infection. The objective of this study was to investigate the effect of oxacillin sub-MICs on agr system expression as the key component in the regulation of virulence in methicillin-susceptible (MSSA) and -resistant S. aureus (MRSA) strains. Furthermore, we studied the genetic basis of the agr locus and their potential association with the expression levels.MethodsWe have examined the expression of RNAIII and agrA mRNA as biomarkers for agr expression in the presence and absence of oxacillin subMICs in 10 MSSA and 4 MRSA clinical strains belonging to 5 clonal complexes (CC45-agrI, CC8-agrI, CC5-agrII, CC15-agrII and CC30-agrIII) causing endovascular complications. The DNA sequences of agr locus were obtained by whole genome sequencing.ResultsOur results revealed that exposure to subMICs of oxacillin had an impact on agr locus expression modifying the relative levels of expression with increases in 11 strains and with decreases in 3 strains. Thereby, the exposure to subMICs of oxacillin resulted in higher levels of expression of agr in CC15 and CC45 and lower levels in CC30. We also observed the presence of mutations in agrC and agrA in 13/14 strains with similar mutation profiles among strains within individual CCs except for strains of CC5. Although, agr expression levels differed among strains within CCs, the presence of these mutations was associated with differences in agr expression levels in most cases.ConclusionsChanges in agr expression induced by exposure to oxacillin subMICs should be considered because they could lead to changes in the virulence modulation and have an adverse effect on the course of infection, especially in certain clonal complexes.

Bacteraemia due to extensively drug-resistant Pseudomonas aeruginosa sequence type 235 high-risk clone: Facing the perfect storm

Predictors of mortality and the impact of multidrug resistance and virulence on patients with Pseudomonas aeruginosa (PA) bacteraemia were evaluated. Patients with PA bacteraemia in a 12-month period were retrospectively analysed. Carbapenemase production, molecular typing and identification of virulence factor ExoU were carried out. The activity of ceftolozane-tazobactam and ceftazidime-avibactam was also investigated. The primary endpoint was 30-day crude mortality. Of 64 patients with bacteraemia, 24 (37.5%) were caused by extensively drug-resistant PA (XDR-PA): 10 (41.7%) cases involved the VIM-2 carbapenemase-producing ST175 clone, 11 (45.8%) the GES-5 carbapenemase-producing ST235 clone, and 3 (12.5%) were non-carbapenemase producers. The exoU genotype was detected in all ST235 strains and in 6 (15%) of the non-XDR isolates. Ceftazidime-avibactam (58.3%) showed greater activity than ceftolozane-tazobactam (12.5%) against XDR-PA isolates, particularly in GES-5 producers (100%). The 30-day crude mortality rate in patients with XDR-PA bacteraemia was higher than in cases caused by susceptible strains (62.5% vs. 30%; P=0.02). Multivariate analysis showed that independent risk factors associated with 30-day crude mortality were Pitt score ≥2 (OR, 42.31; 95% CI, 4.88-366.7; P=0.001) and respiratory source of bacteraemia (OR, 49.13; 95% CI 3.89-620.5; P=0.003). Stratified analysis adjusting for respiratory source revealed a non-significant trend towards higher mortality in patients with bacteraemia caused by the ST235 clone and exoU-producing isolates. These data support the notion that the XDR phenotype associated with the GES-5 carbapenemase-producing ST235 clone and the exoU-positive genotype adversely affects the outcome of patients with PA bacteraemia, particularly those with respiratory tract infections and a severe clinical presentation.

Characterization of mobile genetic elements carrying VIM-1 and KPC-2 carbapenemases in Citrobacter freundii isolates in Madrid

Carbapenemase producing Citrobacter freundii (CPCF) infections are still uncommon in European countries. Here we report a molecular study conducted in a tertiary care facility in southern Madrid, Spain, from 2009 to 2014 to investigate the epidemiology of CPCF. The blaIMP-1,blaIMP-2,blaKPC,blaNDM,blaOXA-48,blaVIM-1 and blaVIM-2 genes were screened by PCR. Molecular typing was carried out by Pulsed-field gel electrophoresis analysis (PFGE) and multilocus sequence typing (MLST). Whole genome sequencing (WGS) was performed to characterize the resistome and the mobile genetic elements associated with the carbapenems resistance of CPCF. A total of 11/521 (2.1%) isolates had reduced susceptibility to carbapenems. PCR amplification revealed the presence of blaVIM-1 in 10 isolates and blaKPC-2 in 2 isolates. One C. freundii isolate co-harbored blaVIM-1 and blaKPC-2 genes. PFGE and MLST assigned 10 different clonal, 4 previously reported (ST11, ST18, ST22 and ST64) and 6 new STs (ST89, ST90, ST91, ST92, ST92 and ST94). The blaVIM-1 gene was part of In624 (intI1-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1). In 3 of these isolates, plasmid-mediated quinolone resistance genes (qnrA1 and qnrB4) were present in its downstream region, taking part of a complex class 1 integron ([In624:ISCR1:qnrB4-blaDHA-1] and [In624:ISCR1:qnrA1]). On the other hand, the blaKPC-2 gene was associated with a Tn3-based transposon. The dissemination of the blaVIM-1 gene among various clones suggests a successful horizontal transfer of integron carrying elements that play a dominant role in the development of multidrug resistance in Enterobacteriaceae.

Draft Whole-Genome Sequence of VIM-1-Producing Multidrug-Resistant Enterobacter cloacae EC_38VIM1

ABSTRACT The VIM-1-producing multidrug-resistant strain Enterobacter cloacae was isolated from blood culture. The strain showed multiple resistances to clinically used antibiotics, including all β-lactams, fluoroquinolones, aminoglycosides, and sulfonamides. Sequence analysis showed the presence of 14 genes associated with resistance to antibiotics, including the metallo-β-lactamase VIM-1 gene, which was located in a class 1 integron.