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.

Isolation of Hafnia paralvei co-harbouring blaNDM-1 and blaVIM-1 in a woman who underwent allogeneic hematopoietic stem cell transplantation.

Metallo-ß-lactamases (MBL) are a threat to public health, since they dramatically limit the use of ß-lactams. We report the isolation of a multidrug-resistant Hafnia paralvei strain from urine and a rectal swab of a female patient after allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome. Antimicrobial susceptibility testing yielded resistance to trimethoprim/sulfamethoxazole, colistin, fosfomycin and all ß-lactams, except cefiderocol. Whole genome sequencing revealed the presence of plasmid-encoded NDM-1 and VIM-1 carbapenemases. This finding highlights the importance of epidemiological surveillance and new therapeutic options for MBL.

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.

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.

Update on the epidemiology of carbapenemases in Latin America and the Caribbean.

INTRODUCTION: Carbapenemases are ß-lactamases able to hydrolyze a wide range of ß-lactam antibiotics, including carbapenems. Carbapenemase production in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp., with and without the co-expression of other ß-lactamases is a serious public health threat. Carbapenemases belong to three main classes according to the Ambler classification: class A, class B, and class D. AREAS COVERED: Carbapenemase-bearing pathogens are endemic in Latin America. In this review, we update the status of carbapenemases in Latin America and the Caribbean. EXPERT OPINION: Understanding the current epidemiology of carbapenemases in Latin America and the Caribbean is of critical importance to improve infection control policies limiting the dissemination of multi-drug-resistant pathogens and in implementing appropriate antimicrobial therapy.

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.

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.

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.

Carbapenemase-producing Enterobacteriaceae: A diagnostic, epidemiological and therapeutic challenge / Enterobacteriaceae productoras de carbapenemasas: Un reto diagnóstico, epidemiológico y terapéutico

The global spread of carbapenemase-producing Enterobacteriaceae (CPE) has become a public health problem. Not all CPE are resistant to carbapenems creating a diagnostic and therapeutic challenge. Furthermore, as resistance to carbapenems can also be mediated by other β-lactamases combined with defects in membrane permeability, their detection can be difficult by microbiology laboratories that lack molecular tools, which may limit and often delay the correct antibiotic selection. There is only limited evidence regarding infection control measures to contain the spread of CPE. However, recomendations have been published from the World Health Organization (WHO) and the European Prevention Center and Disease Control (ECDC). Because of the lack of randomized control trials, treatment regimens are mostly based on observational clinical studies. Several of those studies have reported that combination therapy with two or more in vitro-active agents including a carbapenem is superior to monotherapy. On the other hand, a new β-lactamase inhibitor in combination with ceftazidime has shown clinical efficacy Against KPC and some OXA-type producing Enterobacteriaceae

La diseminación global de las Enterobacteriaceae productoras de carbapenemasas (EPC) se ha convertido en un problema de salud pública. No todas las EPC son resistentes a los carbapenémicos, por lo que representan un reto diagnóstico y terapéutico. Adicionalmente, como la resistencia a los carbapenémicos puede ser mediada por otras β-lactamasas en combinación con cambios de la permeabilidad de la membrana plasmática, su detección puede ser difícil en laboratorios de microbiología que no cuentan con técnicas de diagnóstico molecular, lo que puede restringir y frecuentemente retrasar el inicio de la terapia antimicrobiana adecuada. La evidencia respecto a las medidas para la contención de las EPC es escasa. Sin embargo, existen recomendaciones por parte de la Organización Mundial de la Salud y del European Prevention Center and Disease Control (ECDC). Debido a la ausencia de estudios controlados y aleatorizados, los esquemas terapéuticos se basan en estudios clínicos observacionales. Varios de estos estudios han reportado mejores resultados con la terapia combinada de dos o más agentes activos in vitro, incluyendo a los carbapenémicos, en comparación con la monoterapia. Por otra parte, un nuevo inhibidor de β-lactamasas en combinación con ceftazidime, ha mostrado eficacia clínica contra infecciones por Enterobacteriaceae productoras de KPC y algunas carbapenemasas de tipo OXA.

Infección por Listeria monocytogenes en una mujer gestante con lupus eritematoso sistémico y síndrome HELLP / Listeria monocytogenes infection in a pregnant woman with systemic lupus erythematosus and HELLP syndrome / Infeção por Listeria monocytogenes em uma mulher gestante com antecedente de lúpus eritromatoso sistemático e síndrome HELLP

La listeriosis es una enfermedad transmitida principalmente por alimentos contaminados con Listeria monocytogenes. Se presenta con mayor frecuencia en neonatos, mayores de 65 años, mujeres gestantes y pacientes inmunosuprimidos. La infección por L. monocytogenes durante la gestación se asocia a una importante morbimortalidad materno-fetal.Se reporta el caso de una mujer gestante de 29 años de edad con lupus eritematoso sistémico, a quien se le diagnosticó bacteriemia por L. monocytogenes. Durante la hospitalización, el cuadro clínico se complicó con hipertransaminasemia y, ante la presencia de trombocitopenia, se estableció el diagnóstico presuntivo de síndrome HELLP. El alto riesgo de morbimortalidad llevó a una finalización precoz de la gestación.La importancia de este trabajo clínico radica en presentar la dificultad en el diagnóstico y manejo en una paciente gestante de gran complejidad con una infección relativamente frecuente que puede pasar desapercibida.

Listeriosis is a disease mainly transmitted by food contaminated with bacteria called Listeria monocytogenes. It occurs more often in newborns, elder population, pregnant women and immunosuppressed patients. L. monocytogenes infection during pregnancy is associated to significant maternal mortality and morbidity. The case of a 29-year-old pregnant woman with history of Systemic Lupus Erythematosus is review. The said woman was diagnosed with bacteremia related to L. monocytogenes. During hospitalization, the patient experienced complications with hipertransaminasemia, which led to the presumptive diagnosis of HELLP in presence of thrombocytopenia. Given the high risk of mortality and morbidity, the pregnancy was terminated. The importance of the present clinical work lays in showing the difficulties embedded in diagnosing and handling a high-complexity pregnant patient presenting a frequent infection that would otherwise go undetected.

A listeriosis é uma doença transmitida principalmente por alimentos contaminados com Listeria monocytogenes. Apresentase com maior frequência em neonatos, maiores de 65 anos, mulheres gestantes e pacientes imunossuprimidos. Durante a gestação esta infeção associa-se a uma importante morbimortalidade materno-fetal. Foi reportado o caso de uma mulher gestante de 29 anos com antecedente de lúpus eritematoso sistémico, diagnosticada com bacteriemia por L. monocytogenes, na hospitalização teve complicações com hipertransaminasemia, foi estabelecido um diagnostico presuntivo de síndrome HELLP em presença de trombocitopenia. O alto risco de morbimortalidade levou a uma finalização precoce da gestação. A importância deste trabalho clínico radica em apresentar a dificuldade de diagnóstico e tratamento em pacientes gestantes de alta complexidade com infeção relativamente frequente, que pode passar desapercebida