Rafael Rios

A Prospective Cohort Multicenter Study of Molecular Epidemiology and Phylogenomics of Staphylococcus aureus Bacteremia in Nine Latin American Countries

ABSTRACT Staphylococcus aureus is an important pathogen causing a spectrum of diseases ranging from mild skin and soft tissue infections to life-threatening conditions. Bloodstream infections are particularly important, and the treatment approach is complicated by the presence of methicillin-resistant S. aureus (MRSA) isolates. The emergence of new genetic lineages of MRSA has occurred in Latin America (LA) with the rise and dissemination of the community-associated USA300 Latin American variant (USA300-LV). Here, we prospectively characterized bloodstream MRSA recovered from selected hospitals in 9 Latin American countries. All isolates were typed by pulsed-field gel electrophoresis (PFGE) and subjected to antibiotic susceptibility testing. Whole-genome sequencing was performed on 96 MRSA representatives. MRSA represented 45% of all (1,185 S. aureus) isolates. The majority of MRSA isolates belonged to clonal cluster (CC) 5. In Colombia and Ecuador, most isolates (≥72%) belonged to the USA300-LV lineage (CC8). Phylogenetic reconstructions indicated that MRSA isolates from participating hospitals belonged to three major clades. Clade A grouped isolates with sequence type 5 (ST5), ST105, and ST1011 (mostly staphylococcal chromosomal cassette mec [SCCmec] I and II). Clade B included ST8, ST88, ST97, and ST72 strains (SCCmec IV, subtypes a, b, and c/E), and clade C grouped mostly Argentinian MRSA belonging to ST30. In summary, CC5 MRSA was prevalent in bloodstream infections in LA with the exception of Colombia and Ecuador, where USA300-LV is now the dominant lineage. Clonal replacement appears to be a common phenomenon, and continuous surveillance is crucial to identify changes in the molecular epidemiology of MRSA.

Ceftaroline-Resistant, Daptomycin-Tolerant, and Heterogeneous Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Causing Infective Endocarditis

ABSTRACT We report a case of infective endocarditis (IE) caused by ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant S. aureus (MRSA). Resistance to ceftaroline emerged in the absence of drug exposure, and the E447K substitution in the active site of PBP2a previously associated with ceftaroline resistance was identified. Additionally, we present evidence of patient-to-patient transmission of the strain within the same unit. This case illustrates the difficulties in treating MRSA IE in the setting of a multidrug-resistant phenotype.

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.

The Cefazolin Inoculum Effect Is Associated With Increased Mortality in Methicillin-Susceptible Staphylococcus aureus Bacteremia

Abstract Background Recent studies have favored the use of cefazolin over nafcillin for the treatment of methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. The clinical influence of the cefazolin inoculum effect (CzIE) in the effectiveness of cephalosporins for severe MSSA infections has not been evaluated. Methods We prospectively included patients from 3 Argentinian hospitals with S. aureus bacteremia. Cefazolin minimum inhibitory concentrations (MICs) were determined at standard (105 colony-forming units [CFU]/mL) and high (107 CFU/mL) inoculum. The CzIE was defined as an increase of MIC to ≥16 µg/mL when tested at high inoculum. Whole-genome sequencing was performed in all isolates. Results A total of 77 patients, contributing 89 MSSA isolates, were included in the study; 42 patients (54.5%) had isolates with the CzIE. In univariate analysis, patients with MSSA exhibiting the CzIE had increased 30-day mortality (P = .034) and were more likely to have catheter-associated or unknown source of bacteremia (P = .033) compared with patients infected with MSSA isolates without the CzIE. No statistically significant difference between the groups was observed in age, clinical illness severity, place of acquisition (community vs hospital), or presence of endocarditis. The CzIE remained associated with increased 30-day mortality in multivariate analysis (risk ratio, 2.65; 95% confidence interval, 1.10–6.42; P = .03). MSSA genomes displayed a high degree of heterogeneity, and the CzIE was not associated with a specific lineage. Conclusions In patients with MSSA bacteremia where cephalosporins are used as firstline therapy, the CzIE was associated with increased 30-day mortality. Clinicians should be cautious when using cefazolin as firstline therapy for these infections.

Clonal Emergence of Invasive Multidrug-Resistant Staphylococcus epidermidis Deconvoluted via a Combination of Whole-Genome Sequencing and Microbiome Analyses

Background Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

A Whole Genome Sequencing (WGS) Approach to Predict Daptomycin (DAP) Susceptibility of Enterococcus faecium

Abstract Background We have previously shown that vancomycin-resistant E. faecium (VRE) with DAP MICs close to the breakpoint (4 µg/mL) harbor genetic changes associated with DAP resistance (DAP-R). Further, DAP MIC was a predictor of poor microbiological eradication in patients with VRE bacteremia treated with DAP. Furthermore, DAP-susceptible VRE isolates with DAP MIC of 3–4 µg/mL (Etest) were more likely to fail DAP therapy, independently of the DAP dose used. Here, we used WGS to determine whether mutations associated with DAP-R could predict DAP MICs. Methods We performed WGS to identify potential determinants of DAP-R in 80 E. faecium isolates (62 DAP-S and 18 DAP-R recovered from bloodstream and other infection sites) in diverse US geographical locations. Two modeling strategies were employed with the aim of increasing the robustness of our prediction strategy, (i) a logistic regression model approach to predict the probability of an isolate of exhibiting a DAP MIC of ≥ 3µg/dl based on the presence of relevant mutations, and (ii) a linear regression model to predict a single doubling dilution increase on DAP MIC in the presence or absence of mutations associated with DAP-R, after transforming MICs to a log2 scale. Statistical significance (P value) was set at <0.05. Results Out of 62 genetic determinants examined, the presence of substitutions in LiaFSR or YycFGHI systems were independent predictors of an isolate exhibiting DAP MIC ≥ 3 µg/mL (logistic model, LiaFSR OR 8.9, P < 0.0001 and YycFGHI OR 6.2; P < 0.0001) or of an increase in DAP MIC (lineal model; LiaFSR β 14.6; P < 0.04; YycFGHI β 1.7; P < 0.0001) and were consistent in both models. When we evaluated individual genetic changes within the proteins from both systems, substitutions in YycG were associated with the greatest increase on DAP MIC (8.0-fold; β = 3.0, 95% CI 2.8–4.1 P < 0.0001), followed by LiaF (3.0-fold; β 1.5, 95% CI 0.17–2.9 P = 0.028;), LiaS (2.0-fold; β = 0.9, 95% CI 0.2–1.6; P = 0.006) and LiaR (1.7-fold; β = 0.8, 95% CI 0.1–1.5, P = 0.021). Conclusion Our data indicate that WGS may identify organisms with elevated DAP MIC that, even if not above the clinical breakpoint, may lead to microbiological failure. WGS has the potential of providing a better guidance for DAP therapy. Disclosures M. Zervos, Merck: Investigator, Research grant Genentech: Investigator, Research grant Cempra: Investigator, Research grant Pfizer: Investigator, Research grant

Genomic Pathways Associated With Daptomycin (DAP) Resistance in DAP-Susceptible Enterococcus faecium Harboring Substitutions in LiaFSR

Abstract Background DAP is used off-label for treatment of severe enterococcal infections. DAP resistance (R) in E. faecium has been associated with changes in LiaFSR, a three-component regulatory system that controls the cell envelope stress response to antibiotics. In particular, substitutions in LiaS (T120A) and LiaR (W73C) seem to predispose to development of DAP-R during therapy, without increasing the DAP MIC above the clinical breakpoint. Using a PK/PD model of simulated endocardial vegetations, we evaluated the genomic pathways for DAP-R under different DAP dose schemes. Methods A DAP-susceptible E. faecium (HOU503; MIC 3 mg/ml) harboring the above LiaSR substitutions, was subjected to simulated human doses of 6, 8, and 10 mg/kg/d in the model for 14 days using a starting inoculum of 109 CFU/ml. Sixteen DAP-R isolates were recovered from the SEV model: five isolates from 6 mg/kg (D6 isolates from days 2 to 14); five isolates from 8 mg/kg (D8 isolates from days 2 to 8); and six isolates from 10 mg/kg (D10 isolates from days 1 to 14) which were subjected to whole genome sequencing. Reads from each sequenced isolate were mapped against the HOU503 genome for SNP analyses. Variant calling was done with GATK, SamTools, and the low-frequency variant detector from CLC Genomics Workbench 8.5. Variants detected by the three callers were selected and annotated with SnpEff; then compared among the different groups of isolates accordingly to the DAP doses that were exposed. Results We detected a total of 16 proteins exhibiting substitutions consistently in all the DAP-R sequenced isolates; including mobile genetic elements (9), hypothetical proteins (2), a bacteriocin, a cysteine desulfurase, a N-acetylglucosamine-specific PTS system, a N-acetylmannosamine-6-phosphate 2-epimerase and a MurR/RpiR, which is a transcriptional regulator that represses the operon MurPQ involved in the uptake and degradation of N-acetylmuramic acid. Notably, mutations in cardiolipin synthase were present only in isolates recovered under D8 dose. The LiaRS substitutions remained in all isolates. Conclusion Using a humanized SEV PK/PD model and SNP-based analyses, we were able to uncover possible novel genetic pathways associated with the development DAP-R via the LiaFSR system in enterococci. Disclosures M. J. Rybak, Allergen: Scientific Advisor, Consulting fee

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

Co-occurrence of NDM-5, OXA-181 and CMY-2 in a Clinical E. coli Isolate From a Patient in the United States

Abstract Background Carbapenem-resistant Enterobacteriacae (CRE) are an important public health problem. A mechanism of expansion of the CRE epidemic is transcontinental spread of multidrug-resistant organisms. Here, we present a patient with an intra-abdominal abscess due to Escherichia coli with an unusual multidrug resistance phenotype who was admitted to a tertiary hospital in Houston, TX after treatment in a country from the Middle East. Methods Identification and antimicrobial susceptibility testing were performed by standard methods. Whole-genome sequencing (WGS) was performed on an Illumina platform, with resistance genes identified by ResFinder 2.1 and plasmids recognized using PlasmidFinder 1.3. Results. It is confirmed via PCR, S1 pulsed field gel electrophoresis, and Southern blotting. Carbapenemase activity of bacterial lysates was assayed using 50 µM imipenem in the presence of both EDTA and zinc. Results The E. coli isolate was resistant to all β-lactams, including ceftazidime/avibactam. The organism belonged to ST410 and harbored blaNDM-5, blaOXA-181, blaCMY-2, and blaTEM-1B. The blaOXA-181 gene was located on an IncX3 plasmid of ca. 50 Kb in association with the ISEcp1 mobile element and the qnrS21 gene encoding quinolone resistance. The blaNDM-5 gene was located on an FIA/FIB plasmid of ~100 Kb, in association with ΔISAba125, and upstream of a putative bleomycin resistance gene, a conserved arrangement among NDM expressing Gram-negative organisms. Cell lysate assays showed decreasing carbapenemase activity with increasing concentrations of EDTA and an increase in activity with the addition of zinc, suggesting the NDM-5 metallo-β-lactamase is largely responsible for the observed carbapenemase activity. Comparison with plasmid sequences available suggested convergence of resistance determinants captured from a wide geographic area. Conclusion Plasmid-mediated spread of β-lactamases among Enterobacteriaceae is a rapidly evolving threat, with the introduction of NDM-5 and OXA-181 in the United States being a particularly disturbing development. Introduction of multidrug-resistant organisms from areas of high prevalence of resistance may change the landscape of antimicrobial resistance in the United States. Disclosures All authors: No reported disclosures.