Phytobacter spp: the emergence of a new genus of healthcare-associated Enterobacterales encoding carbapenemases in Argentina: a case series.

Members of the genus Phytobacter (order Enterobacterales) are isolated from the natural environment and clinical settings. Identification of Phytobacter strains based on biochemical characteristics is complicated due to taxonomic confusion, and they are often misidentified by automated identification systems in laboratories. In this study we describe the first three clinical cases associated with Phytobacter spp. reported in Argentina. We describe the identification, the molecular analysis using whole genome sequencing and the potential clinical relevance.

Emergence of monkeypox virus in central Argentina: Epidemiological features and first complete genome sequences in the country.

Monkeypox (Mpox) is a zoonotic disease caused by the monkeypox virus (MPXV). MPXV can be transmitted by close contact with lesions, body fluids, respiratory droplets, and contaminated materials. A new pattern of spread among sexual networks has been recently described. The present work aimed to report the epidemiological and genomic characterization of the 2022 MPXV outbreak in central Argentina. A total of 113 scabs and/or lesion swab specimens were studied. MPXV infection was confirmed in 46.0% of the studied patients, all of whom were men. Varicella-zoster virus infection was the most frequent differential diagnosis. Eight complete viral genomes were obtained by next-generation sequencing. The Argentinian sequences were grouped intermingled with other sequences from the 2022 MPXV outbreak, related to samples from the USA, Europe, and Peru. Taken together, our study provided an initial assessment of the genetic and epidemiological characteristics of the 2022 MPXV outbreak in Córdoba, Argentina.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

[Molecular characterization of Listeria monocytogenes isolates from human and food sources in Argentina, 2018-2023]. / Caracterización molecular de aislamientos de Listeria monocytogenes de origen humano y alimentario en Argentina, 2018-2023.

Human listeriosis is an infectious disease caused by Listeria monocytogenes. The invasive form of this disease leads to a high rate of hospitalizations and fatality. The main mode of transmission is through contaminated ready-to-eat foods such as dairy, vegetables and meat products. The knowledge of the diversity and population dynamics of isolates collected from human and food sources is essential for the detection of clusters and the identification of common sites of infection. The aim of this study was the molecular characterization of L. monocytogenes isolates in Argentina. We sequenced a total of 63 isolates, 35 from human and 28 from food sources, collected between 2018 and 2023. Our genomic study divided the isolates into two lineages, four serogroups, 17 sequence types and 15 clonal complexes (CCs). The hypervirulent clone CC1 (lineage I; serogroup IVb) predominated in human and food samples. The phylogenomic analysis showed a high and possible epidemiological relationship between isolates from human and/or food sources, suggesting the presence of transmission chains in our country. These findings highlight the need to strengthen genomic surveillance of L. monocytogenes in Argentina. The identification of geographic distribution and characteristics of predominant and emerging clones from human and food sources might help to focus action plans and public health policies better directed at the control and prevention of listeriosis.

Genetic and Phenotypic Changes Related to the Development of mec-Independent Oxacillin Non-Susceptibility in ST8 Staphylococcus aureus Recovered after Antibiotic Therapy in a Patient with Bacteremia.

The mec-independent oxacillin non-susceptible S. aureus (MIONSA) strains represent a great clinical challenge, as they are not easily detected and can lead to treatment failure. However, the responsible molecular mechanisms are still very little understood. Here, we studied four clinical ST8-MSSA-t024 isolates recovered during the course of antibiotic treatment from a patient suffering successive episodes of bacteremia. The first isolates (SAMS1, SAMS2, and SAMS3) were susceptible to cefoxitin and oxacillin. The last one (SA2) was susceptible to cefoxitin, resistant to oxacillin, lacked mec genes, and had reduced susceptibility to teicoplanin. SA2 showed higher ß-lactamase activity than SAMS1. However, ß-lactamase hyperproduction could not be linked to oxacillin resistance as it was not inhibited by clavulanic acid, and no genetic changes that could account for its hyperproduction were found. Importantly, we hereby report the in vivo acquisition and coexistence of different adaptive mutations in genes associated with peptidoglycan synthesis (pbp2, rodA, stp1, yjbH, and yvqF/vraT), which is possibly related with the development of oxacillin resistance and reduced susceptibility to teicoplanin in SA2. Using three-dimensional models and PBP binding assays, we demonstrated the high contribution of the SA2 PBP2 Ala450Asp mutation to the observed oxacillin resistance phenotype. Our results should be considered as a warning for physicians and microbiologists in the region, as MIONSA detection and treatment represent an important clinical challenge.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Congruity of genomic and epidemiological data in modelling of local cholera outbreaks.

Cholera continues to be a global health threat. Understanding how cholera spreads between locations is fundamental to the rational, evidence-based design of intervention and control efforts. Traditionally, cholera transmission models have used cholera case-count data. More recently, whole-genome sequence data have qualitatively described cholera transmission. Integrating these data streams may provide much more accurate models of cholera spread; however, no systematic analyses have been performed so far to compare traditional case-count models to the phylodynamic models from genomic data for cholera transmission. Here, we use high-fidelity case-count and whole-genome sequencing data from the 1991 to 1998 cholera epidemic in Argentina to directly compare the epidemiological model parameters estimated from these two data sources. We find that phylodynamic methods applied to cholera genomics data provide comparable estimates that are in line with established methods. Our methodology represents a critical step in building a framework for integrating case-count and genomic data sources for cholera epidemiology and other bacterial pathogens.

Genomic Characterization of Two NDM-5-Producing Isolates of Klebsiella pneumoniae ST11 from a Single Patient.

Two metallo-ß-lactamase-producing Klebsiella pneumoniae (HA30 and HA31) were isolated in a hospital in Argentina during 2018. K. pneumoniae HA30 was isolated from a rectal swab during the epidemiological surveillance for carbapenemase-producing strains, while K. pneumoniae HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing K. pneumoniae strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to E. coli J53 was conducted as previously described. K. pneumoniae HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with blaNDM-5, blaCTX-M-15 and rmtB genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that K. pneumoniae HA30 and HA31 were the same strain. Conjugation assays revealed that K. pneumoniae HA31 strain possessed a transferable plasmid to E. coli J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of blaNDM-5, rmtB and blaCTX-M-15 genes in a K. pneumoniae ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.

Bactericidal killing of meningococcal W strains isolated in Argentina by the sera of adolescents and infants immunized with 4-component meningococcal serogroup B vaccine (4CMenB).

Invasive meningococcal disease (IMD) is a life-threatening disease caused by meningococcal serogroups A, B, C, W, X, and Y, of which B and W are most common in Argentina. The 4-component meningococcal serogroup B (4CMenB) vaccine contains three purified recombinant protein antigens (Neisseria adhesin A [NadA], factor H binding protein [fHbp], and Neisserial Heparin Binding Antigen [NHBA]) and outer membrane vesicles (OMV), which is derived from the New Zealand epidemic strain and contains Porin A 1.4. These antigens are present and conserved in strains that belong to other serogroups. In this study, we show that 10/11 (91%) meningococcal serogroup W (MenW) strains selected to be representative of MenW isolates that caused IMD in Argentina during 2010-2011 were killed in bactericidal assays by the sera of adolescents and infants who had been immunized with the 4CMenB vaccine. We also show that MenW strains that caused IMD in Argentina during 2018-2021 were genetically similar to the earlier strains, indicating that the 4CMenB vaccine would likely still provide protection against current MenW strains. These data highlight the potential of 4CMenB vaccination to protect adolescents and infants against MenW strains that are endemic in Argentina.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

Diagnóstico de un brote de Salmonella Typhimurium en Chinchillas (Chinchilla lanigera) mediante la electroforesis en gel de campo pulsado / Diagnosis of an autbreak of Salmonella Typhimurium in chinchillas (Chinchilla lanigera) by pulsed-fiel gel electrophoresis

Empleando estudios anatomopatológicos y microbiológicos se examinó a un grupo de chinchillas (Chinchilla lanigera) adultas que murieron súbitamente en 2012 en una granja de la ciudad de La Plata (Buenos Aires, Argentina). Se aisló Salmonella enterica serovar Typhimurium (S. Typhimurium) del hígado, el bazo, el corazón, los pulmones, los riñones y los intestinos de los cinco animales evaluados. Los cinco aislamientos estudiados (uno por animal) fueron sensibles a ampicilina, cefalotina, cefotaxima, ácido nalidíxico, gentamicina, estreptomicina, cloranfenicol, fosfomicina, nitrofurantoína y trimetoprima-sulfametoxazol, y resistentes a tetraciclina. El análisis de dichos aislamientos por electroforesis en gel de campo pulsado [pulsed-field gel electrophoresis (PFGE)] con XbaI mostró un perfil electroforético idéntico con 15 bandas, idéntico a su vez al patrón ARJPXX01.0220 del banco nacional argentino de datos de PulseNet, que cuenta con patrones de PFGE de Salmonella. El presente trabajo describe por primera vez el diagnóstico postmortem de un brote de salmonelosis en chinchillas usando un método molecular, como la electroforesis en gel en campo pulsado

Adult chinchillas (Chinchilla lanigera) that had suddenly died in a commercial farm located in La Plata City, Buenos Aires Province, Argentina, in July 2012 were macroscopically, histopathologically, and microbiologically examined. Salmonella enterica serovar Typhimurium (S. Typhimurium) was isolated from the liver, spleen, heart, lungs, kidneys and intestines from each of the five animals evaluated. The five strains were susceptible to ampicillin, cephalotin, cefotaxime, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, nitrofurantoin and trimethoprimsulfamethoxazole, and resistant to tetracycline. Each of the five S. Typhimurium isolates was analyzed by XbaI- pulsed-field gel electrophoresis (PFGE), showing an identical electrophoretic profile with 15 defined bands, which was found to be identical to pattern ARJPXX01.0220 of the PulseNet Argentine National database of Salmonella PFGE patterns. This is the first work describing the postmortem diagnosis of an outbreak of salmonellosis in chinchillas by using molecular methods such as PFGE

Diagnosis of an outbreak of Salmonella typhimurium in chinchillas (Chinchilla lanigera) by pulsed-field gel electrophoresis. / Diagnosis of an outbreak of Salmonella typhimurium in chinchillas (Chinchilla lanigera) by pulsed-field gel electrophoresis.

Adult chinchillas (Chinchilla lanigera) that had suddenly died in a commercial farm located in La Plata City, Buenos Aires Province, Argentina, in July 2012 were macroscopically, histopathologically, and microbiologically examined. Salmonella enterica serovar Typhimurium (S. Typhimurium) was isolated from the liver, spleen, heart, lungs, kidneys and intestines from each of the five animals evaluated. The five strains were susceptible to ampicillin, cephalotin, cefotaxime, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, nitrofurantoin and trimethoprim-sulfamethoxazole, and resistant to tetracycline. Each of the five S. Typhimurium isolates was analyzed by XbaI- pulsed-field gel electrophoresis (PFGE), showing an identical electrophoretic profile with 15 defined bands, which was found to be identical to pattern ARJPXX01.0220 of the PulseNet Argentine National database of Salmonella PFGE patterns. This is the first work describing the postmortem diagnosis of an outbreak of salmonellosis in chinchillas by using molecular methods such as PFGE.

Experiencias en la vigilancia epidemiológica de agentes patógenos transmitidos por alimentos a través de electroforesis en campo pulsado (PFGE) en el Perú / Experiences in the epidemiological surveillance of foodborne pathogens by pulsed field gel electrophoresis (PFGE) in Peru

Las enfermedades transmitidas por alimentos (ETA) y otras enfermedades entéricas infecciosas ocurren a menudo como brotes y son causa de morbilidad y mortalidad en todo el mundo. En el Perú, son un importante problema de salud pública y son causados por una gran variedad de agentes infecciosos. Para la investigación epidemiológica se utiliza una variedad de métodos de tipificación. Una de las herramientas más importantes en la subtipificación molecular de patógenos bacterianos es la técnica de la electroforesis en campo pulsado (PFGE), que es un método altamente resolutivo que permite la discriminación entre diferentes aislamientos bacterianos epidemiológicamente relacionados. El Instituto Nacional de Salud (INS) del Perú integra las redes WHO Global Foodborne Infections Network y la Red PulseNet América Latina y Caribe, con quienes comparte los perfiles genéticos de las cepas patógenas aisladas, permitiendo comparar los genotipos de cepas semejantes halladas en diferentes países y reconocer la ocurrencia de brotes epidémicos en la región, fortaleciendo el sistema de vigilancia epidemiológica regional y generando una rápida respuesta conjunta entre países. Se presenta la experiencia de los dos últimos años sobre los avances en la utilización de estas herramientas estratégicas que nos ha permitido caracterizar patrones de genotipo de principales patógenos implicados en ETA a partir de aislamientos recuperados de la red de laboratorios del Perú.

Foodborne diseases and other enteric infections often occur as outbreaks and cause morbidity and mortality all over the world. In Perú, they represent a serious public health problem, and are caused by a great variety of infectious agents. For epidemiological research, a wide array of typification methods are used. One of the most important tools for the molecular subtyping of bacterial pathogens is the Pulsed Field Gel Electrophoresis (PFGE), which is a highly precise method that allows the discrimination between different bacterial isolates which are epidemiologically related. The Instituto Nacional de Salud del Perú (INS) is part of the WHO Global Foodborne Infections Network (WHO-GFN) and of the PulseNet Latin American and Caribbean Net (PN-AL & C), with whom it shares the genetic profiles of the isolated pathogenic strains, so that it is possible to compare de genotypes of similar strains found in different countries and to identify the occurrence of epidemic outbreaks in the region, strengthening the regional system of epidemiological surveillance and generating a rapid, coordinated response between the countries. We present the two last years´ experience including the advances in the use of these strategic tools that have allowed us to characterize genotype patterns implicated in foodborne diseases from isolates recovered in the laboratory network of Peru.