Natasha Vanegas

Dissemination of Multiple Drug Resistance Genes by Class 1 Integrons in Klebsiella pneumoniae Isolates from Four Countries: a Comparative Study

ABSTRACT A comparative genetic analysis of 42 clinical Klebsiella pneumoniae isolates, resistant to two or more antibiotics belonging to the broad-spectrum β-lactam group, sourced from Sydney, Australia, and three South American countries is presented. The study focuses on the genetic contexts of class 1 integrons, mobilizable genetic elements best known for their role in the rapid evolution of antibiotic resistance among Gram-negative pathogens. It was found that the class 1 integrons in this cohort were located in a number of different genetic contexts with clear regional differences. In Sydney, IS26-associated Tn21-like transposons on IncL/M plasmids contribute greatly to the dispersal of integron-associated multiple-drug-resistant (MDR) loci. In contrast, in the South American countries, Tn1696-like transposons on an IncA/C plasmid(s) appeared to be disseminating a characteristic MDR region. A range of mobile genetic elements is clearly being recruited by clinically important mobile class 1 integrons, and these elements appear to be becoming more common with time. This in turn is driving the evolution of complex and laterally mobile MDR units and may further complicate antibiotic therapy.

Nosocomial infections caused by community-associated methicillin-resistant Staphylococcus aureus in Colombia.

BACKGROUND Community-associated methicillin-resistant Staphylococcus aureus strains (CA-MRSA) have emerged as the causative agent of health care-associated infections. METHODS An observational and prospective study was carried out in 5 hospitals in Bogotá, Colombia; severe MRSA infections were identified, and their origin led to classification as health care-associated (HA-MRSA), community-associated, or nosocomial infections. MRSA isolates were analyzed by SCCmec, pulsed-field gel electrophoresis, multilocus sequence typing, and virulence factors. RESULTS Twenty-six (10.4%) CA-MRSA nosocomial infection-causing strains (eg, USA300) were detected in 250 MRSA infection isolates in mainly primary bacteremia and surgical site infections. The mortality related to nosocomial infection by CA-MRSA was 27%. CONCLUSION The presence of nosocomial infection by CA-MRSA in Colombia was confirmed.

Cryptococcosis in Colombia: Results of the national surveillance program for the years 2006-2010

INTRODUCTION Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are found with increasing the frequency, both in healthy individuals in the community and in hospitalized patients. In Colombia and the Andean region, CA-MRSA isolates have a genetic background that is related to the pandemic USA300 clone. OBJECTIVE Two molecular methods are designed and standardized for the rapid differentiation of Colombian community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates. MATERIALS AND METHODS Two molecular methods were standardized for the identification of CA-MRSA isolates. The first method was based on the differential digestion of the carbamate kinase (arcC)and guanylate kinase (gmk) genes in the sequences type 5 (ST5) in the HA-MRSA isolates and 8 (ST8) in the CA-MRSA isolates. The second method was based on the PCR amplification of 5 specific virulence factors found in CA-MRSA and HA-MRSA isolates. The specificity and precision of each method were evaluated using 237 clinical MRSA isolates. RESULTS The first method identified 100% and 93.2% of the CA-MRSA and HA-MRSA isolates, respectively. The second method also correctly identified the two isolates types (CA-MRSA and HA-MRSA). CONCLUSIONS These two methods are a convenient alternative for the rapid identification of the CA-MRSA isolates, compared with other techniques such as pulsed field gel electrophoresis and multilocus sequence typing, which are time-consuming and more expensive.

Pediatric cases from Colombia caused by a Panton-Valentine Leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus ST8-SCCmecIVc clone.

To the Editor: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection is on the increase throughout the world. Panton-Valentine Leukocidin-positive (PVL) S. aureus strain dissemination in the community represents a public health concern about severe complications in children. Infections for CA-MRSA ST8, clonally related with USA300, have recently appeared in Colombia. We are reporting the first pediatric CAMRSA infection cases that occurred in Bogotá between June 2004 and May 2006. The first case concerned a previously healthy 14-year-old boy who developed severe sepsis with multiorgan failure, infectious endocarditis of the tricuspid valve, and septic pulmonary embolism associated with deep vein thrombosis, bilateral pyomyositis, and osteomyelitis of the lower extremities; he recovered after prolonged medical and surgical treatment. The second case involved a 9-month-old male with congenital Toxoplasma infection who developed respiratory failure associated with pneumonia and cervical cellulitis and responded to antimicrobial therapy. The third case concerned a 16-yearold boy from Bogotá with a history of recurrent furuncles associated with spontaneous drainage in his lower extremities during the previous 3 months. He was admitted because of an acute onset of progressive inflammatory local signs in the left inguinal area that limited his walking; he responded to medical and surgical treatment. The 3 isolates recovered from patients were confirmed as MRSA, the presence of nuc and mecA genes having been detected by polymerase chain reaction. The minimal inhibitory concentrations (MICs) were determined by using the agar diffusion test, according to Clinical and Laboratory Standards Institute. The oxacillin MICs for the 3 isolates were 32, 4, and 8 g/mL, respectively. The third MRSA exhibited the MLSB constitutive phenotype in double-disk diffusion assay (D test), with erythromycin ( 64 g/mL MIC) and clindamycin resistance (32 g/mL MIC). The 3 isolates were positive for the PVL genes (lukF-PV and lukS-PV) and harbored SCCmec type IVc. The 3 MRSA isolates SmaI macrorestriction profiles revealed 3 unique patterns clustering above 87% similarity into 1 pulsed-field gel electrophoresis type by computer analysis. The 3 strains were ascribed to ST8 by multilocus sequence typing belonging to the same sequence type as USA300, a common pulsedfield gel electrophoresis type in the United States with a similarity higher than 75%. These 3 isolates were not epidemiologically related. The clinical spectrum of the cases ranged from mild to severe infection. All cases showed some degree of skin, deep soft tissue, or bone compromise, as has been frequently described in children, and similar to the first report of adult cases from Columbia. Molecular typing identified these 3 isolates as belonging to the ST8-MRSA-IVc clone; they carried the PVL genes often associated with CA isolates. A retrospective report has suggested that CA-MRSA strains have been circulating in Colombia since 2001; they may have been ignored, along with their significant public health implications. Awareness about what is happening in our country will help us to evaluate the current state of our epidemiology regarding CA-MRSA infection and to develop strategies of infection control, following the lead of several other countries.

Detection of a New Community Genotype Methicillin-Resistant Staphylococcus aureus Clone That Is Unrelated to the USA300 Clone and That Causes Pediatric Infections in Colombia

ABSTRACT The dissemination of a clone of community genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) that is related to USA300 has been reported in Latin America. We recently detected isolates of a new clone of CG-MRSA (spa type t1635 and ACME-negative) that was genetically unrelated to the USA300 clone and that causes infections in children in Colombia. This finding indicates the appearance of a new clone of CG-MRSA in our region.

Transcriptional Analysis of the vanC Cluster from Enterococcus gallinarum Strains with Constitutive and Inducible Vancomycin Resistance

ABSTRACT The vanC glycopeptide resistance gene cluster encodes enzymes required for synthesis of peptidoglycan precursors ending in d-Ala-d-Ser. Enterococcus gallinarum BM4174 and SC1 are constitutively and inducibly resistant to vancomycin, respectively. Analysis of peptidoglycan precursors in both strains indicated that UDP-MurNAc-tetrapeptide and UDP-MurNAc-pentapeptide[d-Ser] were synthesized in E. gallinarum SC1 only in the presence of vancomycin (4 μg/ml), whereas the “resistance” precursors accumulated in the cytoplasm of BM4174 cells under both inducing and noninducing conditions. Northern hybridization and reverse transcription-PCR experiments revealed that all the genes from the cluster, vanC-1, vanXYC, vanT, vanRC, and vanSC, were transcribed from a single promoter. In the inducible SC1 isolate, transcriptional regulation appeared to be responsible for inducible expression of resistance. Promoter mapping in E. gallinarum BM4174 revealed that the transcriptional start site was located 30 nucleotides upstream from vanC-1 and that the −10 promoter consensus sequence had high identity with that of the vanA cluster. Comparison of the deduced sequence of the vanSC genes from isolates with constitutive and inducible resistance revealed several amino acid substitutions located in the X box (R200L) and in the region between the F and G2 boxes (D312N, D312A, and G320S) of the putative sensor kinase proteins from isolates with constitutive resistance.

USA300-related methicillin-resistant Staphylococcus aureus clone is the predominant cause of community and hospital MRSA infections in Colombian children

OBJECTIVE Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) isolates are known to be more virulent and clinically aggressive in children. The goal of the present study was characterize the molecular epidemiology of MRSA isolates causing infections in Colombian children. METHODS An observational and prospective study was conducted between April 2009 and June 2011 at 15 hospitals in Bogotá, Colombia. A detailed epidemiological profile was made of 162 children infected with MRSA. The isolates were subjected to antimicrobial susceptibility testing, molecular characterization including 21 virulence genes, SCCmec, spa and agr typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). RESULTS Among all isolates included in the study, 85.8% were obtained from patients whose infectious process was initiated in the community; of these, 69,8% occurred in patients without healthcare-associated risk factors. The molecular characterization of the isolates showed a high proportion (95.1%) containing a community-genotype profile with a high prevalence of SCCmec type IV, PVL-positives, and also related to CC8. Most CG-MRSA isolates (143, 92.9%) were genetically related to the pandemic clone USA300, differing by the presence of SCCmec IVc and the absence of the arginine catabolic mobile element (ACME). CONCLUSIONS An increase in the frequency of CG-MRSA infections has been reported worldwide. In this study we found that almost all MRSA infections in our pediatric population were caused by community-genotype isolates, supporting the success of the CG-MRSA clones.

Genomic Epidemiology of NDM-1-Encoding Plasmids in Latin American Clinical Isolates Reveals Insights into the Evolution of Multidrug Resistance

Abstract Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-β-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.

First Complete Providencia rettgeri Genome Sequence, the NDM-1-Producing Clinical Strain RB151

ABSTRACT Providencia rettgeri is an opportunistic bacterial pathogen of clinical significance due to its association with urinary tract infections and multidrug resistance. Here, we report the first complete genome sequence of P. rettgeri. The genome of strain RB151 consists of a 4.8-Mbp chromosome and a 108-kbp blaNDM-1-positive plasmid.

Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital.

Drug resistant Pseudomonas aeruginosa represents a therapeutic challenge. To assess the diversity of P. aeruginosa antibiotic resistant variants, isolates were recovered from hospital patients in Colombia. Thirty of 60 isolates contained class 1 integrons and five were of Sequence Type ST235 having appeared in a single intensive care unit. All five possessed an unusual integron but showed differences in gene cassette content and the presence/absence of insertion sequence IS26. This showed that differences can arise rapidly, even within a single ICU. Also, the emergence of IS26 in P. aeruginosa is contributing to the evolution of resistance in this bacterium.