First report of KPC variants conferring ceftazidime-avibactam resistance in Colombia: introducing KPC-197.

Resistance to ceftazidime-avibactam (CZA) due to Klebsiella pneumoniae carbapenemase (KPC) variants is increasing worldwide. We characterized two CZA-resistant clinical Klebsiella pneumoniae strains by antimicrobial susceptibility test, conjugation assays, and WGS. Isolates belonged to ST258 and ST45, and produced a KPC-31 and a novel variant KPC-197, respectively. The novel KPC variant presents a deletion of two amino acids on the Ω-loop (del_168-169_EL) and an insertion of two amino acids in position 274 (Ins_274_DS). Continued surveillance of KPC variants conferring CZA resistance in Colombia is warranted. IMPORTANCE: Latin America and the Caribbean is an endemic region for carbapenemases. Increasingly high rates of Klebsiella pneumoniae carbapenemase (KPC) have established ceftazidime-avibactam (CZA) as an essential antimicrobial for the treatment of infections due to MDR Gram-negative pathogens. Although other countries in the region have reported the emergence of CZA-resistant KPC variants, this is the first description of such enzymes in Colombia. This finding warrants active surveillance, as dissemination of these variants could have devastating public health consequences.

Molecular Mechanisms of Resistance to Ceftazidime/Avibactam in Clinical Isolates of Enterobacterales and Pseudomonas aeruginosa in Latin American Hospitals.

Ceftazidime-avibactam (CZA) is the combination of a third-generation cephalosporin and a new non-ß-lactam ß-lactamase inhibitor capable of inactivating class A, C, and some D ß-lactamases. From a collection of 2,727 clinical isolates of Enterobacterales (n = 2,235) and P. aeruginosa (n = 492) that were collected between 2016 and 2017 from five Latin American countries, we investigated the molecular resistance mechanisms to CZA of 127 (18/2,235 [0.8%] Enterobacterales and 109/492 [22.1%] P. aeruginosa). First, by qPCR for the presence of genes encoding KPC, NDM, VIM, IMP, OXA-48-like, and SPM-1 carbapenemases, and second, by whole-genome sequencing (WGS). From the CZA-resistant isolates, MBL-encoding genes were detected in all 18 Enterobacterales and 42/109 P. aeruginosa isolates, explaining their resistant phenotype. Resistant isolates that yielded a negative qPCR result for any of the MBL encoding genes were subjected to WGS. The WGS analysis of the 67 remaining P. aeruginosa isolates showed mutations in genes previously associated with reduced susceptibility to CZA, such as those involved in the MexAB-OprM efflux pump and AmpC (PDC) hyperproduction, PoxB (blaOXA-50-like), FtsI (PBP3), DacB (PBP4), and OprD. The results presented here offer a snapshot of the molecular epidemiological landscape for CZA resistance before the introduction of this antibiotic into the Latin American market. Therefore, these results serve as a valuable comparison tool to trace the evolution of the resistance to CZA in this carbapenemase-endemic geographical region. IMPORTANCE In this manuscript, we determine the molecular mechanisms of ceftazidime-avibactam resistance in Enterobacterales and P. aeruginosa isolates from five Latin American countries. Our results reveal a low rate of resistance to ceftazidime-avibactam among Enterobacterales; in contrast, resistance in P. aeruginosa has proven to be more complex, as it might involve multiple known and possibly unknown resistance mechanisms.

Molecular Analysis of Polymyxin Resistance among Carbapenemase-Producing Klebsiella pneumoniae in Colombia.

Polymyxin resistance in Klebsiella pneumoniae has been attributed to mutations in mgrB, phoPQ, pmrAB, and crrAB and to the presence of mcr plasmid-mediated genes. Herein, we describe the molecular characteristics of 24 polymyxin- and carbapenem-resistant K. pneumoniae isolates recovered from six Colombian cities between 2009 and 2019. Minimum inhibitory concentrations (MICs) to polymyxin were confirmed by broth microdilution, and whole-genome sequencing was performed to determine sequence type, resistome, and mutations in the genes related to polymyxin resistance, as well the presence of mcr. The results showed high-level resistance to polymyxin (MICs ≥ 4 µg/mL). blaKPC-3 was present in the majority of isolates (17/24; 71%), followed by blaKPC-2 (6/24; 25%) and blaNDM-1 (1/24; 4%). Most isolates belonged to the CG258 (17/24; 71%) and presented amino acid substitutions in PmrB (22/24; 92%) and CrrB (15/24; 63%); mutations in mgrB occurred in only five isolates (21%). Additional mutations in pmrA, crrA, and phoPQ nor any of the mcr resistance genes were identified. In conclusion, we found clonal dissemination of polymyxin and carbapenem-resistant K. pneumoniae isolates in Colombia, mainly associated with CG258 and blaKPC-3. Surveillance of this multidrug-resistant clone is warranted due to the limited therapeutic options for the treatment of carbapenem-resistant K. pneumoniae infections.

Genomic Analysis of CTX-M-Group-1-Producing Extraintestinal Pathogenic E. coli (ExPEc) from Patients with Urinary Tract Infections (UTI) from Colombia.

BACKGROUND: The dissemination of the uropathogenic O25b-ST131 Escherichia coli clone constitutes a threat to public health. We aimed to determine the circulation of E. coli strains belonging to O25b:H4-B2-ST131 and the H30-Rx epidemic subclone causing hospital and community-acquired urinary tract infections (UTI) in Colombia. METHODS: Twenty-six nonduplicate, CTX-M group-1-producing isolates causing UTI in the hospital and community were selected for this study. RESULTS: Twenty-two E. coli isolates harboring CTX-M-15, one CTX-M-3, and three CTX-M-55 were identified. Multilocus Sequence Typing (MLST) showed a variety of sequence types (STs), among which, ST131, ST405, and ST648 were reported as epidemic clones. All the E. coli ST131 sequences carried CTX-M-15, from which 80% belonged to the O25b:H4-B2 and H30-Rx pandemic subclones and were associated with virulence factors iss, iha, and sat. E. coli isolates (23/26) were resistant to ciprofloxacin and associated with amino acid substitutions in quinolone resistance-determining regions (QRDR). We detected two carbapenem-resistant E. coli isolates, one coproducing CTX-M-15, KPC-2, and NDM-1 while the other presented mutations in ompC. Additionally, one isolate harbored the gene mcr-1. CONCLUSIONS: Our study revealed the circulation of the E. coli ST131, O25b:H4-B2-H30-Rx subclone, harboring CTX-M-15, QRDR mutations, and other resistant genes. The association of the H30-Rx subclone with sepsis and rapid dissemination warrants attention from the public health and infections control.

Evaluation of Allplex™ Entero-DR assay for detection of antimicrobial resistance determinants from bacterial cultures.

OBJECTIVE: To evaluate the sensitivity and specificity of the Allplex™ Entero-DR, a quantitative PCR-based method, for the detection of ß-lactamase-encoding genes and vancomycin-resistance determinants in 156 previously characterized Gram-negative bacilli and Enterococcus spp. from bacterial cultures. RESULT: The method had 100% sensitivity and between 92 and 100% of specificity for identifying blaKPC, blaVIM, blaIMP, blaNDM, blaOXA-48-like, blaCTX-M and vanA. In nine isolates, unspecific amplifications were detected. The Ct of these false positives was above 33. The Ct of the correctly identified bla and van genes did not surpass 28 and 30, respectively. None of the clinical isolates included as negative controls yielded any amplification. Therefore, the Allplex™ Entero-DR assay is a highly accurate test for the detection of important antibiotic resistance determinants. With this assay, reliable results can be obtained within 3 h. However, according to our data, samples with Ct values greater than 33 should be considered with caution.

In Vitro Susceptibility to Ceftazidime/Avibactam and Comparators in Clinical Isolates of Enterobacterales from Five Latin American Countries.

Background: High rates of resistance to third-generation cephalosporins and carbapenems in Enterobacterales have been reported in Latin America. Ceftazidime/avibactam (CZA) is the combination of a third-generation cephalosporin and a non-ß-lactam ß-lactamase inhibitor, which has shown activity against isolates producing class A, C and D ß-lactamases. Herein, we evaluated the activity of CZA and comparators against clinical isolates of Enterobacterales in Latin America. Methods: The activity of CZA and comparators was evaluated against clinical isolates of Enterobacterales from Argentina, Brazil, Chile, Colombia and Mexico that were collected between January 2016 and October 2017. One specific phenotypic subset was evaluated. A carbapenem non-susceptible (CNS) phenotype was defined as any isolate displaying a minimum inhibitory concentration (MIC) ≥1 mg/L for ertapenem. Results: CZA was active against 95.8% of all isolates and 77.5% of CNS isolates. Fosfomycin (FOS) and tigecycline (TGC) were the second most active antibiotics with 93.4% of Enterobacterales being susceptible. Conclusions: The results of this study underline the potential therapeutic role of CZA in Latin America.

Performance of disk diffusion and broth microdilution for fosfomycin susceptibility testing of multidrug-resistant clinical isolates of Enterobacterales and Pseudomonas aeruginosa.

OBJECTIVES: This study aimed to evaluate the susceptibility of clinical isolates of Enterobacterales and Pseudomonas aeruginosa to fosfomycin and to determine the concordance of disk diffusion (DD) and broth microdilution (BMD) with agar dilution (AD) for fosfomycin susceptibility testing. METHODS: The activity of fosfomycin against 225 clinical isolates of Escherichia coli (n = 64), Klebsiella pneumoniae (n = 68), Enterobacter spp. (n = 28) and P. aeruginosa (n = 65) was tested by AD, DD and BMD. For DD, results were recorded considering and not considering colonies growing within the inhibition halo as recommended by the CLSI and EUCAST, respectively. Escherichia coli breakpoints were used for all Enterobacterales. Results were reported as categorical agreement (CA), major error (ME; false-resistant), very major error (VME; false-susceptible) and minor error (any other discrepancies). RESULTS: Fosfomycin susceptibility of all tested species was >90% by AD. Following CLSI guidelines, DD was the only method reaching ≥90% CA with AD for E. coli and K. pneumoniae, albeit yielding 6% ME. Neither DD nor BMD achieved acceptable CA percentages for Enterobacter spp. Following EUCAST guidelines, none of the methods had CA ≥ 90%. For Enterobacterales, the best performance of DD is achieved when read as indicated by EUCAST but interpreted according the CLSI breakpoints (>97% CA; 0% VME; ≤2% ME). For P. aeruginosa, BMD yielded the best results (89% CA; 0% VME; 11% ME). CONCLUSION: Neither DD or BMD provide accurate results owing to unacceptable ME and VME percentages even when performed as intended by the guidelines.