Complete genome sequence of new bacteriophage phiE142, which causes simultaneously lysis of multidrug-resistant Escherichia coli O157:H7 and Salmonella enterica

Bacterial strains used in the host range spectrum of the bacteriophage phiE142. Phage was assessed for host range by spot testing. (+) indicate positive sensitivity to phage lysis, and (-) indicate negative sensitivity to phage lysis. (DOCX 41 kb

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Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Evaluación de la reacción en cadena de la polimerasa en tiempo real acoplado a separación inmunomagnética (PCRTR-IMS) como método alternativo para la detección rutinaria de Salmonella spp. en carne de res en México

Salmonella is a pathogenic bacterium considered a threat to the food industry, its timely detection being relevant. The objective of the study was to evaluate the real-time polymerase chain reaction coupled to immunomagnetic separation (rtPCR-IMS) as an alternative method to the Official Mexican Standard (NOM-114-SSA1-1994) for the detection of Salmonella in beef. The parameters evaluated were limit of detection, sensitivity, specificity, selectivity (inclusivity and exclusivity) and degree of agreement between both methods for the detection of Salmonella in presumptive and artificially contaminated beef samples. The incidence of Salmonella in presumptive beef samples (n= 60) ranged from 20.0 to 21.6 % by both methods. In the inoculated samples (n= 60), the detection rate of Salmonella by rtPCR-IMS (93.3 %) and NOM-114-SSA1-1994 (98.3 %) showed a match of 56 occasions with a negative deviation. The comparison of rtPCR-IMS and NOM-114-SSA1-1994 in beef reported an accuracy of 98.3 %, sensitivity of 98.2 %, specificity of 100 % and selectivity of 100 %. The limit of detection for both methods was 1-5 CFU·25 g-1 of beef. The statistical analysis indicates that the rtPCR-IMS is equivalent to the reference method for the detection of Salmonella in beef. These results warn of the high incidence of Salmonella in beef and propose rtPCR-IMS as an ideal and fast method for the control of Salmonella in the meat industry.Salmonella es una bacteria patógena considerada una amenaza para la industria alimentaria, siendo relevante su detección oportuna. El objetivo del estudio fue evaluar la reacción en cadena de polimerasa en tiempo real acoplado a separación inmunomagnética (rtPCR-IMS) como método alternativo a la Norma Oficial Mexicana (NOM-114-SSA1-1994) para la detección de Salmonella en carne de res. Los parámetros evaluados fueron límite de detección, sensibilidad, especificidad, selectividad (inclusión y exclusividad), y grado de concordancia entre ambos métodos para la detección de Salmonella en muestras de carne de res presuntivas y contaminadas artificialmente. La incidencia de Salmonella en las muestras presuntivas de carne (n= 60) osciló de 20.0 a 21.6 % por ambos métodos. En las muestras inoculadas (n= 60), la tasa de detección de Salmonella por rtPCR-IMS (93.3 %) y NOM-114-SSA1-1994 (98.3 %) mostró una coincidencia de 56 ocasiones con una desviación negativa. La comparación de rtPCR-IMS y NOM-114-SSA1-1994 en carne de res reportó una exactitud de 98.3 %, sensibilidad de 98.2 %, especificidad de 100 % y selectividad de 100 %. El límite de detección para ambos métodos fue 1-5 UFC·25 g-1 de carne. El análisis estadístico indica que el rtPCR-IMS es equivalente al método de referencia para la detección de Salmonella en carne de res. Estos resultados advierten la alta incidencia de Salmonella en carne de res y proponen al rtPCR-IMS como un método idóneo y rápido para el control de Salmonella en la industria cárnica

Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7

Background Shiga toxin-producing Escherichia coli (STEC) is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. Methods In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. Results Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell) and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. Conclusion These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use

Listeriosis in Mexico: Clinical and epidemiological importance

Listeriosis is caused by Listeria monocytogenes, an important food-borne disease due to its clinical forms, high mortality rate, and the economic impact in both clinical and food production industries. In Mexico, the lack of epidemiological surveillance systems leads to the need of accurate data on the incidence of listeriosis and its association with food-borne disease. In this paper, we present data about the presence of this bacterium in food, reports related to clinical cases of listeriosis, and information of diseases in which L. monocytogenes may be involved. However, in most of these cases the etiology was not established. Given this, there´s a need to inform and warn the appropriate entities, to define strategies for the mandatory search of L. monocytogenes through the whole food production chain and clinical suspects, for the epidemiological importance and control of listeriosis in Mexico

Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli

Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant Escherichia coli strains. Transmission electron microscopy of phiLLS morphology revealed that it belongs to the Siphoviridae family. Furthermore, this phage exhibited a relatively large burst size of 176 plaque-forming units per infected cell. Phage phiLLS significantly reduced the growth of E. coli under laboratory conditions. Analyses of restriction profiles showed the presence of submolar fragments, confirming that phiLLS is a pac-type phage. Phylogenetic analysis based on the amino acid sequence of large terminase subunits confirmed that this phage uses a headful packaging strategy to package their genome. Genomic sequencing and bioinformatic analysis showed that phiLLS is a novel bacteriophage that is most closely related to T5-like phages. In silico analysis indicated that the phiLLS genome consists of 107,263 bp (39.0 % GC content) encoding 160 putative ORFs, 16 tRNAs, several potential promoters and transcriptional terminators. Genome analysis suggests that the phage phiLLS is strictly lytic without carrying genes associated with virulence factors and/or potential immunoreactive allergen proteins. The bacteriophage isolated in this study has shown promising results in the biocontrol of bacterial growth under in vitro conditions, suggesting that it may prove useful as an alternative agent for the control of foodborne pathogens. However, further oral toxicity testing is needed to ensure the safety of phage use

Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and <i>Fol</i> Molecular Markers: Analytical Performance Evaluation

Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance

Agronomic Behavior of Mexican Roselle Cultivars Produced under Protected Agricultural Conditions

Growth dynamics and morphological traits are used to define the characteristics of roselle cultivars Hibiscus sabdariffa. The morpho–agronomic variability of plants was evaluated, as well as the concentration of macro and micronutrients in leaves of Mexican roselle cultivars in hydroponic and greenhouse conditions. The following roselle cultivars were studied: ‘Cruza Negra’, ‘UAN16-2′, ‘Criolla Huajicori’, ‘UAN 6 Puga’, ‘UAN 25-1′ and ‘4Q4′. The relative growth rate of the crops was fitted to a positive cubic, negative quadratic and positive linear model, whereas stem diameter, fitted to a linear model, had a negative quadratic response. The foliar surface of the cultivars ‘4Q4’, ‘Cruza Negra’, ‘UAN16-2’ and ‘Criolla Huajicori’ was directly related to the size of the flower, the calyx and the corolla. Nitrogen and potassium are the elements that showed the highest concentration in the cultivars Criolla Huajicori and Cruza Negra, while in the other four cultivars the highest concentrations of minerals in the leaf were nitrogen and calcium. The leaves of cultivar 4Q4 provided the greatest amount of minerals, with 83,565 mg kg−1 dry basis. Criolla Huajicori was the cultivar that exhibited the tallest height, the most productive branches and the greatest calyx number per plant; however, Cruza Negra exhibited the highest yield by having the longest calyx