Elsa De La Cadena

Novel VIM Metallo-β-Lactamase Variant, VIM-24, from a Klebsiella pneumoniae Isolate from Colombia

ABSTRACT We report the emergence of a novel VIM variant (VIM-24) in a Klebsiella pneumoniae isolate in Colombia. The isolate displays MICs for carbapenems below the resistance breakpoints, posing a real challenge for its detection. The blaVIM-24 gene was located within a class 1 integron carried on a large plasmid. Further studies are needed to clarify its epidemiological and clinical impact.

Initial assessment of the molecular epidemiology of blaNDM-1 in Colombia

ABSTRACT We report complete genome sequences of four blaNDM-1-harboring Gram-negative multidrug-resistant (MDR) isolates from Colombia. The blaNDM-1 genes were located on 193-kb Inc FIA, 178-kb Inc A/C2, and 47-kb (unknown Inc type) plasmids. Multilocus sequence typing (MLST) revealed that these isolates belong to sequence type 10 (ST10) (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumannii and Acinetobacter nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid in E. coli contained a novel complex transposon (Tn125 and Tn5393 with three copies of blaNDM-1) and a recombination “hot spot” for the acquisition of new resistance determinants.

Genomic and Molecular Characterization of Clinical Isolates of Enterobacteriaceae Harboring mcr-1 in Colombia, 2002 to 2016

ABSTRACT Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae. Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1-positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1, including 8 Escherichia coli, 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate. E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S. Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10−5 to 2.07 × 10−1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids.

Estrategias para la implementación y reporte de los puntos de corte CLSI vigentes y pruebas fenotípicas confirmatorias para BLEE y carbapenemasas en bacilos Gram negativos en laboratorios clínicos de Colombia

In 2010, the Clinical and Laboratory Standards Institute (CLSI) began a process to revise and update the breakpoints for broth microdilution and disk diffusion for cephalosporins (Cefazolin, Cefotaxime, Ceftriaxone, Ceftazidime), monobactams (Aztreonam) and carbapenems (Imipenem, Meropenem, Ertapenem and Doripenem). The changes made were based on PK/PD models that attempt to predict clinical outcomes using minimum inhibitory concentration (MIC) and specific dosage regimens, regardless of the resistance mechanism expressed by the organism. The new breakpoints would eliminate the need to perform screening and confirmatory testing for ESBLs and carbapenemases for treatment decisions, and thus they would be used only for infection control purposes. Nevertheless, there are limitations to current methods in Colombia, a lack of knowledge regarding the recent changes and epidemiologic alarm over new B-lactamases spreading in our country. Therefore it was necessary to formulate and issue recommendations for clinical laboratories, with the aim of standardizing the criteria for reports on antibiograms in Gram-negative bacilli, including the current CLSI breakpoints and applying phenotypic confirmatory testing to detect ESBLs and Carbapenemases.

Ceftolozane-Tazobactam Resistance in Multidrug-Resistant Pseudomonas aeruginosa Isolates Not Associated with AmpC Activity

Abstract Background Ceftolozane-tazobactam (CT) is a newly approved cephalosporin/β-lactamase inhibitor combination with excellent in vitro activity against multidrug-resistant (MDR) P. aeruginosa. Unfortunately, CT-resistance (CT-R) has already been reported. In this work, we evaluate mutational pathways associated with high level of CT-R and assess the role of AmpC in a clinical strain-pair of MDR P. aeruginosa. Methods A CT susceptible isolate of P. aeruginosa (2365) and its CT-R derivatives (2366 and 2367) were recovered from the infected device of a patient before and after treatment with CT. Minimum inhibitory concentrations (MICs) to CT were determined by Etest. Resistance mediated by AmpC hyperproduction was evaluated using ceftazidime (CAZ) and meropenem (MER) with and without cloxacillin (CLOX) at concentration of 1 mg/ml. In addition, the β-lactamase hydrolysis activity was determined for crude cell lysate of the isolates using a spectrophotometric assay for nitrocefin degradation. Furthermore, whole genome sequencing of the three strains was performed and compared (2365 vs. 2366 and 2367). Reads from each isolate were mapped against the genome of the reference strain PAO1. Variants identified by GATK, SamTools and CLC Genomics Workbench 8.5 were selected and annotated with SnpEff. Results Strain 2365 had a CT MIC of 0.75 mg/ml while 2366 and 2367 have MICs > 256 mg/ml. AmpC hyperproduction test was positive only for the susceptible isolate (2365). In concordance, the hydrolysis assay showed a lack of nitrocefin degradation by CT-R 2366 compared with its CT-susceptible isolate 2365. Notably, the three strains (S and R) exhibited a truncated AmpD. Comparison of the resistant derivatives vs. 2365 and 2367 showed a 7 amino acid deletion in the Ω-loop of the β-lactamase AmpC in both resistant derivatives and mutations in genes predicted to encode a hypothetical protein, an ABC transporter ATP-binding protein and a multidrug resistance operon repressor MexR. Conclusion Our results suggest that the deletion in the Ω-loop of AmpC in 2366 and 2367 does not contribute to CT-R in these P. aeruginosa strains. Further characterization of AmpC and other predicted proteins identified by WGS are needed to determine the mechanism of CT-R. Disclosures All authors: No reported disclosures.

Plasmid Promiscuity Explains High Endemicity of KPC-2 Among Colombian Enterobacteriaceae

Abstract Background Carbapenemase-producing Enterobacteriaceae (CPE) currently pose a significant global public health threat. KPC carbapenemases are highly endemic in Colombia mostly among Enterobacteriaceae. In Klebsiella pneumoniae (Kpn), horizontal transfer of genes encoding KPC and expansion of isolates belonging to clonal group (CG) 258 resulted in epidemic levels of carbapenemase-producing Kpn. We aimed to understand the dynamics of transmission of KPC-genes among CPE infecting and colonizing patients in an endemic area of Colombia. Methods We conducted a surveillance study in 3 hospitals around Medellin, Antioquia (November 2013 to October 2015) that included patients colonized and infected (by physician criteria) with CPE. The isolates were collected, identified and typed initially using rep-PCR. A subset of isolates were chosen for Whole Genome Sequencing on the Illumina platform based on initial molecular characterization. De-novo assembly and maximum likelihood phylogenetic analyses were performed in all sequenced isolates. Results A total of 131 KPC-producing Enterobacteriaceae isolates were recovered from 77 colonized and 29 infected patients. A total of 76 selected isolates were sequenced. In Kpn, compartmentalization of KPC-3 within CG258 was observed, whereas KPC-2 was identified in different genetic backgrounds but not in CG258. In E. coli, blaKPC-2 was found in two clusters belonging to ST131 and ST349. In one hospital both Kpn (ST36,15,101,140, 502) and E. coli shared a 56 Kb plasmid harboring blaKPC-2 with high degree of identity to the conjugative IncN plasmid N3. The blaKPC-2 gene was found within a variation of Tn4401b harboring ISKpn6 and carrying both a Tn3-transposase and a resolvase. E. cloacae, C. freundii and S. marcescens only harbored KPC-2 (and not KPC-3) within the same Tn4401b structure. Conclusion The KPC epidemic in an area of high endemicity in Colombia is driven by horizontal transfer of plasmids harboring blaKPC-2 among members of the Enterobacteriaceae family. These findings are consistent with a KPC-plasmid epidemic rather than clonal expansion of a successful genetic lineage. Controlling the KPC epidemic in Colombia would be challenging and is likely influenced by antibiotic consumption rather than patient to patient transmission. Disclosures A. M. Rada, COLCIENCIAS: Student, Research grant and Research support; N. Orozco, COLCIENCIAS: Research Contractor, Salary; C. Hernández-Gómez, Merck Sharp and Dohme, Pfizer: Consultant, Consulting fee; C. Pallares, Merck Sharp & Dohme, Pfizer: Consultant, Consulting fee; M. V. Villegas, Merck Sharp & Dohme, Pfizer: Consultant, Consulting fee and Research support; E. Restrepo, COLCIENCIAS: Investigator, Research support

Molecular characterisation of carbapenem-resistant Enterobacter cloacae complex in Colombia: blaKPC and the ‘changing landscape’

OBJECTIVES The aim of this study was to examine the population structure of representative carbapenem-resistant Enterobacter cloacae complex (CR-Ecl) isolates from eight different Colombian regions and to characterise their associated β-lactamases. METHODS A total of 28 CR-Ecl isolates collected in Colombia between 2009-2013 through the Colombian Nosocomial Network were included in this study. Antimicrobial susceptibility testing was performed by the broth microdilution method. Molecular detection of carbapenemase and extended-spectrum β-lactamase (ESBL) genes and the presence of transposon Tn4401 was evaluated by PCR and DNA sequencing. Genetic relatedness was assessed by multilocus sequencing typing (MLST) and repetitive sequence-based PCR (rep-PCR). RESULTS PCR and DNA sequencing revealed that 19/28 (68%) of the CR-Ecl isolates carried blaKPC-2. Analysis of the genetic environment found blaKPC-2 within transposon Tn4401b in 8/19 isolates (42%). Population genetic analysis using rep-PCR revealed four clonal groups. MLST showed a variety of sequence types (STs), among which ST510 was the most common (10/28 isolates; 36%). CONCLUSIONS blaKPC-2 was discovered as the most common mechanism of carbapenem resistance in CR-Ecl and was disseminated among different STs. Although none of the previously reported major clonal complexes were identified, it appears that local strain lineages are associated with the spread of blaKPC within CR-Ecl in various regions of Colombia.

328Clinical Impact of Multidrug-Resistant Organisms (MDRO) causing Healthcare-Associated Infections (HAIs) in 10 Colombian cities

328. Clinical Impact of Multidrug-Resistant Organisms (MDRO) causing Healthcare-Associated Infections (HAIs) in 10 Colombian cities Gabriel Motoa, MD; Cristhian Hernandez, Bsc; Victor M Blanco, MD; Juan S. Muñoz, MD; Adriana Correa, MSc; Elsa De La Cadena, Bsc; Maria V. Villegas, MD, MSc; Colombian Nosocomial Resistance Study Group; Centro Internacional De Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia