Sandra Rincon

Genetic Basis for In Vivo Daptomycin Resistance in Enterococci

BACKGROUND Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. However, the emergence of resistance to daptomycin during therapy threatens its usefulness. METHODS We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 μg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712. RESULTS Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 μg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential. CONCLUSIONS Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).

Dissemination of Methicillin-Resistant Staphylococcus aureus USA300 Sequence Type 8 Lineage in Latin America

BACKGROUND Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. METHODS S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsed-field gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CA-MRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. RESULTS A total of 1570 S. aureus were included; 651 were MRSA (41%)--with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)--and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a < or =6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). CONCLUSION A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Transferable vancomycin resistance in a community-associated MRSA lineage.

We report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecium: a Prospective, Multicenter Study in South American Hospitals

ABSTRACT Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years.

Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs

ABSTRACT Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.

Parallel Epidemics of Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 Infection in North and South America

BACKGROUND The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis, we aimed to understand the relationships between these 2 epidemics. METHODS We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from the United States, Colombia, Venezuela, and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades. RESULTS Phylogenetic analyses revealed 2 dominant clades that segregated by geographical region, had a putative common ancestor in 1975, and originated in 1989, in North America, and in 1985, in South America. Emergence of these parallel epidemics coincides with the independent acquisition of the arginine catabolic mobile element (ACME) in North American isolates and a novel copper and mercury resistance (COMER) mobile element in South American isolates. CONCLUSIONS Our results reveal the existence of 2 parallel USA300 epidemics that shared a recent common ancestor. The simultaneous rapid dissemination of these 2 epidemic clades suggests the presence of shared, potentially convergent adaptations that enhance fitness and ability to spread.

Coregistration of head CT comparison studies: assessment of clinical utility.

RATIONALE AND OBJECTIVES The authors evaluated the clinical utility of image coregistration in the interpretation of follow-up computed tomographic (CT) studies of the head. MATERIALS AND METHODS Fourteen patients with 34 intracranial lesions underwent follow-up head CT. The images were coregistered automatically with proprietary software on a standard personal computer, and all patient demographic data were removed. A neuroradiologist read the coregistered images several days after first reading the nonregistered images. The reading was repeated some months later to assess intraobserver variability, and a second reader was recruited so that interobserver variability also could be assessed. The interpretations of nonregistered images served as controls for the interpretations of coregistered images. Readers were asked to assess changes in lesion size quantitatively and to record the time it took to evaluate each case. Differences in interpretation speed were evaluated with the t test. Univariate analysis was used to measure accuracy; interpretations were compared with those of a nonblinded senior neuroradiologist, which served as the diagnostic standard. Intra- and interindividual variability were assessed with the kappa statistic. RESULTS The time needed to read the studies decreased by an average of 65.6% (P < .05), with statistically significant reductions for each reader. Coregistration also changed the interpretation results in 21.9% of cases. Coregistration increased the accuracy of reading, but not significantly. Intraobserver variability improved from 0.3554 to 0.7328 with coregistration, and interobserver variability improved from 0.2670 to 0.3309. CONCLUSION Image coregistration is technically feasible and accurate. Coregistration of follow-up studies significantly reduces the time needed for comparison and interpretation. It does not detract from the accuracy of interpretation of follow-up studies and tends to decrease intra- and interobserver variability.

Cefazolin high-inoculum effect in methicillin-susceptible Staphylococcus aureus from South American hospitals

OBJECTIVES Clinical failures with cefazolin have been described in high-inoculum infections caused by methicillin-susceptible Staphylococcus aureus (MSSA) producing type A β-lactamase. We investigated the prevalence of the cefazolin inoculum effect (InE) in MSSA from South American hospitals, since cefazolin is used routinely against MSSA due to concerns about the in vivo efficacy of isoxazolyl penicillins. METHODS MSSA isolates were recovered from bloodstream (n = 296) and osteomyelitis (n = 68) infections in two different multicentre surveillance studies performed in 2001-02 and 2006-08 in South American hospitals. We determined standard-inoculum (10(5)cfu/mL) and high-inoculum (10(7) cfu/mL) cefazolin MICs. PFGE was performed on all isolates that exhibited a cefazolin InE. Multilocus sequence typing (MLST) and sequencing of part of blaZ were performed on representative isolates. RESULTS The overall prevalence of the cefazolin InE was 36% (131 isolates). A high proportion (50%) of MSSA isolates recovered from osteomyelitis infections exhibited the InE, whereas it was observed in 33% of MSSA recovered from bloodstream infections. Interestingly, Ecuador had the highest prevalence of the InE (45%). Strikingly, 63% of MSSA isolates recovered from osteomyelitis infections in Colombia exhibited the InE. MLST revealed that MSSA isolates exhibiting the InE belonged to diverse genetic backgrounds, including ST5, ST8, ST30 and ST45, which correlated with the prevalent methicillin-resistant S. aureus clones circulating in South America. Types A (66%) and C (31%) were the most prevalent β-lactamases. CONCLUSIONS Our results show a high prevalence of the cefazolin InE associated with type A β-lactamase in MSSA isolates from Colombia and Ecuador, suggesting that treatment of deep-seated infections with cefazolin in those countries may be compromised.

The Use of Magnetic Resonance Spectroscopy in the Evaluation of Epilepsy

Magnetic resonance spectroscopy (MRS) is indicated in the imaging protocol of the patient with epilepsy to screen for metabolic derangements such as inborn errors of metabolism and to characterize masses that may be equivocal on conventional magnetic resonance imaging for dysplasia versus neoplasia. Single-voxel MRS with echo time of 35 milliseconds may be used for this purpose as a quick screening tool in the epilepsy imaging protocol. MRS is useful in the evaluation of both focal and generalized epilepsy.

Nosocomial outbreak of Enteroccocus gallinarum: untaming of rare species of enterococci

An unusual increase in infections caused by vancomycin-resistant Enterococcus gallinarum (VREG) was identified in May 2004, in a Colombian tertiary care teaching hospital. A case-control study was subsequently designed to identify risk factors associated with the development of infections due to these organisms. All VREG isolates were subjected to antimicrobial susceptibility testing, vancomycin resistance gene detection and pulsed-field gel electrophoresis (PFGE) typing. Additionally, the presence of genes associated with an acquired pathogenicity island of E. faecalis and a hyl-like gene of E. faecium was assessed by hybridisation assays. Eleven cases of VREG were identified between May through June 2004. VREG was isolated from blood (N=4), surgical secretions (N=4), paranasal sinus secretion (N=1), lung abscess (N=1) and urine (N=1). Infections with VREG were associated with mucositis, hospitalisation in the haematology ward and surgical unit, length of hospital stay prior to culture and invasive procedures within 30 days prior to the culture. Logistic regression found that female sex and hospitalisation in the surgical unit were independent factors for VREG infection. All isolates were identified as E. gallinarum, harboured the vanC1 gene and exhibited indistinguishable restriction patterns by PFGE. Virulence-associated genes were not detected. This is the first documented hospital-wide outbreak of VREG and highlights the fact that uncommon species of enterococci are capable of nosocomial dissemination.