Dataset Information

Species identification and molecular typing of human Brucella isolates from Kuwait.

ABSTRACT: Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

SUBMITTER: Mustafa AS

PROVIDER: S-EPMC5553756 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Species identification and molecular typing of human Brucella isolates from Kuwait.

Mustafa Abu S AS Habibi Nazima N Osman Amr A Shaheed Faraz F Khan Mohd W MW

PloS one 20170811 8

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this stu ...[more]

PMID: 28800594

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Project description:Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.

Project description:BACKGROUND: Within the genus Aspergillus, A. flavus is the second most important species of clinical significance. It is predominantly associated with infections involving sinuses, eye and skin, mostly in geographic regions with hot and arid climate, including the Middle East. Recent reports on emergence of resistance to triazoles among Aspergillus spp. is a cause of concern for treatment of patients with invasive aspergillosis. In this study we present data on genetic characterization and antifungal susceptibility profile of clinical and environmental isolates of A. flavus. METHODS: Ninety-nine Aspergillus section Flavi isolates, originating from clinical (n=92) and environmental (n=7) sources, initially identified by morphological characteristics, were analyzed by partial sequencing of ?-tubulin and calmodulin gene fragments and their susceptibilities to six antifungal agents was determined by Etest on RPMI1640 and Muller-Hinton agar media. Etest minimum inhibitory concentrations (MICs) of amphotericin B and voriconazole were also compared with zone of inhibition diameters obtained by disc diffusion test on RPMI agar medium. RESULTS: The identity of all clinical and environmental isolates was confirmed as A. flavus species by combined analysis of ?-tubulin and calmodulin genes. The mean MIC90 (?g/ml) values on RPMI medium for amphotericin B, voriconazole, posaconazole, anidulafungin, micafungin and caspofungin were 3, 0.25, 0.25, 0.002, 0.002 and 0.032, respectively. No environmental isolate exhibited MIC value of >2 ?g/ml for amphotericin B. For clinical isolates, the zone of inhibition diameters for amphotericin B and voriconazole ranged from 7-16 mm and 24-34 mm, respectively. Linear regression analysis between Etest MIC values and disk diffusion diameters revealed a significant inverse correlation with amphotericin B (p <0.001) and voriconazole (p<0.003). CONCLUSIONS: The ?-tubulin and calmodulin gene sequences confirmed that all 92 clinical isolates identified phenotypically belonged to A. flavus taxon, thus suggesting that the other species within Aspergillus section Flavi are of little clinical significance. Triazoles and echinocandins showed very good in vitro activity against the A. flavus, however, 10% clinical isolates showed MICs of >2 ?g/ml for amphotericin B.

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Project description:Brucellosis, caused by several species of the genus Brucella, is a zoonotic disease that affects humans and animal species worldwide. Information on the Brucella species circulating in different hosts in Kenya is largely unknown, thus limiting the adoption of targeted control strategies. This study was conducted in multi-host livestock populations in Kenya to detect the circulating Brucella species and assess evidence of host-pathogen associations. Serum samples were collected from 228 cattle, 162 goats, 158 sheep, 49 camels, and 257 humans from Narok and Marsabit counties in Kenya. Information on age, location and history of abortion or retained placenta were obtained for sampled livestock. Data on age, gender and location of residence were also collected for human participants. All samples were tested using genus level real-time PCR assays with primers specific for IS711 and bcsp31 targets for the detection of Brucella. All genus positive samples (positive for both targets) were further tested with a speciation assay for AlkB and BMEI1162 targets, specific for B. abortus and B. melitensis, respectively. Samples with adequate quantities aggregating to 577 were also tested with the Rose Bengal Test (RBT). A total of 199 (33.3%) livestock and 99 (38.5%) human samples tested positive for genus Brucella. Animal Brucella PCR positive status was positively predicted by RBT positive results (OR = 8.3, 95% CI 4.0-17.1). Humans aged 21-40 years had higher odds (OR = 2.8, 95% CI 1.2-6.6) of being Brucella PCR positive compared to the other age categories. The data on detection of different Brucella species indicates that B. abortus was detected more often in cattle (OR = 2.3, 95% CI 1.1-4.6) and camels (OR = 2.9, 95% CI 1.3-6.3), while B. melitensis was detected more in sheep (OR = 3.6, 95% CI 2.0-6.7) and goats (OR = 1.7, 95% CI 1.0-3.1). Both B. abortus and B. melitensis DNA were detected in humans and in multiple livestock host species, suggesting cross-transmission of these species among the different hosts. The detection of these two zoonotic Brucella species in humans further underpins the importance of One Health prevention strategies that target multiple host species, especially in the multi-host livestock populations.

Dataset Information

Species identification and molecular typing of human Brucella isolates from Kuwait.

Publications

Species identification and molecular typing of human Brucella isolates from Kuwait.

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