Antimicrobial stewardship programs in seven Latin American countries: facing the challenges.

BACKGROUND: Studies have shown that more than 50% of the antibiotics used in hospitals are unnecessary or inappropriate and, that antimicrobial resistance may cost up to 20 billion USD in excess medical costs each year. On the other hand, Antimicrobial Stewardship Programs (ASP) significantly reduce inappropriate antimicrobial use, emergence of antimicrobial resistance, healthcare associated infections, and costs in hospital settings. OBJECTIVE: To evaluate the development of ASP and antibiotic savings in 7 Latin American hospitals using standardized quantitative indicators in all the participating health care institutions. METHODS: An interventional study was conducted, where pre- and post- evaluations were performed using a standardized score tool adapted from the Joint Commission International accreditation standards and, the Colombian Institute of Technical Standards and Certification. We evaluated ASP from 7 Latin American hospitals between 2019 and 2020. A pre-intervention evaluation was done in each hospital to quantify the degree of development of the ASP (ASP Development score). Based on these results, tailored on-site training was implemented in each hospital, followed by a post-intervention evaluation to quantify improvement of ASP-development indicators. In addition, monetary savings in antimicrobials derived from the ASP intervention were estimated. RESULTS: In the pre-intervention evaluation, the average ASP development score for the 7 institutions was 65.8% (40-94.3%). The items with the lowest development score were those related to monitoring and communicating the ASP progress and success. For the post-intervention evaluation, 2 institutions couldn't participate due to the pressure imposed by the COVID-19 pandemic. For the remaining 5/7 hospitals, the average ASP development score was 82.3% with an increase of 12.0% when compared to the pre-intervention measurement of the same institutions (average pre-intervention score 70.3% (48.2%-94.3%) The items with a significant increase were key performance indicators, AMS education and training of the prescribers. Three of the seven (3/7) hospitals reported antibiotic monetary savings associated to the ASP intervention. CONCLUSIONS: The use of the tool described shown to be useful to evaluate specific areas of ASP-development that were lacking and tailor interventions for the participating hospitals, consequently, it helped improve ASP-development in the institutions that underwent pre- intervention and post-intervention analysis. In addition, the strategies showed monetary savings on antimicrobial costs when measured.

Impact of antimicrobial stewardship programs on antibiotic consumption and antimicrobial resistance in four Colombian healthcare institutions.

BACKGROUND: Antimicrobial stewardship programs (ASPs) have become a fundamental pillar in optimizing antimicrobial usage, improving patient care, and reducing antimicrobial resistance (AMR). Herein we evaluated the impact of an ASP on antimicrobial consumption and AMR in Colombia. METHODS: We designed a retrospective observational study and measured trends in antibiotic consumption and AMR before and after the implementation of an ASP using interrupted time series analysis over a 4-year period (24 months before and 24 months after ASP implementation). RESULTS: ASPs were implemented according to the available resources in each of the institutions. Before ASP implementation, there was a trend toward an increase in the antibiotic consumption of all measured antimicrobials selected. Afterward, an overall decrease in antibiotic consumption was observed. The use of ertapenem and meropenem decreased in hospital wards, while a decrease in the use of ceftriaxone, cefepime, piperacillin/tazobactam, meropenem, and vancomycin was observed in intensive care units. After ASP implementation, the trend toward an increase of oxacillin-resistant Staphylococcus aureus, ceftriaxone-resistant Escherichia coli, and meropenem-resistant Pseudomonas aeruginosa was reversed. CONCLUSIONS: In our study, we showed that ASPs are a key strategy in tackling the emerging threat of AMR and have a positive impact on antibiotic consumption and resistance.

Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

An Analysis of the Epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: Convergence of Two Evolutionary Mechanisms Creates the "Perfect Storm".

Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005. Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates. Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3. Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia.

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

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.

Initial Assessment of the Molecular Epidemiology of blaNDM-1 in Colombia.

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.

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.

Identification of mcr-1 Genes and Characterization of Resistance Mechanisms to Colistin in Escherichia coli Isolates from Colombian Hospitals.

We report the presence of the mcr-1 gene among 880 Escherichia coli clinical isolates collected in 13 hospitals from 12 Colombian cities between 2016 and 2019. Seven (0.8%) isolates were colistin resistant (MIC ≥ 4 µg/mL). These colistin-resistant isolates were screened for the presence of the mcr-1 gene; five carried the gene. These five isolates were subjected to whole genome sequencing (WGS) to identify additional resistomes and their ST. In addition, antimicrobial susceptibility testing revealed that all E. coli isolates carrying mcr-1 were susceptible to third generation-cephalosporin and carbapenems, except one, which carried an extended-spectrum ß-lactamase (CTX-M-55), along with the fosfomycin resistance encoding gene, fosA. WGS indicated that these isolates belonged to four distinct sequence types (ST58, ST46, ST393, and a newly described ST14315) and to phylogroups B1, A, and D. In this geographic region, the spread of mcr-1 in E. coli is low and has not been inserted into high-risk clones such as ST131, which has been present in the country longer.

Update of antimicrobial resistance in level III and IV health institutions in Colombia between January 2018 and December 2021 / Actualización sobre la resistencia antimicrobiana en instituciones de salud de nivel III y IV en Colombia entre enero del 2018 y diciembre del 2021

Introduction: Antimicrobial resistance surveillance is a fundamental tool for the development, improvement, and adjustment of antimicrobial stewardship programs, therapeutic guidelines, and universal precautions to limit the cross-transmission of resistant bacteria between patients. Since the beginning of 2020, the SARS-CoV-2 pandemic profoundly challenged the health system and, according to some reports, increased the rates of antimicrobial resistance. Objective: To describe the behavior of antimicrobial resistance of the most frequent bacterial pathogens in twenty Colombian hospitals from January 2018 to December 2021. Materials and methods: We conducted a descriptive study based on the microbiological information recorded from January 2018 to December 2021 in twenty levels III and IV health institutions in twelve Colombian cities. We identified the species of the ten most frequent bacteria along with their resistance profile to the antibiotic markers after analyzing the data through WHONET. Results: We found no statistically significant changes in most pathogens' resistance profiles from January 2018 to December 2021. Only Pseudomonas aeruginosa had a statistically significant increase in its resistance profile, particularly to piperacillin/tazobactam and carbapenems. Conclusions: The changes in antimicrobial resistance in these four years were not statistically significant except for P. aeruginosa to piperacillin/tazobactam and carbapenems.

Introducción: El comportamiento de la resistencia antimicrobiana es fundamental en el mejoramiento y ajuste de los programas de optimización de uso de antimicrobianos, la implementación de las guías terapéuticas y las precauciones que limitan la transmisión cruzada de bacterias resistentes entre pacientes. Desde el inicio del 2020, la pandemia del SARS-CoV-2 desafió profundamente al sistema de salud y, según algunos reportes, aumentó las tasas de resistencia antimicrobiana. OBJETIVO: Describir el comportamiento de la resistencia antimicrobiana en los microrganismos más frecuentes en veinte hospitales colombianos durante el periodo 2018-2021. Materiales y métodos: Se trata de un estudio descriptivo basado en la información microbiológica reportada por veinte instituciones de salud de nivel III y IV, entre enero de 2018 y diciembre de 2021, en doce ciudades de Colombia, las cuales hacen parte del "Grupo para el estudio de la resistencia nosocomial en Colombia", liderado por la Universidad El Bosque. La identificación de género y especie de los microorganismos más frecuentes, junto con su perfil de resistencia frente a antibióticos marcadores, se determinaron mediante el análisis de los datos vía WHONET. RESULTADOS: En general, los 10 microorganismos más frecuentes analizados a lo largo de los 4 años no presentaron cambios estadísticamente significativos en sus perfiles de resistencia durante los cuatro años del periodo evaluado, de 2018 a 2021. En contraste, Pseudomonas aeruginosa aumentó su resistencia frente a piperacilina-tazobactam y carbapenémicos, lo cual fue estadísticamente significativo. CONCLUSIONES: Los cambios en la resistencia antimicrobiana en estos años no han sido estadísticamente significativos, excepto para P. aeruginosa, bacteria que mostró un incremento en las tasas de resistencia a piperacilina-tazobactam y carbapenémicos.

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.

Comparative In Vitro Activity of Ceftolozane/Tazobactam against Clinical Isolates of Pseudomonas aeruginosa and Enterobacterales from Five Latin American Countries.

BACKGROUND: Ceftolozane/tazobactam (C/T) is a combination of an antipseudomonal oxyiminoaminothiazolyl cephalosporin with potent in vitro activity against Pseudomonas aeruginosa and tazobactam, a known ß-lactamase inhibitor. The aim of this study was to evaluate the activity of C/T against clinical isolates of P. aeruginosa and Enterobacterales collected from five Latin American countries between 2016 and 2017, before its clinical use in Latin America, and to compare it with the activity of other available broad-spectrum antimicrobial agents. METHODS: a total of 2760 clinical isolates (508 P. aeruginosa and 2252 Enterobacterales) were consecutively collected from 20 hospitals and susceptibility to C/T and comparator agents was tested and interpreted following the current guidelines. RESULTS: according to the CLSI breakpoints, 68.1% (346/508) of P. aeruginosa and 83.9% (1889/2252) of Enterobacterales isolates were susceptible to C/T. Overall, C/T demonstrated higher in vitro activity than currently available cephalosporins, piperacillin/tazobactam and carbapenems when tested against P. aeruginosa, and its performance in vitro was comparable to fosfomycin. When tested against Enterobacterales, it showed higher activity than cephalosporins and piperacillin/tazobactam, and similar activity to ertapenem. CONCLUSIONS: these results show that C/T is an active ß-lactam agent against clinical isolates of P. aeruginosa and Enterobacterales.

Molecular mechanisms leading to ceftolozane/tazobactam resistance in clinical isolates of Pseudomonas aeruginosa from five Latin American countries.

Objectives: Identify molecular mechanisms responsible for the in vitro non-susceptibility to ceftolozane/tazobactam (TOL) in a group of 158 clinical isolates of Pseudomonas aeruginosa from five Latin American countries collected before the introduction of TOL into the clinical practice. Methods: Clinical isolates of P. aeruginosa (n = 504) were collected between January 2016 and October 2017 from 20 hospitals located in Argentina, Brazil, Chile, Colombia, and Mexico. Minimum inhibitory concentrations (MICs) to TOL were determined by standard broth microdilution and interpreted according to CLSI breakpoints. Initially, production of carbapenemases in TOL non-susceptible isolates was assessed by Rapidec® followed by qPCR to detect bla KPC, bla NDM-1, bla VIM, and bla IMP. Illumina® WGS was performed for isolates in which non-susceptibility to TOL was not mediated by carbapenemases. Results: A total of 158 (31.3%) isolates were non-susceptible to TOL. In 74 (46.8%) of these isolates, non-susceptibility to TOL was explained by the production of at least one carbapenemase. WGS revealed that some isolates carried ESBLs, mutated bla PDC and ampD, associated with decreased susceptibility to TOL. Conclusion: Substitutions found in PDC and carbapenemase production were the most common presumed mechanisms of resistance to TOL detected in this study. This study shows that epidemiological surveillance is warranted to monitor the emergence of novel mechanisms of resistance to TOL that might compromise its clinical utility.

Novel VIM metallo-beta-lactamase variant, VIM-24, from a Klebsiella pneumoniae isolate from Colombia.

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.

Microbiological and Clinical Aspects of Raoultella spp.

The genus Raoultella was established in 2001. Species of Raoultella and Klebsiella share many ecological, biochemical, clinical, and microbiological features. Given the shortcomings of available technology for species identification in the clinical microbiology laboratory, are practically indistinguishable. Since the late 2000s there has been an increase in case reports of human Raoultella infections. Therefore, several authors are postulating that Raoultella spp. are rare and/or emerging pathogens. Conclusions:Raoultella spp. are very similar to Klebsiella spp. The epidemiology and the clinical relevance of the human Raoultella spp. infections is uncertain and further studies are required. The previous difficulties in the identification of Raoultella spp. and the introduction of more precise identification techniques may explain the recent increase in the number of case reports. Raoultella spp. might be rather underdiagnosed than rare or emerging pathogens.

Genetic Diversity of Multidrug-Resistant Pseudomonas aeruginosa Isolates Carrying bla VIM-2 and bla KPC-2 Genes That Spread on Different Genetic Environment in Colombia.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with an increase in the frequency of infections caused by multidrug resistant (MDR) and extensively drug resistant (XDR) strains, limiting the available therapeutic options. The most troublesome resistance is the acquisition and production of carbapenemases such as Verona integron-encoded metallo-ß-lactamases (VIM), the most frequent and widespread, and the Klebsiella pneumoniae carbapenemases (KPC), which has continuously spread in the last decade. Its dissemination is linked to their location on mobile genetic elements (MGEs). In Colombia, VIM and KPC have been increasing in its frequency showing major successful dissemination. In this article, we molecularly characterized and analyzed the genetic context of bla VIM and bla KPC in carbapenem-resistant P. aeruginosa (CRPA) isolates from infected and colonized patients in two tertiary-care hospitals, one in Medellín and the other in a municipality close to Medellín, both areas with high carbapenemase endemicity in Colombia (2013-2015). Using whole-genome sequencing (WGS), we identified a remarkable variety of genetic backgrounds in these MDR P. aeruginosa isolates carrying bla KPC- 2 and bla VIM- 2. There were a diversity of class 1 integron and variations in the gene cassettes associated to bla VIM- 2, as well as a possible event of spread of bla KPC- 2 mediated by a plasmid that contained part of Tn4401b in one infection case. The dissemination of bla VIM- 2 and bla KPC- 2 in P. aeruginosa in this area in Colombia has been strongly influenced by successful international clones, carrying these genes and additional determinants of resistance on MGEs, accompanied by gene rearrangement under an antimicrobial selection pressure. These findings emphasize the need to implement control strategies based on rational antibiotic use.

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.

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.

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.

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.

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.