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1.
Int J Food Microbiol ; 343: 109091, 2021 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-33639477

RESUMEN

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.


Asunto(s)
Farmacorresistencia Bacteriana/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética , Factores de Virulencia/genética , Adhesinas Bacterianas/genética , Animales , Antibacterianos/farmacología , Brasil , Heces/microbiología , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Genoma Bacteriano/genética , Islas Genómicas/genética , Humanos , Filogenia , Plásmidos/genética , Aves de Corral/microbiología , Enfermedades de las Aves de Corral/microbiología , Salmonella enterica/aislamiento & purificación , Salmonella enterica/patogenicidad , Serotipificación , Virulencia/genética , Secuenciación Completa del Genoma
2.
Nat Commun ; 12(1): 1102, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597521

RESUMEN

The four-dengue virus (DENV) serotypes infect several hundred million people annually. For the greatest safety and efficacy, tetravalent DENV vaccines are designed to stimulate balanced protective immunity to all four serotypes. However, this has been difficult to achieve. Clinical trials with a leading vaccine demonstrated that unbalanced replication and immunodominance of one vaccine component over others can lead to low efficacy and vaccine enhanced severe disease. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a live attenuated tetravalent DENV vaccine (TV003), which is currently being tested in phase 3 clinical trials. Here we report, our study to determine if TV003 stimulate balanced and serotype-specific (TS) neutralizing antibody (nAb) responses to each serotype. Serum samples from twenty-one dengue-naive individuals participated under study protocol CIR287 (ClinicalTrials.gov NCT02021968) are analyzed 6 months after vaccination. Most subjects (76%) develop TS nAbs to 3 or 4 DENV serotypes, indicating immunity is induced by each vaccine component. Vaccine-induced TS nAbs map to epitopes known to be targets of nAbs in people infected with wild type DENVs. Following challenge with a partially attenuated strain of DENV2, all 21 subjects are protected from the efficacy endpoints. However, some vaccinated individuals develop post challenge nAb boost, while others mount post-challenge antibody responses that are consistent with sterilizing immunity. TV003 vaccine induced DENV2 TS nAbs are associated with sterilizing immunity. Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Dengue/prevención & control , Dengue/virología , Vacunas contra el Dengue/administración & dosificación , Virus del Dengue/clasificación , Epítopos/inmunología , Humanos , Serotipificación , Especificidad de la Especie , Resultado del Tratamiento , Vacunación/métodos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología
3.
Int J Food Microbiol ; 337: 108941, 2021 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-33181420

RESUMEN

Whole genome sequencing (WGS) has made impressive progress in the field of molecular biology. Its most common application for public health is in the area of surveillance of food-borne diseases. WGS has the potential for providing a large amount of information, such as the identification of the strain type, the characterization of antibiotic resistance and virulence, and phylogeny. In our study, thirty-nine non-typhoidal Salmonella strains were isolated from diverse sources in Tunisia. Non-typhoidal Salmonella are among the most common pathogens contaminating food animals. The presence of virulence and antimicrobial resistance determinants in those strains were investigated using whole genome sequencing (WGS) and appropriate data analysis. The genomes were screened for several Salmonella virulence genes using the Virulence Factor Database VFDB. Twelve different virulence profiles, which correspond to the 12 identified serovars, were recognized. Several antimicrobial resistance genes were also detected: aac (6')-Iaa, sul1, tetA, bla-TEM and qnrS genes. Phylogenetic relationships among the strains were further assessed by a cgMLST analysis. The resulting phylogenetic tree consisted of several clusters consistently with the in silico multilocus sequence typing (MLST) and serotyping. Our findings demonstrated that WGS and subsequent data analysis provided an accurate tool for genetic characterization of bacterial strains compared to usual molecular typing techniques. To the best of our knowledge, this is the first report of an application of WGS for genetic characterization of food-borne Tunisian strains.


Asunto(s)
Genoma Bacteriano/genética , Filogenia , Salmonella , Virulencia/genética , Animales , Farmacorresistencia Bacteriana/genética , Tipificación de Secuencias Multilocus/métodos , Salmonella/clasificación , Salmonella/genética , Salmonella/patogenicidad , Serogrupo , Serotipificación , Túnez , Secuenciación Completa del Genoma
4.
Methods Mol Biol ; 2220: 57-78, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975766

RESUMEN

For assessing isolates of Listeria monocytogenes, serotype designation is the first subtyping method used. Methodologies used to assign serotype are currently evolving and will eventually be replaced with whole genome sequencing. Traditionally, serotyping has been done with agglutination reactions; however, alternative methods utilizing enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are common. Described here are the three non-genomic methods and the advantages and disadvantages of each.


Asunto(s)
Pruebas de Aglutinación/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Serotipificación/métodos , Humanos , Listeria monocytogenes/clasificación , Listeriosis/diagnóstico
5.
Int J Food Microbiol ; 339: 109023, 2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33341686

RESUMEN

The aim of the present study is to investigate the prevalence and genetic diversity of Listeria monocytogenes in various fresh and frozen vegetable products available in Poland. The samples were collected at retail market within the framework of national official control and monitoring program. In the years 2016-2019 a total of 49 samples out of 8712 collected vegetable samples were positive for L. monocytogenes. Our findings demonstrated that the occurrence of L. monocytogenes in various vegetable products was generally low, on average only 0.56% in the studied years. All isolates were susceptible to 11 antimicrobial agents: penicillin, ampicillin, meropenem, erythromycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, ciprofloxacin, chloramphenicol, gentamicin, vancomycin, and tetracycline. All of them harbored virulence-associated genes (inlA, inlC, and lmo2672), 82% harbored inlJ gene and few of them (22%) also possessed the llsX gene. The majority of collected isolates (65%) belonged to molecular serogroup 1/2a-3a, followed by 4ab-4b-4d-4e (33%), and only one to serogroup 1/2b-3b-7 (2%). Isolates yielded 18 different restriction profiles, revealing a large cluster of contamination linked to frozen corn (21 strains) and distributed in 3 pulsotypes. MLST analysis classified selected isolates into nine clonal complexes (CCs). The obtained results contribute to characterizing the diversity of L. monocytogenes isolated from various vegetable products in Poland and their impact on food safety and public health.


Asunto(s)
Microbiología de Alimentos , Variación Genética , Listeria monocytogenes/genética , Verduras/microbiología , Antibacterianos/farmacología , Microbiología de Alimentos/estadística & datos numéricos , Humanos , Incidencia , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Tipificación de Secuencias Multilocus , Polonia , Serogrupo , Serotipificación , Virulencia/genética
6.
Int J Food Microbiol ; 339: 109026, 2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33360877

RESUMEN

The abundant information provided by the pan-genome analysis approach reveals the diversity among Listeria monocytogenes serotypes. The objective of this study was to mine novel target genes using pan-genome analysis for multiplex PCR detection and differentiation of the major L. monocytogenes serotypes present in food. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: one for lineage I, two for serogroup I.1, one for serogroup I.2, two for lineage II, one for serogroup II.1, three for lineage III. Primers for the novel targets were highly specific in individual reactions. The detection limits were 103-104 colony-forming units (CFU)/mL in pure bacterial cultures, meeting the requirements of molecular detection. Based on these novel targets, two new "lineage" multiplex PCR assays were developed to simultaneously distinguish between three lineages (I, II, and III) and five major serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c) of L. monocytogenes. The detection limits of lineage I and lineage II&III mPCRs were 0.771 pg/µL and 1.76 pg/µL genomic DNA, respectively. The specificity of the mPCRs was robustly verified using other L. monocytogenes and non-L. monocytogenes serotypes. These results suggest that the two "lineage" multiplex PCRs based on novel targets offer a promising approach for accurate, sensitive, and rapid identification of L. monocytogenes serotypes.


Asunto(s)
Listeria monocytogenes/genética , Reacción en Cadena de la Polimerasa Multiplex , Serotipificación/métodos , Cartilla de ADN/genética , Microbiología de Alimentos , Genoma Bacteriano/genética , Listeriosis/microbiología , Serogrupo
7.
PLoS One ; 15(12): e0244713, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33382795

RESUMEN

The prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) was determined by evaluating its presence in faecal samples from 155 heifers, and 254 dairy cows in 21 farms at North of Portugal sampled between December 2017 and June 2019. The prevalence of STEC in heifers (45%) was significantly higher than in lactating cows (16%) (p<0.05, Fisher exact test statistic value is <0.00001). A total of 133 STEC were isolated, 24 (13.8%) carried Shiga-toxin 1 (stx1) genes, 69 (39.7%) carried Shiga-toxin 2 (stx2) genes, and 40 (23%) carried both stx1 and stx2. Intimin (eae) virulence gene was detected in 29 (21.8%) of the isolates. STEC isolates belonged to 72 different O:H serotypes, comprising 40 O serogroups and 23 H types. The most frequent serotypes were O29:H12 (15%) and O113:H21 (5.2%), found in a large number of farms. Two isolates belonged to the highly virulent serotypes associated with human disease O157:H7 and O26:H11. Many other bovine STEC serotypes founded in this work belonged to serotypes previously described as pathogenic to humans. Thus, this study highlights the need for control strategies that can reduce STEC prevalence at the farm level and, thus, prevent food and environmental contamination.


Asunto(s)
Adhesinas Bacterianas/genética , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Heces/microbiología , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Animales , Bovinos , Infecciones por Escherichia coli/microbiología , Femenino , Portugal , Serogrupo , Serotipificación , Escherichia coli Shiga-Toxigénica/genética , Virulencia
8.
PLoS One ; 15(12): e0244450, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33382792

RESUMEN

OBJECTIVES: To study Group B Streptococcus (GBS) isolates associated with different clinical syndromes: asymptomatic carriage in pregnant women, intrauterine fetal death (IUFD), and early onset disease (EOD) in the newborn. METHODS: GBS isolates were collected from asymptomatic pregnant women admitted for labor, IUFD cases, and neonates with EOD. Serotypes and antibiotic susceptibilities were determined. Multilocus sequence typing (MLST) was performed to assess genetic epidemiology. RESULTS: GBS carriage rate was 26.1% (280/1074). The dominant serotype among asymptomatic pregnant women was VI [98/240 women (40.8%)], followed by serotypes III, V and IV in 42/240 (17.5%), 30/240 (12.5%) and 28/240 (11.7%) women, respectively. The dominant serotype in IUFD cases was serotype VI [10/13 (76.9%)]. In contrast the prevalent serotype among EOD cases was III [16/19 (84.2%)]. ST-1 was associated with IUFD [7/13 (53.8%)], ST-17 was associated with serotype III and EOD in the newborn 14/19 (73.7%)]. Erythromycin and clindamycin resistance reached 36.8%, 7.7% and 20.0%among EOD, vaginal carriage and IUFD, respectively. CONCLUSIONS: Serotypes VI and ST-1 were dominant among asymptomatic pregnant women and in IUFD cases while EOD was associated with serotype III and ST-17. Invasive mechanisms thus may differ between IUFD and EOD in the newborn and virulence may be related to capsule serotype. Resistance rates to erythromycin and clindamycin were high in EOD cases.


Asunto(s)
Portador Sano/diagnóstico , Muerte Fetal , Sepsis Neonatal/diagnóstico , Complicaciones Infecciosas del Embarazo/diagnóstico , Infecciones Estreptocócicas/diagnóstico , Streptococcus agalactiae/genética , Adulto , Edad de Inicio , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Portador Sano/tratamiento farmacológico , Portador Sano/epidemiología , Portador Sano/microbiología , Clindamicina/farmacología , Clindamicina/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genética , Eritromicina/farmacología , Eritromicina/uso terapéutico , Femenino , Humanos , Recién Nacido , Tipificación de Secuencias Multilocus , Sepsis Neonatal/tratamiento farmacológico , Sepsis Neonatal/epidemiología , Sepsis Neonatal/microbiología , Polisacáridos Bacterianos/genética , Embarazo , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/epidemiología , Complicaciones Infecciosas del Embarazo/microbiología , Serogrupo , Serotipificación , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/aislamiento & purificación , Streptococcus agalactiae/patogenicidad , Virulencia/genética , Adulto Joven
9.
PLoS One ; 15(10): e0240143, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33007026

RESUMEN

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 54) and environmental samples (n = 38) in Huzhou were analyzed by serological typing, virulence gene detection, antibiotic resistance testing, and pulsed-field gel electrophoresis (PFGE) for molecular typing. O3:K6 was the main serotype and tlh+tdh+trh- was the most frequently detected virulence genotype in clinical strains. O2:Kut was the main serotype and tlh+tdh-trh- was the most frequently detected virulence genotype in environmental strains. Antibiotic resistance testing indicated that the isolates were highly resistant to ampicillin (90.76%), followed by gentamicin and tetracycline. Following the restriction enzyme NotI digestion, the 91 strains yielded 81 PFGE patterns, and 16 clones had similarity values of > 85.00%, indicating a high level of diversity. Finally, there may be cross-contamination between freshwater and seawater products, so it is necessary to strengthen supervision of food processing.


Asunto(s)
Microbiología Ambiental , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/aislamiento & purificación , China , Análisis por Conglomerados , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Electroforesis en Gel de Campo Pulsado , Genes Bacterianos , Humanos , Filogenia , Serotipificación , Vibrio parahaemolyticus/clasificación , Virulencia/genética
10.
PLoS One ; 15(9): e0239457, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32997676

RESUMEN

This study aimed to detect Salmonella from retail meat collected from nine wet markets in Metro Manila, and identify the subtypes of Salmonella isolates using molecular serotyping assays from previously developed primers. Of the 720 collected meat samples, 57.64% were found to be Salmonella-contaminated. The most predominant serogroup was Salmonella O:3, and Salmonella serogroups O:4, O:6,7, O:8, O:9, and undetermined serogroups were also found. Most frequently detected isolates in bovine meat were S. 3:e,h:1,6 (putative identity: S. Anatum) and S: 4:e,h:1,2 (putative identity: S. Saintpaul), in porcine meat was S. 3:e,h:1,6 (putative identity: S. Anatum), and S. 8:i:z6 (putative identity: S. Kentucky) was common in poultry products. This study also demonstrated retail meat samples were contaminated with multiple Salmonella serogroups and serovars. This is the first Philippine study that utilized PCR-based assays to characterize Salmonella isolates down to a serovar level and provides baseline information regarding Salmonella prevalence and serovar distribution in retail meat. Molecular serotyping performed in this study can be used as an alternative approach to traditional serotyping in surveillance of Salmonella in the Philippines since the latter is expensive, time-consuming, and requires skilled technicians.


Asunto(s)
Carne/microbiología , Reacción en Cadena de la Polimerasa , Salmonella/genética , Salmonella/aislamiento & purificación , Animales , Bovinos , Pollos/microbiología , Contaminación de Alimentos , Filipinas , Serogrupo , Serotipificación/métodos , Porcinos/microbiología
11.
J Med Microbiol ; 69(10): 1249-1252, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32924920

RESUMEN

Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a variety of animals. In humans, in contrast to the cutaneous form called erysipeloid, which is an occupational disease and common in individuals who handle raw meat and fish, invasive systemic infections are unusual. E. rhusiopathiae expresses an immunogenic surface protein, Spa (surface protective antigen), which is involved in virulence. Among the antigenically different Spa proteins (SpaA, B and C), which are mostly associated with serovars, SpaA is by far the most prevalent in E. rhusiopathiae isolates from diseased animals. However, the Spa type has not been examined for human isolates, and it is unknown whether SpaB- or SpaC-possessing isolates can cause disease in humans. A Gram-positive, rod-shaped bacterium isolated from a case of human pyogenic spondylitis was analysed. The bacterium was identified as E. rhusiopathiae by a routine biochemical test and MS, and ultimately confirmed by an E. rhusiopathiae-specific PCR assay. Spa typing by sequencing revealed the SpaB type, and the serovar of the strain was identified as untypeable by a conventional agar gel precipitation test, but determined to be serovar 6 by a serotyping PCR assay. Sequence analysis of the serovar-defining chromosomal region revealed that the isolate displayed the same gene organization as the serovar 6 reference strain, but the region was disrupted by an insertion sequence element, suggesting that the isolate originated from a serovar 6 strain. These results highlight that unusual, spaB-possessing E. rhusiopathiae strains can potentially pose serious risks to humans.


Asunto(s)
Antígenos de Superficie/inmunología , Infecciones por Erysipelothrix/microbiología , Erysipelothrix/genética , Anciano de 80 o más Años , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos de Superficie/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Erysipelothrix/metabolismo , Erysipelothrix/patogenicidad , Femenino , Humanos , Proteínas de la Membrana/genética , Reacción en Cadena de la Polimerasa/métodos , Serogrupo , Serotipificación , Virulencia
12.
Int J Food Microbiol ; 335: 108857, 2020 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-32947144

RESUMEN

Cronobacter species (Cronobacter spp.) are important foodborne pathogens that can infect and cause serious life-threatening diseases in infants and immunocompromised elderly. This study aimed to acquire data on Cronobacter spp. contamination of aquatic products in China from 2011 to 2016. In total, 800 aquatic products were tested, and the overall contamination rate for Cronobacter spp. was 3.9% (31/800). The average contamination level of the positive samples was 2.05 MPN/g. Four species and nine serotypes were identified among 33 isolates, of which the C. sakazakii serogroup O1 (n = 9) was the primary serotype. The majority of Cronobacter spp. strains harbored highest resistance against cephalothin (84.8%), followed by tetracycline (6.1%), trimethoprim/sulfameth-oxazole (3.0%) and chloramphenicol (3.0%). Two isolates were resistant to three antibiotics. In total, 26 sequence types and 33 CRISPR types (including 6 new STs and 26 new CTs) were identified, which indicates the extremely high diversity of Cronobacter spp. in aquatic products. Pathogenic C. sakazakii ST4, ST1, and C. malonaticus ST7 were also observed. Overall, this large-scale study revealed the relatively low prevalence and high genetic diversity of Cronobacter spp. in aquatic products in China, and the findings provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp. in aquatic products during production processes.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cronobacter/clasificación , Cronobacter/aislamiento & purificación , Farmacorresistencia Bacteriana , Alimentos Marinos/microbiología , Antibacterianos/farmacología , Cefalotina/farmacología , China , Cloranfenicol/farmacología , Cronobacter sakazakii/clasificación , Cronobacter sakazakii/aislamiento & purificación , Contaminación de Alimentos , Microbiología de Alimentos , Variación Genética , Tipificación de Secuencias Multilocus , Prevalencia , Serotipificación , Tetraciclina/farmacología , Trimetoprim/farmacología
13.
Int J Food Microbiol ; 335: 108885, 2020 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-32947145

RESUMEN

Worldwide, Listeria monocytogenes is a common foodborne pathogen and serotyping is necessary for traceability and surveillance. Due to high accuracy and speed, PCR is commonly used for serotyping by targeting specific genes, such as lmo1118 and ORF2110 for 1/2c-3c and 4b-4d-4e, respectively. Single nucleotide polymorphisms (SNPs) are single base alterations at specific loci which are associated with various phenomena. In this study, a method was explored to mine specific SNPs from 253 L. monocytogenes genomes with known serotypes by writing Python programming language script. After blasting all the CDS, seventeen candidate genes with specific SNPs were obtained and these genes contained multiple types of SNPs, including lineages I, II, III, 1/2a-3a, 1/2b-3b-7, and 1/2c-3c specific SNPs. The sensitivity and specificity of these candidate SNP sites are higher than 85% in the genomes examined. Combining lineage I-specific, lineage II-specific, 1/2b-3b-7-specific, and 1/2c-3c-specific SNP sites together, using allele-specific multiplex PCR, we could serotype major L. monocytogenes serotypes. This method was used for 60 L. monocytogenes strains isolated from various food samples and the serotyping results were 100% identical to those of the currently accepted method with the reduced primers from ten to eight. Our results indicate that allele-specific multiplex PCR after mining specific SNPs from common genome database can be used for bacterial typing.


Asunto(s)
Microbiología de Alimentos/métodos , Listeria monocytogenes/aislamiento & purificación , Listeriosis/diagnóstico , Polimorfismo de Nucleótido Simple/genética , Serotipificación/métodos , Alelos , Humanos , Listeria monocytogenes/genética , Reacción en Cadena de la Polimerasa Multiplex , Sensibilidad y Especificidad , Serogrupo
14.
PLoS Negl Trop Dis ; 14(8): e0008573, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32841248

RESUMEN

BACKGROUND: Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. CONCLUSIONS/SIGNIFICANCE: This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.


Asunto(s)
Genotipo , Leptospira/clasificación , Leptospira/genética , Leptospira/aislamiento & purificación , Leptospirosis/microbiología , Tipificación de Secuencias Multilocus/métodos , Adolescente , Adulto , Anciano , Pruebas de Aglutinación , Animales , Niño , ADN Bacteriano/análisis , Reservorios de Enfermedades , Femenino , Humanos , Leptospirosis/epidemiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Análisis de Secuencia de ADN , Serogrupo , Serotipificación , Sri Lanka/epidemiología , Adulto Joven
15.
J Food Sci ; 85(9): 2889-2895, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32794185

RESUMEN

This study was aimed to investigate the presence of Listeria monocytogenes in raw water buffalo milk and milk products, besides determining its serotype and the extent of its resistance against various antibiotics. A total of 188 samples of raw water buffalo milk and milk products were collected from Samsun Province, Turkey between November 2012 and May 2013. The classical culture technique was used to isolate and identify L. monocytogenes, as described in EN ISO 11290-1. The isolates were confirmed as L. monocytogenes by using PCR with (hylA) primers specific for the hemolysin gene. The antimicrobial susceptibility test was achieved by using the VITEK 2 compact system and VITEK 2 AST-P640 card. L. monocytogenes was found in 7 (3.7%) of the 188 samples. Four of them were obtained from cheese and three from milk samples. Whereas, L. monocytogenes was not detected in any of the clotted cream samples. A total of 13 isolates were confirmed by PCR as L. monocytogenes. Among these isolates, one was 1/2c (or 3c) (7.6%), three were 4b (or 4d, 4e) (23%), four were 1/2b (or 3b) (30.7%), and the other five isolates were serotype 1/2a (or 3a) (38.4%). The highest antimicrobial resistance was recorded against fosfomycine (100%) followed by oxacillin (92%), penicillin (84%), and erythromycin (69%). However, no resistance was determined against ciprofloxacin, gentamicin, and tigecycline. PRACTICAL APPLICATION: This study showed that some samples of raw buffalo milk and the milk products were contaminated with Listeria monocytogenes. The serotype with the highest prevalence was determined as L. monocytogenes 1/2a. This study also demonstrated that most of the L. monocytogenes isolates had developed multiresistance to many frequently used medical antimicrobial agents.


Asunto(s)
Queso/microbiología , Farmacorresistencia Bacteriana , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/aislamiento & purificación , Leche/microbiología , Animales , Antibacterianos/farmacología , Búfalos , Cartilla de ADN/genética , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Listeria monocytogenes/clasificación , Listeria monocytogenes/genética , Reacción en Cadena de la Polimerasa , Serotipificación , Turquia
16.
PLoS Negl Trop Dis ; 14(8): e0008535, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32813703

RESUMEN

Dengue fever occurs worldwide and about 1% of cases progress to severe haemorrhage and shock. Dengue is endemic in Guatemala and its surveillance system could document long term trends. We analysed 17 years of country-wide dengue surveillance data in Guatemala to describe epidemiological trends from 2000 to 2016.Data from the national dengue surveillance database were analysed to describe dengue serotype frequency, seasonality, and outbreaks. We used Poisson regression models to compare the number of cases each year with subsequent years and to estimate incidence ratios within serotype adjusted by age and gender. 91,554 samples were tested. Dengue was confirmed by RT-qPCR, culture or NS1-ELISA in 7097 (7.8%) cases and was IgM ELISA-positive in 19,290 (21.1%) cases. DENV1, DENV2, DENV3, and DENV4 were detected in 2218 (39.5%), 2580 (45.9%), 591 (10.5%), and 230 (4.1%) cases. DENV1 and DENV2 were the predominant serotypes, but all serotypes caused epidemics. The largest outbreak occurred in 2010 with 1080 DENV2 cases reported. The incidence was higher among adults during epidemic years, with significant increases in 2005, 2007, and 2013 DENV1 outbreaks, the 2010 DENV2 and 2003 DENV3 outbreaks. Adults had a lower incidence immediately after epidemics, which is likely linked to increased immunity.


Asunto(s)
Dengue/diagnóstico , Dengue/epidemiología , Adolescente , Adulto , Anticuerpos Antivirales/inmunología , Niño , Preescolar , Dengue/inmunología , Virus del Dengue/inmunología , Brotes de Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Guatemala/epidemiología , Humanos , Incidencia , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Serogrupo , Serotipificación/métodos , Adulto Joven
17.
PLoS One ; 15(8): e0237247, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32790696

RESUMEN

Streptococcus pneumoniae is one of the leading causes of death worldwide. It disseminates through colonizers and causes serious infections. Aims of this study are to determine pneumococcal carriage rate, resistance, serotype distribution, and coverage of pneumococcal conjugate vaccines from children attending day care centers from Irbid and Madaba in Jordan. Nasopharyngeal swabs were collected from day care centers (DCCs) of healthy Jordanian children 2-4 years of age from four regions of Madaba (n = 596), and from eastern Irbid (n = 423). Swabs were cultivated on Columbia blood agar base supplemented with 5% sheep blood and incubated for 18-24 hours at 37°C with 5% CO2. Alpha-hemolytic isolates were tested for optochin sensitivity and bile solubility for identification. Isolates were analyzed for antimicrobial susceptibility by the Vitek2 system and E-test (BioMérieux). Serotyping was performed using the Neufeld Quellung method. A total of 341 pneumococcal strains were isolated from 1019 nasopharyngeal (NP) samples of healthy children attending DCCs for two winter seasons from 2017-2019. Carriage rate in eastern Irbid for both seasons was 29.6% and for Madaba 37.9%. Resistance rates for Irbid and Madaba, respectively, were as follows: Penicillin (86.3%; 94.4%), erythromycin (57.0%; 78.2%), clindamycin (30.8%; 47.2%), trimethoprim-sulfamethoxazole (68.6%; 86.6%), and tetracycline (45.7%; 51.9%). Predominant serotypes for Irbid were 19F (20.8%), 23F (12.0%), 6A (10.4%), and 6B (9.6%); whereas for Madaba were 19F (24.5%), 14 (7.4%), 6A (6.9%) and 23F (6.5%). Serotype coverage of the thirteen valent pneumococcal conjugate vaccine (PCV13) was about 65% for both regions. Over 96% of isolates with PCV13 serotypes in this study were resistant to penicillin with the exception of serotypes 3 and 5. As a conclusion resistance and carriage rates among the age group 2 to 4 years reached an alarming rate especially among vaccine types, which can be controlled by pneumococcal conjugate vaccination strategies.


Asunto(s)
Infecciones Neumocócicas/diagnóstico , Streptococcus pneumoniae/aislamiento & purificación , Animales , Antibacterianos/uso terapéutico , Preescolar , Farmacorresistencia Bacteriana , Femenino , Humanos , Jordania/epidemiología , Masculino , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/epidemiología , Estaciones del Año , Serotipificación , Ovinos , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/efectos de los fármacos
18.
PLoS One ; 15(8): e0237384, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32764809

RESUMEN

Dengue fever is one of the major public health problems in Lao PDR. Over the last decade, dengue virus (DENV) epidemics were characterized by a novel predominant serotype accompanied by at least two other serotypes. Since 2008, DENV-2 circulated at a low level in Lao PDR but its epidemiologic profile changed at the end of 2018. Indeed, the number of confirmed DENV-2 cases suddenly increased in October 2018 and DENV-2 became predominant at the country level in early 2019. We developed a Genotype Screening Protocol (GSP) to determine the origin(s) of the Lao DENV-2 and study their genetic polymorphism. With a good correlation with full envelope gene sequencing data, this molecular epidemiology tool evidence the co-circulation of two highly polymorphic DENV-2 genotypes, i.e. Asian I and Cosmopolitan genotypes, over the last five years, suggesting multiple introductions of DENV-2 in the country. GSP approach provides relevant first line information that may help countries with limited laboratory resources to reinforce their capabilities to DENV-2 and to follow the epidemics progresses and assess situations at the regional level.


Asunto(s)
Virus del Dengue/genética , Técnicas de Genotipaje/métodos , Encuestas y Cuestionarios , Virus del Dengue/aislamiento & purificación , Virus del Dengue/fisiología , Humanos , Laos , Serotipificación , Factores de Tiempo , Población Urbana/estadística & datos numéricos
19.
PLoS One ; 15(7): e0234475, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32663215

RESUMEN

BACKGROUND: Neisseria meningitidis is a significant cause of morbidity and mortality worldwide. Meningococcal isolates have a highly dynamic population structure and can be phenotypically and genetically differentiated into serogroups and clonal complexes. The aim of this study was to describe the phenotypic and genotypic characteristics of invasive isolates recovered in Colombia from 2013 to 2016. METHODOLOGY: A total of 193 invasive isolates were analyzed. Phenotypic and genotypic characteristics were determined by serotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. RESULTS: Based on the results, meningococcal serogroups C, B and Y were responsible for 47.9%, 41.7%, and 9.4% of cases, respectively, and the distribution of serogroups B and C changed over time. Fifteen clonal groups and 14 clonal complexes (cc) were identified by PFGE and genome sequencing. The main clonal group included serogroup B isolates with sequence type (ST)-9493 and its four single-locus variants, which has only been identified in Colombian isolates. The clonal population structure demonstrates that the isolates in this study mainly belong to four clonal complexes: ST-11 cc, ST-32 cc, ST-35 cc and ST-41/44 cc. Thirty-eight penA alleles were identified, but no correlation between MICs and specific sequences was observed. CONCLUSION: This study shows that most meningococcal isolates recovered from patients with invasive meningococcal disease in Colombia are strains associated with distinct globally disseminated hyperinvasive clones.


Asunto(s)
Infecciones Meningocócicas/epidemiología , Infecciones Meningocócicas/genética , Neisseria meningitidis/genética , Adolescente , Adulto , Técnicas de Tipificación Bacteriana/métodos , Niño , Preescolar , Colombia/epidemiología , Electroforesis en Gel de Campo Pulsado/métodos , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Neisseria meningitidis/aislamiento & purificación , Neisseria meningitidis/patogenicidad , Serogrupo , Serotipificación
20.
BMC Infect Dis ; 20(1): 472, 2020 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-32616018

RESUMEN

BACKGROUND: Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. METHODS: Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. RESULTS: The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antimicrobial resistant plasmids and a virulent plasmid. The antimicrobial resistant plasmid expressing blaKPC-2, blaCTX-M-15, aph(3″)-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. CONCLUSION: Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/patogenicidad , Brotes de Enfermedades , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Carbapenémicos/uso terapéutico , China/epidemiología , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Infecciones por Klebsiella/sangre , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/aislamiento & purificación , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Tipificación de Secuencias Multilocus , Plásmidos/genética , Serogrupo , Serotipificación , Virulencia/genética , Secuenciación Completa del Genoma , Adulto Joven
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