Genomic Surveillance of Salmonella from the Comunitat Valenciana (Spain).

Salmonella enterica subspecies enterica is one of the most important foodborne pathogens and the causative agent of salmonellosis, which affects both humans and animals producing numerous infections every year. The study and understanding of its epidemiology are key to monitoring and controlling these bacteria. With the development of whole-genome sequencing (WGS) technologies, surveillance based on traditional serotyping and phenotypic tests of resistance is being replaced by genomic surveillance. To introduce WGS as a routine methodology for the surveillance of food-borne Salmonella in the region, we applied this technology to analyze a set of 141 S. enterica isolates obtained from various food sources between 2010 and 2017 in the Comunitat Valenciana (Spain). For this, we performed an evaluation of the most relevant Salmonella typing methods, serotyping and sequence typing, using both traditional and in silico approaches. We extended the use of WGS to detect antimicrobial resistance determinants and predicted minimum inhibitory concentrations (MICs). Finally, to understand possible contaminant sources in this region and their relationship to antimicrobial resistance (AMR), we performed cluster detection combining single-nucleotide polymorphism (SNP) pairwise distances and phylogenetic and epidemiological data. The results of in silico serotyping with WGS data were highly congruent with those of serological analyses (98.5% concordance). Multi-locus sequence typing (MLST) profiles obtained with WGS information were also highly congruent with the sequence type (ST) assignment based on Sanger sequencing (91.9% coincidence). In silico identification of antimicrobial resistance determinants and minimum inhibitory concentrations revealed a high number of resistance genes and possible resistant isolates. A combined phylogenetic and epidemiological analysis with complete genome sequences revealed relationships among isolates indicative of possible common sources for isolates with separate sampling in time and space that had not been detected from epidemiological information. As a result, we demonstrate the usefulness of WGS and in silico methods to obtain an improved characterization of S. enterica enterica isolates, allowing better surveillance of the pathogen in food products and in potential environmental and clinical samples of related interest.

Listeria monocytogenes, a silent foodborne pathogen in Ecuador.

Listeria monocytogenes is a foodborne pathogen that can produce serious, even fatal, infections. Among other foods, it can be found in unpasteurized dairy and ready-to-eat products. Surveillance of L. monocytogenes is of great interest since sources of infection are difficult to determine due to the long incubation period, and because the symptoms of listeriosis are similar to other diseases. We performed a genomic study of L. monocytogenes isolated from fresh cheeses and clinical samples from Ecuador. Sixty-five isolates were evaluated and sequenced, 14 isolates from cheese samples and 20 from clinical listeriosis cases from the National Institute of National Institute of Public Health Research, and 31 isolates from artisanal cheese samples from 8 provinces. All isolates exhibited heterogeneous patterns of the presence of pathogenicity islands. All isolates exhibited at least 4 genes from LIPI-1, but all references (26 L. monocytogenes closed genomes available in the NCBI database) showed the complete island, which encompasses 5 genes but is present in only two Ecuadorian isolates. Most isolates lacked gene actA. Genes from LIPI-2 were absent in all isolates. LIPI-3 and LIPI-4 were present in only a few references and isolates. With respect to the stress survival islets, our samples either presented SSI-1 or SSI-F2365, except for one isolate that presented SSI-F2365 and also one gene from SSI-1. None of the samples presented SSI-2. The predominant ST (sequence type) was ST2 (84.62% 55/65), and the only ST found in food (93.33% 42/45) and clinical samples (65% 13/20). Isolates were not grouped according to their sampling origin, date, or place in a phylogenetic tree obtained from the core alignment. The presence of ST2 in food and clinical samples, with high genomic similarity, suggests a foodborne infection risk linked to the consumption of fresh cheeses in Ecuador.

Identification of human leukocyte antigen in precancerous and cancerous cervical lesions from Ecuadorian women.

Cervical cancer is the fourth most common type of cancer in women. Worldwide, it is a public health problem with around 604,127 women diagnosed per year and 341,831 deaths. Cervical cancer and persistent high-risk human papillomavirus (HPV) infection are highly associated. However, other factors are also involved, such as viral load, HPV variants, sexual behavior, and genetic factors. The host immune response against HPV has been widely studied and it has shown associations with development of cervical cancer. The human leukocyte antigen (HLA) genes are related to the persistence of HPV infection and progression to cervical cancer because of their role in controlling T-cell mediated immune response to clear the infection. In Ecuador, there is scarce information about HLA and HPV infection with high-risk genotypes in the population. This study aimed to identify host-specific HLA alleles in women with cervical intraepithelial neoplasia (CIN) II and III, and cancer infected with HPV-16, 58, and 52. In this study, we included 51 samples previously identified as positive for HPV-16, 58, and 52 from 12 Ecuadorian provinces. As a result, we found that HLA-A*02, HLA-B*35, HLA-C*04, HLA-DRB1*04, and HLA-DQB1*03 alleles were the most frequent, these alleles have been associated with cervical cancer in previous studies; nevertheless, we did not find a statistically significant association between HLA alleles, HPV genotype, and histopathological lesion. This is a baseline study to uncover possible relationships between HLA and HPV to elucidate why this virus can develop a persistent infection in some women leading to the development of cervical cancer.

The impact of genetic recombination on pathogenic Leptospira.

Leptospirosis is the most common zoonosis worldwide, and is increasingly common in poor urban communities, where there is inadequate sewage disposal and abundance of domestic and peridomestic animals. There are many risk factors associated with the disease, such as contaminated water exposure, close contact with animals, floods, recreational activities related to water, wet agriculture. The symptoms of leptospirosis are common to other infectious diseases and, if not treated, it can lead to meningitis, liver failure, kidney damage and death. Leptospirosis is caused by 38 pathogenic species of Leptospira, which are divided in almost 30 serogroups and more than 300 serovars. The serological classification (serogroups and serovars) is based on the expression of distinct lipopolysaccharide (LPS) antigens. These antigens are also associated to protective immunity; antibodies against a serovar protect from any member of the same serovar. Serologic and phylogenetic analyses are not congruent probably due to genetic recombination of LPS genes among different leptospiral species. To analyze the importance of recombination in leptospiral evolution, we performed a gene-by-gene tree topology comparison on closed genomes available in public databases at two levels: among core genomes of pathogenic species (34 recombinant among 1213 core genes), and among core genomes of L. interrogans isolates (178/798). We found that most recombinant genes code for proteins involved in translation, ribosomal structure and biogenesis, but also for cell wall, membrane and envelope biogenesis. Besides, our final results showed that half of LPS genes are recombinant (18/36). This is relevant because serovar classification and vaccine development are based on these epitopes. The frequent recombination of LPS-associated genes suggests that natural selection is promoting the survival of recombinant lineages. These results may help understanding the factors that make Leptospira a successful pathogen.

Detection and genotyping of Listeria monocytogenes in artisanal soft cheeses from Ecuador

Abstract Listeria monocytogenes is one of the most important bacteria associated with food-borne diseases, soft cheese being an important L. monocytogenes vehicle. In Ecuador, softcheese is consumed in 84.3% of urban households. We determined the prevalence of L. mono-cytogenes and serogroups in 260 fresh artisanal soft cheese samples collected in 18 of 24Ecuadorian provinces. Listeria spp. detection was carried out by culture-dependent and inde-pendent methods; 14.23% of samples were positive for L. monocytogenes. Serogroup IVb wasfound in 83.78% of the food isolates. Serogroups IIb and IIa were present in 8.11% of our isolates.To our knowledge, this is the first report of L. monocytogenes serogroups associated with foodin Ecuador; we also found serogroup similarities between cheese isolates and clinical isolates.

Resumen Listeria monocytogenes es una de las bacterias más importantes asociadas conenfermedades transmitidas por alimentos, y el queso fresco es un importante vehículo de trans-misión de L. monocytogenes. En Ecuador, el consumo de quesos frescos se produce en el 84,3%de los hogares urbanos. Determinamos la prevalencia de L. monocytogenes y sus serogrupos en260 muestras de queso fresco artesanal recolectadas en 18 de las 24 provincias ecuatorianas.La detección de Listeria spp. se llevó a cabo mediante métodos independientes y dependientesde cultivo; el 14,23% de las muestras fueron positivas para L. monocytogenes.

Detection and genotyping of Listeria monocytogenes in artisanal soft cheeses from Ecuador.

Listeria monocytogenes is one of the most important bacteria associated with foodborne diseases, soft cheese being an important L. monocytogenes vehicle. In Ecuador, soft cheese is consumed in 84.3% of urban households. We determined the prevalence of L. monocytogenes and serogroups in 260 fresh artisanal soft cheese samples collected in 18 of 24 Ecuadorian provinces. Listeria spp. detection was carried out by culture-dependent and independent methods; 14.23% of samples were positive for L. monocytogenes. Serogroup IVb was found in 83.78% of the food isolates. Serogroups IIb and IIa were present in 8.11% of our isolates. To our knowledge, this is the first report of L. monocytogenes serogroups associated with food in Ecuador; we also found serogroup similarities between cheese isolates and clinical isolates.

Determining the microbial and chemical contamination in Ecuador's main rivers.

One major health issue is the microbial and chemical contamination of natural freshwater, particularly in Latin American countries, such as Ecuador, where it is still lacking wastewater treatment plants. This study analyzed the water quality in twelve rivers of Ecuador (Coastal, Andean, and Amazonian regions). All rivers showed levels of E. coli and total coliforms above the maximum limit according to International and Ecuadorian legislations. The most polluted rivers were Zamora, Esmeraldas and Machángara. Also, E. coli pathotypes were found in six rivers. Several physicochemical and metal parameters were detected in high levels, such as CODTOTAL (in eight rivers), TSS (in six rivers), TS (in two rivers), Al (in nine rivers), Zn (in eight rivers), Pb (in three rivers), Cu (in three rivers), Fe (in two rivers), and Mn (in Machángara River). Our results agree with other studies in Latin America (such as Colombia, Brazil, and Peru) reporting similar contamination in water resources used for agriculture, livestock, and human consumption. Overall, Guayas, Guayllabamba, and Machángara Rivers showed the highest levels of physicochemical parameters (such as CODTOTAL and TSS) and metal concentrations (such as copper, zinc, aluminum, iron, and manganese). Further studies should evaluate contamination sources and public health impact.

One is not enough: On the effects of reference genome for the mapping and subsequent analyses of short-reads.

Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.

High Occurrence of Multiresistant Salmonella Infantis in Retail Meat in Ecuador.

Salmonella enterica is among the most important foodborne pathogens. In Ecuador, there is limited information about non-typhoidal S. enterica occurrence in raw meats, its serotype distribution, and antimicrobial resistance. In this study, we addressed this issue in 1095 retail fresh meats (chicken, pork, veal, lamb, beef, and turkey) in Quito by performing a traditional culture methodology and molecular detection. We found that S. enterica was present in 38.1% of the samples, and Salmonella Infantis was the most common serotype showing a high antibiotic resistance and a wide host range. Some host-adapted serotypes were found in uncommon sources of meat, suggesting cross-contamination and the need to implement good manufacturing practices in meat processing. High levels of multidrug resistance were found in all serotypes. There is an urgent need to identify Salmonella serotypes in food to compare with clinical data and to carry out epidemiological studies to control and prevent outbreaks and infections.

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.

Reducción embrionaria en embarazo gemelar / Embryo reduction in twin pregnancy

La reducción embrionaria es la interrupción selectiva del desarrollo de uno o varios fetos en el primer trimestre del embarazo. El embarazo gemelar se presenta aproximadamente en uno de cada 100 nacimientos y se considera como una entidad con alto riesgo materno y fetal. Los embarazos múltiples tienen un impacto mayor en los sistemas de salud, debido a la mayor frecuencia de complicaciones. La rotura prematura de membranas causa aproximadamente el 40 por ciento de los partos pretérmino y, como consecuencia, aportan un 10 por ciento de la mortalidad perinatal según la Sociedad Española de Ginecología y Obstetricia. En este caso clínico se observó que una actitud expectante con los pertinentes controles ecográficos (índice del líquido amniótico), analíticos (recuento leucocitario y reacción en cadena de la polimerasa) y clínicos (frecuencia cardiaca y temperatura) pueden llevar a una buena evolución posnatal que justificó al menos en esta ocasión, una actitud conservadora(AU)

Embryonic reduction is the selective interruption of the development of one or several fetuses in the first trimester of pregnancy. Twin pregnancy occurs in approximately one in every 100 births. It is considered an entity with high maternal and fetal risk. Multiple pregnancies have greater impact on health systems due to the higher frequency of complications. Premature rupture of membranes causes approximately 40 percent of preterm births and, consequently, it contributes 10 percent of perinatal mortality according to the Spanish Society of Gynecology and Obstetrics. In this clinical case it was observed that an expectant attitude with the relevant ultrasound (index of amniotic fluid), analytical (leukocyte count and polymerase chain reaction) and clinical (heart rate and temperature) controls can lead to good postnatal evolution, justified at least on this occasion, a conservative attitude(AU)

Prevalence of human papillomavirus types in cervical cancerous and precancerous lesions of Ecuadorian women.

Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of uterine cancer. In Ecuador there is limited information about HPV types (and variants) in cancerous lesions; however, identifying the type-specific HPV prevalence in cervical lesions of women living in Ecuador is important to better predict the impact of HPV prophylactic vaccination in this country. We studied the prevalence of HPV types in cervical cancerous or precancerous lesions from 164 Ecuadorian women and found that 86.0% were HPV positive. The most common types were HPV16 (41.8%) and HPV58 (30.5%). Interestingly, HPV18 was detected only in 2.8% of the HPV-positive samples. Fifteen DNA sequences (genes E6 and L1) from 16 samples positive for HPV16 belonged to the European lineage, considered one of the least carcinogenic lineages, and 1 (6.25%) to the Asian-American lineage. Similar analysis in 12 HPV58 positive samples showed that 10 (83.3%) sequences grouped in sublineage A2, which belongs to the oldest HPV58 lineage, 1 belonged to A3 and 1 to lineage C. This study suggests that the currently used HPV vaccines (bivalent and tetravalent) may have lower effectiveness in Ecuador than in other geographic locations where HPV18 is more prevalent.

Healthcare-associated respiratory tract infection and colonization in an intensive care unit caused by Burkholderia cepacia isolated in mouthwash.

OBJECTIVES: Burkholderia cepacia has been linked to healthcare-associated infections and colonization caused by contamination of alcohol-free mouthwash used in patients undergoing mechanical ventilation. The purpose of our study was to establish the source of a clustering of healthcare-associated B. cepacia isolates in patients on mechanical ventilation in the intensive care unit (ICU). METHODS: During April 2012 the Infection Control Committee became concerned when B. cepacia was isolated from tracheal aspirate cultures of three ICU patients. The medical records for the years 2011 and 2012 were reviewed to identify further cases. Cultures of potential reservoirs were done. Isolates from patients and an alcohol-free mouthwash were submitted to multilocus sequence typing (MLST) analysis and antimicrobial resistance testing. RESULTS: Four patients with positive cultures for B. cepacia were identified before the review of the medical records for the years 2011 and 2012. Nine further cases were identified in the review, defined as a patient with pneumonia who had a culture of respiratory secretions that was positive for B. cepacia. Three were cases of infection and 10 were colonizations. All of the isolates from patients (J, K, L, and M) and mouthwash samples (B19, B20, and B21) were genetically identical by MLST analysis. CONCLUSIONS: Our findings strongly suggest that alcohol-free mouthwash solution intrinsically contaminated with B. cepacia was the source of these colonizations and infections involving adults in the ICU.

Localization of a bacterial protein in starch granules of transgenic maize kernels.

The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling/electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.