Molecular Epidemiology and Antifungal Susceptibility Profile of Candidozyma Isolates From Argentina.

BACKGROUND: Epidemiological surveillance of Candidozyma sp. has become important because many species of this new genus have been reported to be responsible for nosocomial outbreaks and to exhibit elevated minimal inhibitory concentrations (MIC) to one or more classes of antifungal drugs. OBJECTIVES: To describe the genetic relationships among Argentinian clinical isolates belonging to the Candidozyma genus and to study the molecular mechanisms associated with antifungal resistance. METHODS: We performed whole-genome sequencing of 41 isolates. Identification was based on ribosomal DNA sequencing and susceptibility testing was determined according to the EUCAST document. Phylogenetic analysis, non-synonymous mutations in genes associated with antifungal resistance and the presence of copy number variations (CNVs) were investigated. RESULTS: We identified 12 Candidozyma haemuli, 11 Candidozyma haemuli var. vulneris, 5 Cz. haemuli/ Cz. haemuli var. vulneris ITS hybrids, 8 Candidozyma duobushaemuli and 5 Candidozyma cf. pseudohaemuli. Phylogenetic analysis, together with clinical data, demonstrated nosocomial transmission events. In addition, Cz. haemuli and Cz. haemuli var. vulneris were not separated in the phylogenetic tree; the Cz. cf. pseudohaemuli isolates clustered distantly from the Cz. pseudohaemuli type strain. Most isolates were resistant to amphotericin B, and two Cz. haemuli isolates showed fluconazole resistance and Y132F mutation in ERG11. We did not find CNV in genes associated with antifungal resistance. CONCLUSIONS: These findings highlight the need for epidemiological surveillance of these species and the study of molecular mechanisms associated with antifungal resistance. Furthermore, we propose a taxonomic revision for Cz. haemuli var. vulneris and Cz. pseudohaemuli based on genomic data.

Hypermutability bypasses genetic constraints in SCV phenotypic switching in Pseudomonas aeruginosa biofilms.

Biofilms are critical in the persistence of Pseudomonas aeruginosa infections, particularly in cystic fibrosis patients. This study explores the adaptive mechanisms behind the phenotypic switching between Small Colony Variants (SCVs) and revertant states in P. aeruginosa biofilms, emphasizing hypermutability due to Mismatch Repair System (MRS) deficiencies. Through experimental evolution and whole-genome sequencing, we show that both wild-type and mutator strains undergo parallel evolution by accumulating compensatory mutations in factors regulating intracellular c-di-GMP levels, particularly in the Wsp and Yfi systems. While wild-type strains face genetic constraints, mutator strains bypass these by accessing alternative genetic pathways regulating c-di-GMP and biofilm formation. This increased genetic accessibility, driven by higher mutation rates and specific mutational biases, supports sustained cycles of SCV conversion and reversion. Our findings underscore the crucial role of hypermutability in P. aeruginosa adaptation, with significant implications for managing persistent infections in clinical settings.

Genomic characteristics and molecular epidemiology of MRSA from medical centers in Mexico: Results from the Invifar network.

INTRODUCTION: The methicillin-resistant Staphylococcus aureus (MRSA) genome varies by geographical location. This study aims to determine the genomic characteristics of MRSA using whole-genome sequencing (WGS) data from medical centers in Mexico and to explore the associations between antimicrobial resistance genes and virulence factors. METHODS: This study included 27 clinical isolates collected from sterile sites at eight centers in Mexico in 2022 and 2023. Antibiotic susceptibility testing was performed using VITEK 2. In addition, WGS was performed using a NovaSeq platform, and a bioinformatic analysis was conducted using several tools. RESULTS: In this study, 21 strains were CC5, five were CC8, and one was CC93. Moreover, six strains were identified as ST5(CC5)-MRSA-IIa- t895, four strains were found to be ST1011(CC5)-MRSA-IIa-t895, five strains were found to be ST1011(CC5)-MRSA-IIa-t9364, one strain was found to be ST1011(CC5)-MRSA-IIa-t8116, another was found to be ST1011(CC5)-MRSA-IIa-t62, three were found to be ST8(CC8)-MRSA-IVa-t8, one strain was ST5(CC5)-MRSA-IVa-t2, one strain was as ST93(CC93)-MRSA-IVa-t3949, two strains were ST9003(CC8)-MRSA-IVa-t18492, and three strains were ST9034(CC5)-MRSA-V-t2. All SCCmec IIa strains showed resistance to levofloxacin and ciprofloxacin, and all but two strains were resistant to clindamycin. Among the strains that harbored the type IIa cassette, most had the aadD, blaZ, and ermA_SDS genes and the erm A gene. Multiple genes for adhesion, enzymes, immune evasion, and secretion system were detected, regardless of SCCmec type. Of the SCCmec IVa strains, most harbored the Panton-Valentine leukocidin encoding genes. CONCLUSION: In this study, the most frequently detected CC was CC5, followed by CC8, and CC93, and the most frequently detected MRSA ST was ST1011, followed by ST5. Most SCCmec elements were found to be type IIa, followed by type IVa. High MIC values were observed for ciprofloxacin, erythromycin, and clindamycin, particularly within SCCmec IIa. Of the SCCmec IVa strains, most harbored the lukS-PV and lukF-PV genes.

Whole-Genome Sequencing of Resistance, Virulence and Regulation Genes in Extremely Resistant Strains of Pseudomonas aeruginosa.

BACKGROUND/OBJECTIVES: Pseudomonas aeruginosa is a clinically significant opportunistic pathogen, renowned for its ability to acquire and develop diverse mechanisms of antibiotic resistance. This study examines the resistance, virulence, and regulatory mechanisms in extensively drug-resistant clinical strains of P. aeruginosa. METHODS: Antibiotic susceptibility was assessed using the Minimum Inhibitory Concentration (MIC) method, and whole-genome sequencing (WGS) was performed on the Illumina NovaSeq platform. RESULTS: The analysis demonstrated a higher prevalence of virulence genes compared to resistance and regulatory genes. Key virulence factors identified included secretion systems, motility, adhesion, and biofilm formation. Resistance mechanisms observed comprised efflux pumps and beta-lactamases, while regulatory systems involved two-component systems, transcriptional regulators, and sigma factors. Additionally, phenotypic profiles were found to correlate with resistance genes identified through genotypic analysis. CONCLUSIONS: This study underscores the significant resistance and virulence of the clinical P. aeruginosa strains analyzed, highlighting the urgent need for alternative strategies to address infections caused by extensively drug-resistant bacteria.

Genomic characterization of antimicrobial-resistance and virulence factors in Salmonella isolates obtained from pig farms in Antioquia, Colombia.

BACKGROUND: Occurrence of antimicrobial-resistant Salmonella strains has been reported worldwide, because of inappropriate use of antimicrobial products in either humans or animals. The presence of multidrug resistant Salmonella in pig production systems had been reported in Antioquia, Colombia. AIM: To identify antimicrobial resistance genes (ARG) in different Salmonella spp. strains isolated from pig productions in Antioquia, Colombia. Methods: Samples were received at the Diagnostic Unit of the Faculty of Agrarian Sciences at the University of Antioquia, from January 1, 2019, to January 2021. A total of 28 isolates of Salmonella spp. were included, which presented phenotypic resistance to more than one antibiotic used in pig farms. Whole genome sequencing (WGS) was performed in the Unit of Genomic of Agrosavia using an automated pipeline from the GHRU- Sanger Institute, employing the Illumina MiSeq platform. RESULTS: WGS revealed 34 ARGs among these isolates. In 25 isolates (89%) more than two ARGs were found. Genes encoding resistance were found for 10 different groups of antibiotics (beta-lactam, aminoglycosides, chloramphenicol, rifampicins, lincosamides, fluoroquinolones, tetracyclines, sulfonamides and trimethoprim). The most frequently observed MDR profile in Typhimurium isolates was AMP-CEX-CEP-CEF-EFT-CEQ-FLU-ENR-TE-FFC-SXT. CONCLUSION: The presence of multi-drug resistant Salmonella strains in pigs destined for human consumption in Antioquia, Colombia was determined. This research emphasizes the utmost importance of epidemiological tools to understand the presence and spreading of antimicrobial resistance genes in pig farms. Additionally, it highlights the critical need for developing educational programs and public policies to help reduce the spread of antimicrobial resistance in production systems.

Potential links between human bloodstream infection by Salmonella enterica serovar Typhimurium and international transmission to Colombia.

Salmonella enterica serovar Typhimurium is a prevalent food-borne pathogen that is usually associated with gastroenteritis infection. S. Typhimurium is also a major cause of bloodstream infections in sub-Saharan Africa, and is responsible for invasive non-typhoidal Salmonella (iNTS) disease. The pathogen also causes bloodstream infection in Colombia, but there has been a lack of information about the S. Typhimurium isolates that were responsible. Here, we investigated the genomic characteristics of 270 S. Typhimurium isolates from bloodstream infection patients in Colombia, collected between 1997 and 2017. We used whole-genome sequencing to analyse multidrug-resistant (MDR) profiles, plasmid distribution, and to define phylogenetic relationships. The study identified the distinct sequence types and phylogenetic clusters of S. Typhimurium prevalent in Colombia. The majority of isolates (90.8%) were ST19, which is distinct from the iNTS-associated S. Typhimurium in sub-Saharan Africa (ST313). The two prominent clusters of MDR S. Typhimurium were either DT104 or closely related to the LT2 reference strain. We used a phylogenetic approach to associate the Colombian clusters with global S. Typhimurium isolates from public databases. By putting the Colombian S. Typhimurium isolates in the context of the global spread of DT104, ST313 and LT2-related variants, we found that the Colombian clusters were introduced to the country via multiple independent events that were consistent with international transmission. We suggest that the acquisition of quinolone and chloramphenicol resistance by the Colombian S. Typhimurium isolates was driven by horizontal gene transfer. Three ST313 isolates that caused bloodstream infection in Colombia were identified. These ST313 isolates were related to the Malawian ST313 lineage 3 & UK ST313, and shared a similarly high invasiveness index. To our knowledge, this is the first report of ST313 in Colombia.

Resistome and plasmidome genomic features of mcr-1.1-harboring Escherichia coli: a One Health approach.

AIMS: This study evaluated the phenotypic and genotypic traits of mcr-1.1-harboring Escherichia coli isolates from chickens, pigs, humans, and farm environments. The resistome and the mobile genetic elements associated with the spread of mcr-1.1 in Southern Brazil were also characterized. METHODS AND RESULTS: The 22 mcr-1.1-harboring E. coli isolates from different origins were selected for antimicrobial susceptibility testing and whole genome sequencing for characterization of the resistome, plasmids, and sequence types. All isolates presented several resistance genes and harbored the mcr-1.1 gene in a highly similar IncX4 plasmid. Furthermore, the mcr-1.1 gene co-occurred with the mcr-3.12 gene in a multidrug-resistant isolate from the farm environment. CONCLUSIONS: These findings demonstrate that the mcr-1.1 gene in E. coli isolates from Brazil is spreading mainly by horizontal transfer of the IncX4 plasmid. The co-occurrence of mcr-1.1 and mcr-3.12 highlights pig farming as an important reservoir of colistin resistance.

Salmonella enterica Serovar Infantis KPC-2 Producer: First Isolate Reported in Ecuador.

Antimicrobial resistance is currently considered a public health threat. Carbapenems are antimicrobials for hospital use, and Enterobacterales resistant to these ß-lactams have spread alarmingly in recent years, especially those that cause health care-associated infections. The blaKPC gene is considered one of the most important genetic determinants disseminated by plasmids, promoting horizontal gene transfer. This study describes, for the first time in Ecuador, and worldwide, the presence of a blaKPC-2 gene in an isolate of Salmonella enterica serovar Infantis from a clinical sample. Through whole-genome sequencing, we characterized the genetic determinants of antimicrobial resistance in this Salmonella ST-32 strain. Our results showed the presence of several resistance genes, including blaCTX-M-65, and a conjugative plasmid Kpn-WC17-007-03 that may be responsible for the horizontal transference of these resistance mechanisms.

Characterization of Enterobacter phage vB_EcRAM-01, a new Pseudotevenvirus against Enterobacter cloacae, isolated in an urban river in Panama.

The Enterobacter cloacae complex, a prominent bacterium responsible worldwide for most bloodstream infections in the hospital environment, has shown broad-spectrum antibiotic resistance, including carbapenems. Therefore, bacteriophages have again attracted the attention of the science and medical community as an alternative to control Multidrug resistant bacteria. In this study, water samples from Río Abajo River, in Panama City, Panama, were collected, for phage isolation, purification, characterization and propagation against the E. cloacae complex. As result, a phage produced clear and round plaque-forming units indicating a lytic phage was isolated. Further analyses concluded that this phage is stable at temperatures between 25°C and 50°C, it remains infective in a pH range between 7 to 11, with high sensitivity to Ultraviolet light. Remarkedly, it exhibits a narrow host specificity only infecting E. cloacae. Whole genome sequencing revealed that is a myovirus with a genome size of 178,477 bp, a G-C content of 45.8%, and containing approximately 294 genes. Among them, protein-encoding genes involved in morphology, inactivation, adsorption to cells, DNA injection and lytic enzymes were identified. Additionally, the genome contained two tRNA sequences. Genes that encode holins and endolysins, typical of lytic bacteriophages, were also present. A whole-genome sequencing analysis indicated that, according to the genus demarcation criteria, this phage belongs to a novel species within the Family Straboviridae, called genus Pseudotevenvirus.

Genomic analysis of blaNDM-1-carrying-Pseudomonas aeruginosa ST2407 in the chromosome from Brazil.

Pseudomonas aeruginosa, an opportunistic pathogen often found in Healthcare-associated infections (HAI), has shown increased resistance to carbapenems (imipenem, meropenem, doripenem), the primary treatment options. We've seen a rise in carbapenemase-producing P. aeruginosa in Brazil, including NDM-producers. This study characterises an isolate carrying blaNDM-1 from a patient's skin fragment in a Brazilian hospital. The whole genomic sequence (WGS) of P. aeruginosa CCBH26428 was extracted and sequenced using Illumina and minION platforms. The assembly used MinION results mapped with Illumina reads, and annotation was performed by the RAST server. Resistance genes and clonality were identified using the CABGen platform. Additional information was carried out by manual annotation using Geneious software and BLAST tool. The genomic analysis revealed a genome of 6.995.008 bp and G+C 65.9 %. P. aeruginosa CCBH26428 belongs to ST2407. The blaNDM gene, associated with ISAba125, was found in a 63.862 pb genomic region flanked by IS26 insertion sequences. This region also contained the repA of the plasmid incompatibility group IncC2 and other resistance genes, suggesting it is a possible "translocation unit". Additionally, 17 resistance genes, mutations in OprD and GyrA, and several virulence genes were detected, potentially exacerbating the infection. This study is report a WGS analysis of P. aeruginosa carrying blaNDM-1 in Brazil, highlighting the role of IS26 in the acquisition and spread of resistance genes between plasmids and chromosomes.

The complete genome sequence of "Candidatus Liberibacter asiaticus" strain 9PA and the characterization of field strains in the Brazilian citriculture.

"Candidatus Liberibacter asiaticus" (CLas) is associated with citrus huanglongbing, a severe disease with global importance that affects citrus production in Brazil. This study reports the first complete genome of a Brazilian strain of CLas. The genomic structure comparison of strain 9PA with those of 13 complete CLas genomes revealed 9,091 mismatches and 992 gaps/insertions, highlighting eight locally colinear blocks, among which six are in the prophage region. Phylogenetic analysis categorized 13 CLas genomes into two clusters with 9PA clustered with strains from China and the United States. Whole-genomic comparison identified diverse hypervariable genomic regions (HGRs). Three HGRs in the chromosomal region and three in the prophage region were selected and investigated by polymerase chain reaction. HGRs assessed from 68 samples, from medium- to high-huanglongbing incidence areas in Sao Paulo state, were grouped into haplotypes A to P. Haplotype A, which includes strain 9PA, is the second most prevalent, representing 19.1% of the samples. Haplotype B, the most common, accounts for 42.6%. Together with haplotype C, these make up 72% of the evaluated samples. The 9PA strain has prophage P-9PA-1, both integrated and circularized, and P-9PA-3, only found in a circularized form. Prophages show high identity with SC1 (83%) and P-JXGC-3 (98%). Co-occurrence of both type 1 and 3 prophages was observed in field samples. The approach employed provides insights into the Brazilian CLas population, providing markers for population studies and highlighting the prevalence of type 1 and 3 prophages in the population. IMPORTANCE: CLas is a destructive pathogen responsible for causing the severe citrus disease known as huanglongbing. Our study presents the first fully sequenced Brazilian strain of CLas, designated as 9PA, and includes an analysis of two prophages occurring in this strain. The main objective of our research was to compare the genome features of this Brazilian strain with other fully sequenced genomes and to identify its hypervariable genetic regions. These regions were subsequently used to assess genomic variability within both the chromosomal and prophage regions in Brazilian isolates of CLas. Our findings offer valuable insights into the diversified adaptation of CLas.

Clonal outbreak of Candida vulturna in a paediatric oncology ward in Maranhão, Brazil.

OBJECTIVE: To describe an outbreak due to Candida vulturna, a newly emerging Candida species belonging to the Candida haemulonii species complex in the Metschnikowiaceae family. METHODS: In this retrospective cohort study we genotyped 14 C. vulturna bloodstream isolates, occurring in a 4-month-period in paediatric cancer patients in a Brazilian hospital. To prove an outbreak, ITS sequence analysis and whole genome sequencing (WGS) was done. Antifungal susceptibility was performed with the reference CLSI method and the commercial Sensititre YeastOne (SYO) YO10 plates. A control C. vulturna isolate from another region in Brazil was included in all analyses. RESULTS: MALDI-TOF-MS identified isolates as C. pseudohaemulonii and C. duobushaemulonii albeit with low scores and therefore molecular methods were required for accurate identification. ITS sequence analyses clearly differentiated C. vulturna from other species in the C. haemulonii species complex. WGS proved the presence of a clonal outbreak with C. vulturna involving 14 paediatric patients. Antifungal susceptibility testing (AFST) with two methods showed the isolates had low MICs of commonly available antifungals. CONCLUSION: This study describes an outbreak due to the rare yeast C. vulturna, related to C. auris, during a four-month period in patients admitted to a paediatric oncology ward in a Brazilian hospital. In contrast to previous studies the yeast was susceptible to all antifungals and patient outcome was good.

Myroides species, pathogenic spectrum and clinical microbiology sight in Mexican isolates.

INTRODUCTION: Myroides is a bacterial genus of opportunistic bacteria responsible for diverse infections including in the skin and soft tissues, urinary tract, cardiovascular system, and bacteremia, although the incidence of its reported infections is low, it is increasing, likely due the use of better bacterial identification methods, but also perhaps due an increase in its prevalence. In addition, their pathogenic role is limited in terms of reporting their microbial physiology, so the present work provides information in this regard in addition to the information that is available in the international literature. OBJECTIVE: To describe the microbiological and genetic characteristics of seven different Myroides spp. clinical strains and comment on their phylum, pathogenic and resistance characteristics. METHODS: Seven Myroides spp., strains associated with infections were included from 1/January/2012 to 1/January/20 and identified by miniaturized biochemistry and MALDI-ToF. Susceptibility tests were performed according to CLSI recommendations by broth microdilution. Whole genome sequencing was performed for each strain and bioinformatics analysis were performed. RESULTS: Strains were identified at genus level by two methodologies. Our results revealed that likely four strains belong to the species Myroides odoratimimus, while the other two may be undescribed ones. Remarkably, all isolates harbored several genes encoding antibiotic resistance determinants for ß-lactams, aminoglycosides and glycopeptides and in concordance, presented high levels of resistance, against these antibiotics (AK and GN both 100%, ATM, CAZ and FEP 100%, e.g.); moreover, the presences of carbapenemases were evidenced by meropenem (mCIM) and imipenem (CARBA NP) degrading activity in six isolates and two strains possessed plasmids harboring mainly ribosomal RNA genes, tRNAs and genes encoding proteins with unknown functions. CONCLUSIONS: Our study increases the knowledge about the biology of this understudied genus and highlights the potential of Myroides to emerge as a broader cause of recalcitrant opportunistic infections.

Polyphasic identification of Vibrio species from aquatic sources using mass spectrometry, housekeeping gene sequencing and whole genome analysis.

Accurate bacterial identification is essential for determining the causative agent of an infection, thus facilitating appropriate treatment and management strategies in both human and animal health contexts. Some species in the Vibrio genus are recognized pathogens, associated with a variety of infections. However, identification of these bacteria is oftentimes controversial. Therefore, we aimed to evaluate different identification approaches in terms of their reliability in distinguishing Vibrio species. To achieve this, we selected a set of 40 Vibrio isolates previously recovered from water and floating plastic samples in a large bay environment and identified them employing MALDI-TOF mass spectrometry, and rrs and pyrH gene sequencing. A subset of isolates was also submitted to whole genome sequencing. Overall, MALDI-TOF was found to be a fast-screening methodology for identification, notably at genus-level. However, for better species discrimination, pyrH gene sequencing stood out as a more reliable tool in contrast to rrs gene sequencing and MALDI-TOF, as corroborated by whole genome sequencing analysis.

Linking intraspecies variability of Salmonella enterica isolates under acidic conditions to genotype.

There is a lack of information about Salmonella enterica strains under acidic conditions and their association with their genome. This study characterized intraspecies variability in the growth of 167 S. enterica isolates under two acid conditions (pH 4 and 5) and linked to the whole genome sequencing (WGS) data. A total of 1002 curves for each condition were obtained using turbidimetry measurements, and Baranyi and Roberts model was used to estimate the maximum rate of change (rcmax; OD600 nm h-1). Strains were categorized into slow, intermediate, and fast; and associations with their WGS data were performed. Huge variability in r c max ¯ $\overline {{\mathrm{r}}{{{\mathrm{c}}}_{{\mathrm{max}}}}} $ was observed at both conditions (pH 5 = 0.016-0.066 OD600nm h-1 and pH 4 = 0.003-0.028 OD600nm h-1). The majority of isolates was classified as intermediate r c max ¯ $\overline {{\mathrm{r}}{{{\mathrm{c}}}_{{\mathrm{max}}}}} $ (59.5% at pH 5 and 46.1% at pH 4). Strains classified as fast had a low frequency of allABCD genes at both pHs, and any of them having the presence of pefABCD, spvBCR, aadA2, dfrA12, and gyrA_D87G genes were linked to virulence or antimicrobial resistance. This study suggests that strains with fast capacity for growth under acidic conditions could have a fitness cost in their virulence or resistance potential. PRACTICAL APPLICATION: Data presented in this study could be used to select representative strains to evaluate the exposure assessment in different food items, mainly the growth and survival in acidic foods.

Whole-genome sequencing of Klebsiella pneumoniae MDR circulating in a pediatric hospital setting: a comprehensive genome analysis of isolates from Guayaquil, Ecuador.

BACKGROUND: Klebsiella pneumoniae is the major cause of nosocomial infections worldwide and is related to a worsening increase in Multidrug-Resistant Bacteria (MDR) and virulence genes that seriously affect immunosuppressed patients, long-stay intensive care patients, elderly individuals, and children. Whole-Genome Sequencing (WGS) has resulted in a useful strategy for characterizing the genomic components of clinically important bacteria, such as K. pneumoniae, enabling them to monitor genetic changes and understand transmission, highlighting the risk of dissemination of resistance and virulence associated genes in hospitals. In this study, we report on WGS 14 clinical isolates of K. pneumoniae from a pediatric hospital biobank of Guayaquil, Ecuador. RESULTS: The main findings revealed pronounced genetic heterogeneity among the isolates. Multilocus sequencing type ST45 was the predominant lineage among non-KPC isolates, whereas ST629 was found more frequently among KPC isolates. Phylogenetic analysis suggested local transmission dynamics. Comparative genomic analysis revealed a core set of 3511 conserved genes and an open pangenome in neonatal isolates. The diversity of MLSTs and capsular types, and the high genetic diversity among these isolates indicate high intraspecific variability. In terms of virulence factors, we identified genes associated with adherence, biofilm formation, immune evasion, secretion systems, multidrug efflux pump transporters, and a notably high number of genes related to iron uptake. A large number of these genes were detected in the ST45 isolate, whereas iron uptake yersiniabactin genes were found exclusively in the non-KPC isolates. We observed high resistance to commonly used antibiotics and determined that these isolates exhibited multidrug resistance including ß-lactams, aminoglycosides, fluoroquinolones, quinolones, trimetropins, fosfomycin and macrolides; additionally, resistance-associated point mutations and cross-resistance genes were identified in all the isolates. We also report the first K. pneumoniae KPC-3 gene producers in Ecuador. CONCLUSIONS: Our WGS results for clinical isolates highlight the importance of MDR in neonatal K. pneumoniae infections and their genetic diversity. WGS will be an imperative strategy for the surveillance of K. pneumoniae in Ecuador, and will contribute to identifying effective treatment strategies for K. pneumoniae infections in critical units in patients at stratified risk.

Influenza outbreak during the surge of SARS-CoV-2 omicron in a metropolitan area from southern Brazil: genomic surveillance.

Influenza circulation was significantly affected in 2020-21 by the COVID-19 pandemic. During this time, few influenza cases were recorded. However, in the summer of 2021-22, an increase in atypical influenza cases was observed, leading to the resurgence of influenza in the southernmost state of Brazil, Rio Grande do Sul (RS). The present study aimed to identify the circulation of FLUAV, FLUBV and SARS-CoV-2 and characterize the influenza genomes in respiratory samples using high-throughput sequencing technology (HTS). Respiratory samples (n = 694) from patients in RS were selected between July 2021 and August 2022. The samples were typed using reverse transcriptase real-time PCR (RT-qPCR) and showed 32% (223/694) of the samples to be positive for SARS-CoV-2, 7% for FLUAV (H3) (49/694). FLUBV was not detected. RT-qPCR data also resulted in FLUAV and SARS-CoV-2 co-infections in 1.7% (4/223) of samples tested. Whole genome sequencing of FLUAV produced 15 complete genomes of the H3N2 subtype, phylogenetically classified in the 3C.2a1b.2a.2a.3 subclade and revealing the dominance of viruses in the southern region of Brazil. Mutation analysis identified 72 amino acid substitutions in all genes, highlighting ongoing genetic evolution with potential implications for vaccine effectiveness, viral fitness, and pathogenicity. This study underscores limitations in current surveillance systems, advocating for comprehensive data inclusion to enhance understanding of influenza epidemiology in southern Brazil. These findings contribute valuable insights to inform more effective public health responses and underscore the critical need for continuous genomic surveillance.

Impact of Vaccination on Intra-Host Genetic Diversity of Patients Infected with SARS-CoV-2 Gamma Lineage.

The high transmissibility, rapid evolution, and immune escape of SARS-CoV-2 variants can influence the course of infection and, in turn, morbidity and mortality in COVID-19, posing a challenge in controlling transmission rates and contributing to the emergence and spread of new variants. Understanding the factors that shape viral genetic variation is essential for comprehending the evolution and transmission of SARS-CoV-2, especially in vaccinated individuals where immune response plays a role in the progression and spread of this disease. In this context, we evaluated the impact of immunity induced by the CoronaVac vaccine (Butantan/Sinovac) on intra-host genetic diversity, analyzing 118 whole-genome sequences of SARS-CoV-2 from unvaccinated and vaccinated patients infected with the Gamma variant. Vaccination with CoronaVac favors negative selection at the intra-host level in different genomic regions. It prevents greater genetic diversity of SARS-CoV-2, reinforcing the importance of vaccination in reducing the emergence of new mutations and virus transmission.

Full Genome Characterization of the First Oropouche Virus Isolate Imported in Europe from Cuba.

On 27 May 2024, the Cuban Ministry of Health reported the first outbreak of Oropouche fever on the island. The etiologic agent, Oropouche virus (OROV), is a poorly understood arbovirus that has been known since the 1960s and represents a public health burden in Latin America. We report the whole-genome characterization of the first European OROV isolate from a returning traveler from Cuba with Oropouche fever-like symptoms. The isolate was obtained from the patient's serum; whole-genome sequencing was performed by next-generation sequencing, followed by phylogenetic analysis and genetic variability studies. The analysis showed that the most closely related sequence was from the French Guiana 2020 outbreak. Interestingly, our isolate is a reassortant virus, included in a highly supported monophyletic clade containing recent OROV cases (Brazil 2015-Colombia 2021), separated from the other four previously known genotypes. More deeply, it was found to be included in a distinct branch containing the sequences of the Brazil 2022-2024 outbreak. The reassortment event involved the S and L segments, which have high similarity with sequences belonging to a new cluster (here defined as OROV_SCDC_2024), while the M segment shows high similarity with older sequences. These results likely describe the viral strain responsible for the current outbreak in Cuba, which may also reflect the ongoing outbreak in Latin America. Further studies are needed to understand how OROV evolves towards traits that facilitate its spread and adaptation outside its original basin, and to track its spread and evolution in the European continent.

Long-term genomic surveillance reveals the circulation of clinically significant Salmonella in lymph nodes and beef trimmings from slaughter cattle from a Mexican feedlot.

This longitudinal study characterized Salmonella circulating in lymph nodes (LN, n = 800) and beef trimmings (n = 745) from slaughter cattle from a Mexican feedlot. During two years, LN and beef trimming samples were collected 72-96 h post-slaughter, and we obtained 77 isolates of the serovars Anatum (n = 23), Reading (n = 22), Typhimurium (n = 10), London (n = 9), Kentucky (n = 6), Fresno (n = 4), Give, Muenster, and monophasic 1,4,[5],12:i- (n = 1 each). These isolates were subjected to whole genome sequencing, single-nucleotide polymorphism (SNP)-based phylogenetic analysis, reconstruction of their ancestral isolation sources through evolutionary analysis, and virulence profiling. Although LN and beef trimmings were not mixed, evolutionary analysis estimated that the common ancestor of all study isolates was likely of LN origin. Moreover, isolates from both sources were highly clonal (0-21 SNP distance), highlighting the complexity of Salmonella transmission dynamics. The pathogen persisted across cattle cohorts, as shown by clonality between isolates collected in different years (1-20 SNP distance). Major virulence genes were highly conserved (97-100% identity to the reference sequences) and most isolates carried a conserved version of pathogenicity islands 1-5, 9, 11, and 12. Typhimurium strains carried the Salmonella plasmid virulence operon (spvRABCD), and a Muenster isolate carried the st313td gene, both of which are associated with invasive phenotypes. Most isolates (49/77) were genetically similar (1-43 SNPs) to strains involved in human salmonellosis, highlighting their public health significance. Further research is needed on Salmonella transmission dynamics in cattle and the mechanisms determining subclinical infection and persistence in farm environments.