Genomic characterization of a WHO critical priority isolate Enterobacter kobei ST2070 harboring OXA-10, KPC-2, and CTX-M-12 recovered from a water irrigation channel in Ecuador.

The discharge of untreated or partially treated wastewater can have detrimental impacts on the quality of water bodies, posing a significant threat to public health and the environment. In Ecuador, previous research indicates a high prevalence of antimicrobial resistant (AMR) bacteria in surface waters affected by human activities, including irrigation channels. In this study, we analyzed sediment samples collected from an irrigation channel utilized for agricultural purposes in northern Ecuador, using microbiological techniques and whole-genome sequencing (WGS). Our investigation revealed the first documented occurrence of E. kobei in Ecuador and the initial report of environmental E. kobei ST2070. Furthermore, we identified the coexistence of OXA-10-type class D ß-lactamase and KPC-2-type class A ß-lactamase in the E. kobei isolate (UTA41), representing the first report of such a phenomenon in this species. Additionally, we detected various antibiotic resistance genes in the E. kobei UTA41 isolate, including blaCTX-M-12, fosA, aac(6')-lb, sul2, msr(E), and mph(A), as well as virulence genes such as bacterial efflux pump and siderophore biosynthesis genes. We also identified two intact prophage regions (Entero_186 and Klebsi_phiKO2) in the isolate. Our study presents the first evidence of E. kobei isolate containing two carbapenemase-encoding genes in environmental samples from Latin America. This finding indicates the potential spread of critical-priority bacteria in water samples originating from anthropogenic sources, such as urban wastewater discharges and livestock facilities.

Emergence of Raoultella ornithinolytica in human infections from different hospitals in Ecuador with OXA-48-producing resistance.

Purpose: The purpose of this study was to highlight the clinical and molecular features of 13 Raoultella ornithinolytica strains isolated from clinical environments in Ecuador, and to perform comparative genomics with previously published genomes of Raoultella spp. As Raoultella is primarily found in environmental, clinical settings, we focused our work on identifying mechanisms of resistance that can provide this bacterium an advantage to establish and persist in hospital environments. Methods: We analyzed 13 strains of Raoultella ornithinolytica isolated from patients with healthcare associated infections (HAI) in three hospitals in Quito and one in Santo Domingo de Los Tsáchilas, Ecuador, between November 2017 and April 2018. These isolates were subjected to phenotypic antimicrobial susceptibility testing, end-point polymerase chain reaction (PCR) to detect the presence of carbapenemases and whole-genome sequencing. Results: Polymerase chain reaction revealed that seven isolates were positive isolates for blaOXA-48 and one for blaKPC-2 gene. Of the seven strains that presented the blaOXA-48 gene, six harbored it on an IncFII plasmid, one was inserted into the bacterial chromosome. The blaKPC gene was detected in an IncM2/IncR plasmid. From the bioinformatics analysis, nine genomes had the gene blaOXA-48, originating from Ecuador. Moreover, all R. ornithinolytica strains contained the ORN-1 gene, which confers resistance for ß-lactams, such as penicillins and cephalosporins. Comparative genome analysis of the strains showed that the pangenome of R. ornithinolytica is considered an open pangenome, with 27.77% of core genes, which could be explained by the fact that the antibiotic resistance genes in the ancestral reconstruction are relatively new, suggesting that this genome is constantly incorporating new genes. Conclusion: These results reveal the genome plasticity of R. ornithinolytica, particularly in acquiring antibiotic-resistance genes. The genomic surveillance and infectious control of these uncommon species are important since they may contribute to the burden of antimicrobial resistance and human health.

Epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples, Ecuador, 2018.

Objective: To compare the epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples in Ecuador. Methods: A secondary analysis was done of data on bacteria isolated from inpatient and outpatient samples. Data were taken from the 2018 national antimicrobial resistance surveillance database of the National Reference Center for Antimicrobial Resistance. The variables included were: age, sex, inpatient versus outpatient setting, type of specimen, bacterial species identified, pattern of resistance to antibiotics, and geographic area. Results: Data from 57 305 bacterial isolates were included in the study: 48.8% were from hospitalized patients, 55.7% were from women, and 60.1% were from patients older than 45 years. Urine (42.9%) and blood (12.4%) were the most common clinical samples. Overall, 77.1% of bacterial isolates were gram-negative (83% and 71% in outpatients and inpatients, respectively). The most common gram-positive and gram-negative species were Staphylococcus aureus and Escherichia coli, respectively. Antimicrobial resistance levels were high (up to 80% for some antimicrobial drugs), and were higher in hospitalized patients compared with outpatients. A variety of carbapenemases were found to confer resistance to carbapenems (antibiotics of last resort) in gram-negative bacteria. Conclusions: The study findings provide an important baseline on antimicrobial resistance in Ecuador. This will allow the strengthening of guidelines of the surveillance system, the creation of public policies for standardization of laboratory methodologies, the proper handling of information, and the development of empirical therapy guidelines based on local epidemiology.

Epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples, Ecuador, 2018 / Características epidemiológicas de la resistencia antimicrobiana en cepas bacterianas aisladas en pacientes de servicios hospitalarios y ambulatorios de Ecuador, 2018 / Epidemiologia da resistência aos antimicrobianos em bactérias isoladas de amostras hospitalares e ambulatoriais no Equador, 2018

ABSTRACT Objective. To compare the epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples in Ecuador. Methods. A secondary analysis was done of data on bacteria isolated from inpatient and outpatient samples. Data were taken from the 2018 national antimicrobial resistance surveillance database of the National Reference Center for Antimicrobial Resistance. The variables included were: age, sex, inpatient versus outpatient setting, type of specimen, bacterial species identified, pattern of resistance to antibiotics, and geographic area. Results. Data from 57 305 bacterial isolates were included in the study: 48.8% were from hospitalized patients, 55.7% were from women, and 60.1% were from patients older than 45 years. Urine (42.9%) and blood (12.4%) were the most common clinical samples. Overall, 77.1% of bacterial isolates were gram-negative (83% and 71% in outpatients and inpatients, respectively). The most common gram-positive and gram-negative species were Staphylococcus aureus and Escherichia coli, respectively. Antimicrobial resistance levels were high (up to 80% for some antimicrobial drugs), and were higher in hospitalized patients compared with outpatients. A variety of carbapenemases were found to confer resistance to carbapenems (antibiotics of last resort) in gram-negative bacteria. Conclusions. The study findings provide an important baseline on antimicrobial resistance in Ecuador. This will allow the strengthening of guidelines of the surveillance system, the creation of public policies for standardization of laboratory methodologies, the proper handling of information, and the development of empirical therapy guidelines based on local epidemiology.

RESUMEN Objetivo. Comparar las características epidemiológicas de la resistencia a los antimicrobianos en cepas bacterianas aisladas de muestras de pacientes de servicios hospitalarios y ambulatorios en Ecuador. Métodos. Se realizó un análisis secundario de los datos sobre cepas bacterianas aisladas en muestras de pacientes de servicios hospitalarios y ambulatorios. Se recogieron los datos de la base de datos nacional del 2018 para la vigilancia de la resistencia a los antimicrobianos del Centro de Referencia Nacional para la Resistencia a los Antimicrobianos. Las variables incluidas fueron: edad, sexo, entorno hospitalario frente a entorno ambulatorio, tipo de muestra, especies bacterianas detectadas, patrón de resistencia a los antibióticos y zona geográfica. Resultados. En el estudio se incluyeron datos de 57 305 cepas aislamientos bacterianos: 48,8% fueron de pacientes hospitalizados, 55,7% fueron de mujeres y 60,1% fueron de pacientes mayores de 45 años. La orina (42,9%) y la sangre (12,4%) fueron las muestras clínicas más comunes. En general, 77,1% de las cepas bacterianas aisladas fueron gramnegativas (83% y 71% en pacientes de servicios ambulatorios y hospitalarios, respectivamente). Las especies grampositivas y gramnegativas más comunes fueron Staphylococcus aureus y Escherichia coli, respectivamente. Los niveles de resistencia a los antimicrobianos fueron elevados (hasta 80% en el caso de algunos fármacos antimicrobianos) y fueron más elevados en los pacientes de servicios hospitalarios en comparación con los pacientes de servicios ambulatorios. Se encontró que una variedad de carbapenemasas confiere resistencia a los carbapenémicos (antibióticos de último recurso) en bacterias gramnegativas. Conclusiones. Los resultados del estudio proporcionan una línea de base importante sobre la resistencia a los antimicrobianos en Ecuador, que permitirá el fortalecimiento de las directrices del sistema de vigilancia, la creación de políticas públicas para la estandarización de los métodos de laboratorio, una adecuada gestión de la información y la elaboración de orientaciones de tratamiento empírico basadas en las características epidemiológicas locales.

RESUMO Objetivo. Comparar a epidemiologia da resistência aos antimicrobianos em bactérias isoladas de amostras hospitalares e ambulatoriais no Equador. Métodos. Foi feita uma análise secundária de dados sobre bactérias isoladas de amostras hospitalares e ambulatoriais. Os dados foram obtidos do banco de dados nacional de vigilância da resistência aos antimicrobianos de 2018 do Centro Nacional de Referência para a Resistência aos Antimicrobianos. As variáveis incluídas foram: idade, sexo, ambiente hospitalar versus ambiente ambulatorial, tipo de espécime, espécies bacterianas identificadas, padrão de resistência a antibióticos e área geográfica. Resultados. Foram incluídos no estudo os dados de 57 305 isolados bacterianos: 48,8% eram de pacientes hospitalizados, 55,7% eram de mulheres e 60,1% eram de pacientes com mais de 45 anos. As amostras clínicas mais comuns foram urina (42,9%) e sangue (12,4%). No total, 77,1% dos isolados bacterianos eram gram-negativos (83% e 71% em pacientes ambulatoriais e pacientes internados, respectivamente). As espécies gram-positivas e gram-negativas mais comuns foram Staphylococcus aureus e Escherichia coli, respectivamente. Os níveis de resistência aos antimicrobianos foram elevados (até 80% para alguns antimicrobianos) e foram mais elevados em pacientes hospitalizados em comparação com pacientes ambulatoriais. Foram encontradas várias carbapenemases que conferem resistência aos carbapenêmicos (antibióticos de último recurso) em bactérias gram-negativas. Conclusões. Os resultados do estudo fornecem uma importante linha de base sobre a resistência aos antimicrobianos no Equador. Isto permitirá o fortalecimento das diretrizes do sistema de vigilância, a criação de políticas públicas para padronização de metodologias laboratoriais, o manejo adequado de informações e o desenvolvimento de diretrizes para a antibioticoterapia empírica com base na epidemiologia local.

Increased Detection of Carbapenemase-Producing Enterobacterales Bacteria in Latin America and the Caribbean during the COVID-19 Pandemic.

During 2020-2021, countries in Latin America and the Caribbean reported clinical emergence of carbapenemase-producing Enterobacterales that had not been previously characterized locally, increased prevalence of carbapenemases that had previously been detected, and co-production of multiple carbapenemases in some isolates. These increases were likely fueled by changes related to the COVID-19 pandemic, including empirical antibiotic use for potential COVID-19-related bacterial infections and healthcare limitations resulting from the rapid rise in COVID-19 cases. Strengthening antimicrobial resistance surveillance, epidemiologic research, and infection prevention and control programs and antimicrobial stewardship in clinical settings can help prevent emergence and transmission of carbapenemase-producing Enterobacterales.

Characterization of blaKPC-2-Harboring Klebsiella pneumoniae Isolates and Mobile Genetic Elements from Outbreaks in a Hospital in Ecuador.

Aim: To investigate the mobile genetic elements harboring blaKPC gene in carbapenem-resistant Klebsiella pneumoniae recovered during a 6-month outbreak in a high-complexity hospital from Ecuador. Results: A total of 62 isolates belonging to ST258 pilv-I-positive (n = 45), ST25 serotype K2 (n = 8), ST348 (n = 6), ST42 (n = 1), ST196 (n = 1), and ST1758 (n = 1) were collected from intensive care unit (ICU), neurosurgery, burn unit, internal medicine, pneumology, and neurology. Pulsed-field gel electrophoresis analysis showed two major clusters of ST258 and ST25 related to bloodstream infections and pneumonia circulating in ICU. The PCR assay showed that in non-ST258 isolates, the blaKPC-2 gene were located on the Tn4401a transposon inserted in the transferable pKpQIL-like IncFIIK2 plasmid; the whole-genome sequencing of ST258 clone showed two plasmids, the blaKPC-2 gene was located on nonconjugative IncR plasmid, whereas the IncFIB/IncFII plasmid lacked ß-lactamase genes. We found an IncM plasmid in blaKPC-2-harboring Klebsiella pneumoniae ST1758 clone. Conclusions: These findings highlight the presence of pKpQIL-like plasmids in non-ST258 and nonconjugative plasmids in ST258 isolates causing hospital outbreaks.

Broiler Farms and Carcasses Are an Important Reservoir of Multi-Drug Resistant Escherichia coli in Ecuador.

Antimicrobial resistance (AMR) is a major health threat for public and animal health in the twenty-first century. In Ecuador, antibiotics have been used by the poultry industry for decades resulting in the presence of multi-drug resistant (MDR) bacteria in the poultry meat production chain, with the consequent risk for public health. This study evaluated the prevalence of ESBL/AmpC and mcr genes in third-generation cephalosporin-resistant Escherichia coli (3GC-R E. coli) isolated from broiler farms (animal component), broiler carcasses (food component), and human enteritis (human component) in Quito-Ecuador. Samples were collected weekly from November 2017 to November 2018. For the animal, food, and human components, 133, 335, and 302 samples were analyzed, respectively. Profiles of antimicrobial resistance were analyzed by an automated microdilution system. Resistance genes were studied by PCR and Sanger sequencing. From all samples, 122 (91.7%), 258 (77%), and 146 (48.3%) samples were positive for 3GC-R E. coli in the animal, food, and human components, respectively. Most of the isolates (472/526, 89.7%) presented MDR phenotypes. The ESBL bla CTX-M-55, bla CTX-M-3, bla CTX-M-15, bla CTX-M-65, bla CTX-M-27, and bla CTX-M-14 were the most prevalent ESBL genes while bla CMY-2 was the only AmpC detected gene. The mcr-1 gene was found in 20 (16.4%), 26 (10.1%), and 3 (2.1%) of isolates from animal, food, and human components, respectively. The implication of poultry products in the prevalence of ESBL/AmpC and mcr genes in 3GC-R must be considered in the surveillance of antimicrobial resistance.

Genomic Epidemiology of Salmonella Infantis in Ecuador: From Poultry Farms to Human Infections.

Salmonella enterica is one of the most important foodborne pathogens around the world. In the last years, S. enterica serovar Infantis has become an important emerging pathogen in many countries, often as multidrug resistant clones. To understand the importance of S. enterica in the broiler industry in Ecuador, we performed a study based on phenotypic and WGS data of isolates from poultry farms, chicken carcasses and humans. We showed a high prevalence of S. enterica in poultry farms (41.4%) and chicken carcasses (55.5%), but a low prevalence (1.98%) in human samples. S. Infantis was shown to be the most prevalent serovar with a 98.2, 97.8, and 50% in farms, foods, and humans, respectively, presenting multidrug resistant patterns. All sequenced S. Infantis isolates belonged to ST32. For the first time, a pESI-related megaplasmid was identified in Ecuadorian samples. This plasmid contains genes of antimicrobial resistance, virulence factors, and environmental stress tolerance. Genomic analysis showed a low divergence of S. Infantis strains in the three analyzed components. The results from this study provide important information about genetic elements that may help understand the molecular epidemiology of S. Infantis in Ecuador.

Fosfomycin, Applying Known Methods and Remedies to A New Era.

The exponential increase in the numbers of isolates of Carbapenem-Resistant Enterobacteriaceae (CRE) creates the need for using novel therapeutic approaches to save the lives of patients. Fosfomycin has long been considered a rational option for the treatment of CRE to be used as part of a combined therapy scheme. However, the assessment of fosfomycin susceptibility in the laboratory presents a great challenge due to the discrepancies found between different methodologies. Thus, our goal was to evaluate fosfomycin susceptibility in a group of 150 Enterobacteriaceae bacterial isolates using agar dilution as the gold standard technique to compare the results with those obtained by disk diffusion. We found a fosfomycin susceptibility of 79.3% in general terms. By comparing both methodologies, we reported a categorical agreement of 96% without Very Major Errors (VMEs) or Major Errors (MEs) and 4% of minor Errors (mEs). Our results suggest that fosfomycin could provide a rational alternative treatment for those patients that are infected by a Multidrug-Resistant (MDR) microorganism that is currently untreatable and that the disk diffusion and classical agar dilution techniques are adequate to assess the resistance profile of CRE to fosfomycin.

Diverse Escherichia coli lineages from domestic animals carrying colistin resistance gene mcr-1 in an Ecuadorian household.

OBJECTIVE: The aim of this study was to detect potential animal reservoirs of Escherichia coli carrying the mcr-1 gene in an Ecuadorian household. METHODS: The mobile colistin-resistance gene, mcr-1, was first detected in Ecuador in a commensal E. coli isolate from a boy. A cross-sectional study was performed to detect the possible source of colistin-resistant E. coli in the boy's household. Faecal swabs and soil faecal samples were collected from companion animals. Samples were plated on selective media to isolate colistin-resistant E. coli and isolates were submitted to PCR detection of mcr-1, pulsed field gel electrophoresis (PFGE), and multi-locus sequences typing (MLST). Moreover, the genomes of all the isolates were sequenced. RESULTS: Three different colistin-resistant E. coli sequence types (ST3941, 1630 and 2170), corresponding to three PFGE patterns, were obtained from a chicken and two dogs; these isolates were different from the human isolate (ST609). By whole-genome sequencing, the mcr-1.1 gene was found on IncI2 plasmids with very high nucleotide identity. CONCLUSIONS: Our results indicate a polyclonal dissemination of mcr-1.1 in the environment surrounding the first MCR-producing E. coli strain reported in Ecuador. Our findings support the idea of lateral dissemination of mcr-1.1 gene between unrelated E. coli isolates.

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 µm of diameter and 3 µm thick with more than 10 bacteria in each cut.•Lysis with 30 µL of tissue lysis buffer and 20 µL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 µL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/µL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.