Greater Genotypic Diversity Research Articles

Genomic characterization of MRSA recovered from people with cystic fibrosis during two Spanish multicentre studies (2013 and 2021).

Chronic bronchopulmonary infection due to MRSA in people with cystic fibrosis (pwCF) has been associated with accelerated decline in lung function, increased hospitalizations and increased mortality. We studied microbiological and genomic characteristics of MRSA isolates recovered from pwCF in two Spanish multicentre studies (2013, 2021). Antimicrobial susceptibility was performed. WGS was carried out to determine population structure [MLST, spa-typing, staphylococcal cassette chromosome mec (SCCmec)], resistome and virulome. Clinical charts of MRSA-infected and MRSA-non-infected pwCF were also reviewed. MRSA infection prevalence decreased between 2013 (29/341, 8.5%) and 2021 (21/326, 6.4%) (P = 0.378). Differences in lung function were observed between infected and non-infected patients (P < 0.005). A higher prevalence of hospital-acquired (HA) clones was found compared with community-acquired (CA) clones (2013: 67% versus 33%; and 2021: 71% versus 29%). Overall, we noted clustering of isolates based on year of sampling, type of acquisition and clonal complex (CC). HA-MRSA population was dominated by CC5, with ST125-MRSA-IVc-t067 the most prevalent lineage (37%). A higher clonal diversity was detected among CA-MRSA. One Panton-Valentine leucocidin (PVL)-positive strain (ST8-MRSA-IV) and three strains of porcine origin (two ST398-MRSA-V-t011, one ST398-MRSA-V-t8567) were found. Additionally, acquired resistance genes (n = 24) were detected, including the cfr gene conferring linezolid resistance. A higher gentamicin resistance was found in 2021 (42%) compared with 2013 (7%) (P = 0.046), associated with the aac(6')-aph(2″) gene. Despite a decrease in MRSA prevalence, we showed its potential impact on CF severity and progression. Moreover, we observed great genotypic and phenotypic diversity in MRSA isolates from pwCF as well as an MDR trait.

Variation in the Photosynthetic Leaf Pigments of Different Basil (Ocimum spp.) Genotypes under Varying Conditions at the Flowering Stage

Basil is a culinary herb in high demand all year round, either fresh, dried, or frozen. Field basil crops are commercially predominant, while greenhouse crops can cover the demand for fresh basil during the off-season. The leaf aspect of basil has great importance for the food industry, and a great diversity of genotypes has been created. The aim of this research was to conduct a comparative characterization of 12 basil genotypes (green and purple leaf) that present interest for breeding programs from a national germplasm collection. The study examines the accumulation of photosynthetic pigments in basil cultivated in field versus greenhouse conditions as indicators of plant performance and herb quality from the perspective of ensuring fresh plant material all year round. The experimental median indicated that photosynthetic leaf pigments accumulated in higher concentrations in the field relative to the greenhouse conditions, in descending order: β-carotene, lutein, chlorophyll a, and chlorophyll b. The trend was not as consistent for chlorophyll b, since four out of twelve genotypes presented higher levels in the greenhouse than in the field, while the overall values were not much lower in the greenhouse than the field (16.82%). All genotypes accumulated much higher carotenoid contents in field conditions relative to greenhouse conditions (&gt;200%) and could also provide better nutritional advantages given their demonstrated health benefits. The differences in photosynthetic leaf pigments have both nutritional (the carotenoids), shelf-life, and processing relevance (chlorophylls) and serve as quality markers.

Diversity and genetic structure of yellow passion fruit in Boyacá-Colombia using microsatellite DNA markers.

The Yellow passion fruit belongs to the Passifloraceae family with great economic, nutritional and social importance in Colombia. It presents a great phenotypic and genotypic diversity, which has not yet been explored or used in genetic improvement programs. The objective of this study was to evaluate the genetic diversity of 84 cultivars of Passiflora edulis f. flavicarpa from nine farms in the municipality of Miraflores, Boyacá, using eight microsatellite markers (SSR). On the basis of this information, estimates of genetic diversity parameters, molecular variance analysis (AMOVA), genetic distances, and cluster of cultivars were obtained. Low levels of genetic differentiation between cultivars were observed in the Bayesian analysis using Structure software, as well as the absence of correlation between genetic and geographic distances. The observed heterozygosity (0.50) was greater than the expected heterozygosity (0.43), suggesting a significant number of heterozygous individuals. The number of alleles per locus varied from 2 to 4, with a mean 2.88. In general, SSR were classified as informative (0.36). The average value of the Shannon Index was 0.71, which shows moderate variability in this cultivar. AMOVA showed higher diversity within cultivars (98%). The gene flow (Nm=28.4) was moderate, this can be explained by the flow of pollen between the different cultivars, the reproduction system of the species, self-incompatibility and the introduction of genotypes from other sites by farmers. The genetic diversity identified in this study is sufficient to initiate breeding programs aimed at identifying cultivars with higher yields.

First study of genital HPV infection among women living with HIV recruited from May to September 2018 in Eastern Algeria.

Genital HPV infection is recognized worldwide as one of the most common, if not the most common, sexually transmitted infection. Several studies have shown a higher prevalence of HPV infection in women living with HIV (WLWH). We aimed to determine the prevalence of HPV, the circulating genotypes, and its association with risk factors among Algerian WLHIV. Cervical specimens were collected from 100 WLHIV. The detection of HPV infection was performed by Roche Linear Array test. The overall prevalence of HPV infection was determined at 32%, all including high risk HPV (HR-HPV). We found a great diversity of HR-HPV genotypes, the most common individually were HPV52, HPV16, HPV18 and HPV58, The highest prevalence was found in groups of genotype 52, 25%. HPV16 and 18 were less common (16%). Cervical cytology was abnormal in 66% (81.3% in the HPV + population), with a predominance of inflammatory lesions (75% HPV +), The most important determining risk factor for HPV infection in this series was a low CD4 T cell count <200/mm3 found in 72% of the HPV positive participants. Our study is an initial database, to be completed by a multicenter study to determine the most frequent genotypes in Algerian WLHIV, in order to discuss the introduction of the vaccine against HPV in Algeria, especially among WLHIV.

Characterization of yellow pitahaya (Selenicereus megalanthus Haw.) genotypes under two productive systems in Colombia.

Selenicereus megalanthus Haw. It is an exotic fruit tree, with productive and nutritional potential. In Colombia, there is a great phenotypic and genotypic diversity, but its genetic studies are scarce. The objective was to characterize morphologically 15 selected yellow pitahaya genotypes, under two productive systems in the open field and under cover, in the municipalities of Miraflores and Zetaquira, in Boyacá, Colombia. Quantitative characters were evaluated: plant height (PH), number of vegetative sprouts (NVS), sub-sprouts (SS), longest sprouts length (LSL), distance between areoles (DBA), width of the ribs in the apical region (WRA), width of the ribs in the middle region (WRM), width of the ribs in the basal region (WRB), height of undulations between successive areoles in a rib (HUA), number of spines per areole (NSA) and longest spine length (LSP). The results showed under the two productive systems and the evaluated localities that the variables with the highest coefficient of variation (greater than 90%) were the number of sub-sprouts, height of the undulations between successive areoles (HUA) and the longest spine length (LSP). High positive correlations were obtained between the distances areoles, the width of the ribs and the length of the spines (r>0.7). The conglomerate showed that the characteristics that define the groupings are height of the plant, the texture of the cladodes, the width of the ribs and the height of the undulations. Characters associated with the shoots and cladodes were identified, which directly influence the vegetative propagation and therefore the yield of the yellow pitahaya.

GENETIC DIVERSITY OF Lutzomyia (Nyssomyia) intermedia IN AN ENDEMIC AREA OF AMERICAN CUTANEOUS LEISHMANIASIS, STATE OF PARANÁ, BRAZIL

Lutzomyia intermedia (Diptera: Psychodidae) features as one of the main vectors that are involved in the transmission of American cutaneous leishmaniasis (ACL) in the Neotropical region. However, genetic studies involving this taxon are still incipient and important for understanding the level of variability of different populations, their role, and implications as vectors. The aim of this study was to determine the level of genetic diversity of L. intermedia present in the Ribeira River Valley, an area of ACL transmission in the state of Paraná, Brazil, through the Random amplified polymorphic DNA (RAPD). Two municipalities were chosen to collect sand flies: Cerro Azul (new transmission area of the ACL) and Adrianópolis (endemic area of the ACL). The insects were captured in the house, in the peridomicile and in the wild (forest). Two of the used markers made it possible to estimate the polymorphism of the studied populations, resulting in 40 genotypes, most of them from peridomicile. The dendrogram generated by the analysis with the primer A10 showed different degrees of similarity, suggesting that there may be gene flow in the studied populations. The Principal Coordinate Analysis (PCO) with the A2 primer, was useful in grouping L. intermedia according to its ecological and geographical origin. There was no distinction between the lineages composing the L. intermedia complex. The results of this study, with the record of great genotypic diversity in L. intermedia, may contribute to explain the maintenance of the life cycle of Leishmania braziliensis (Kinetoplastida: Trypanosomatidae) in the region.

Detection of Alpha, Beta, Gamma, and Unclassified Human Papillomaviruses in Cervical Cancer Samples From Mexican Women.

Background: Cervical cancer (CC) is associated to high-risk human papillomavirus (HPV) infections, for this reason it is crucial to have sensitive and accurate HPV diagnostic tests. To date, most research is focused on HPVs within the Alphapapillomavirus (α-PVs) genus and little attention has been paid to cervical infections with other HPV genotypes, like those of the Betapapillomavirus (β-PVs) and Gammapapillomavirus (γ-PVs) genera. The aim of this study was to determine the HPV genotypes from different genera in women with CC using Next-Generation Sequencing (NGS).Methods: The study comprised 48 HPV positive CC samples evaluated with the Linear Array HPV Genotyping test and individually sequenced by 454 NGS using PGMY09/11 and FAP primers. To determine the HPV genotypes present in each sample, the obtained sequences were compared with all HPV L1 gene reference sequences from the Papillomavirus Episteme database (PaVE). Moreover, 50 HPV positive low-grade cervical lesion samples individually genotyped with NGS were also included to determine the genotypes present preferentially in CC patients.Results: Among the 48 CC samples, 68.75% consisted of multiple HPV infections, 51 different genotypes were detected, of which 7 are still unclassified, 28 belong to α-PVs (6, 11, 16, 18, 26, 30, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 54, 59, 62, 66, 68, 69, 70, 71, 74, 81, 102, 114), 10 to β-PVs (5, 12, 21, 37, 38b, 47, 80, 107, 118, 122), and 6 to γ-PVs (101, 103, 123, 135, 147, 214). Among them, HPV16 was the most prevalent genotype (54.2%), followed by HPV18 (16.7%), HPV38b (14.6%), and HPVs 52/62/80 (8.3%). Some genotypes were exclusively found in CC when compared with Cervical Intraepithelial Neoplasia grade 1 (CIN1) samples, such as HPVs 5, 18, 38b, 107, 122, FA39, FA116, mSK_120, and mSK_136.Conclusions: This work demonstrates the great diversity of HPV genotypes detected by combining PGMY and FAP primers with NGS in cervical swabs. The relatively high attribution of β- and γ- PVs in CC samples suggest their possible role as carcinogenic cofactors, but deeper studies need to be performed to determine if they have transforming properties and the significance of HPV-coinfections.

Genotypic diversity of Streptococcus mutans associated with the risk factors for dental caries in children

The association between the genotypic diversity of Streptococcus mutans and risk factors for dental caries in children attending an educational program in the public sector was investigated. Twenty-one children (2-7 years old) who presented at least three risk factors were allocated into two groups: caries free (n = 12); with caries activity (n = 9). Initially, 210 isolates of S. Mutans were analyzed through AP-PCR and in the second intervention (after 12 months), new evaluation of risk factors and dmf-t index was carried out, followed by statistical analysis of the data (Simple Logistic Regression and Chi-square Test). There was an association between genotype diversity and caries (p = 0.05). It was found that 66,6% of the caries-free children had one genotype, while 77.7% in the group with caries had two or more genotypes. Having two or more genotypes increased by seven times the chance of injury. Genotypic diversity was associated with inadequate eating habits and oral hygiene practices. The dmf-t index of children with two or more genotypes increased from 2.64 to 4.64. These findings suggest that harmful habits of oral hygiene and diet may favor colonization by S. mutans and greater genotypic diversity, potentializing the risk of dental caries in the children evaluated.

Molecular characterization and antimicrobial susceptibility pattern of Streptococcus agalactiae isolated from clinical mastitis in dairy cattle.

The objectives of this study were to: (a) genotypically characterize Streptococcus agalactiae isolates recovered from clinical mastitis (CM) cases in dairy cows and, (b) determine the association of antimicrobial susceptibility (AMS) and genotypes of Strep. agalactiae clustered according to the genetic similarity. A total of 89 Strep. agalactiae isolates recovered from bovine CM were genotyped using random amplified polymorphic DNA (RAPD) analysis. In addition, the AMS of the isolates was determined using a commercial broth microdilution test composed of 10 antimicrobials (penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin/novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine). Descriptive analysis was used to report the frequency of RAPD-types and genotypic clusters within herd, housing system, season and CM severity scores. The minimal antimicrobial concentrations that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were calculated and survival analysis was completed to verify the differences of AMS among genotypic clusters. Results of RAPD showed a great genotypic diversity of Strep. agalactiae (45 RAPD-types) and three clusters (Ia, Ib and II) were created based on the genetic similarity among genotypes. After clustering, a high genetic similarity was observed within and between herds. Overall, Strep. agalactiae showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the AMS among clusters were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulfadimethoxine and tetracycline. In conclusion, Strep. agalactiae is still highly susceptible to most antimicrobials, although differences in susceptibility to certain antimicrobials were observed among genotypic clusters.

Genetic diversity in maize under nitrogen restriction conditions

Because of the great diversity of corn genotypes and the effects of sowing seasons and nitrogenous fertilizers, in plant development, the identification of promising genotypes in these conditions allows you to provide information to determine the best strategies to be adopted in future breeding programs. Thus, this study aimed to effect of the genetic divergence among corn genotypes in different levels of nitrogen and sowing seasons in the State of Tocantins. In this sense, were conduced six maize populations of competition tests in Palmas-TO, distributed in two sowing seasons (01.21.2014 and 02.19.2014). In each sowing season, the essays were conduced under conditions of high nitrogen (N), mean and low N The experimental design used in each essay was a randomized block with three replications and 12 treatments. Genetic divergence was evaluated by method of Tocher. The characteristics ear weight with straw (Low N), humidity (Average N), number of grains in the row (High N), ear weight with straw (first and second sowing season), they are those that contributed for genetic divergence.

Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds

ABSTRACTKlebsiella pneumoniae is considered one of the most important Gram-negative opportunistic pathogens. The contact between humans and birds poses health risks to both. The aim of this study was to investigate the resistance and virulence of K. pneumoniae isolates from psittacines and passerines, seized from illegal trade in Brazil. We analysed 32 strains isolated from birds of the orders Psittaciformes and Passeriformes by polymerase chain reaction (PCR) for virulence factor genes. Antibiotic resistance was assessed by disk diffusion assay and PCR. The results indicated that fimH (100%), uge (96.8%), kfu (81.2%) and irp-2 (68.7%) were the most common virulence genes, followed by kpn (46.8%), K2 (43.7%), mrkD (34.3%) and iroN (15.6%). The combination of virulence genes resulted in a great diversity of genotypes and the heterogeneity of the strains is also confirmed in the analysis by amplified fragment length polymorphism. The susceptibility profiles of the K. pneumoniae showed 25% of multiple antibiotic resistance strains. We identified seven strains that presented non-extended spectrum beta lactamase blaSHV variants SHV-1 and SHV-11 and one strain positive to the blaTEM-1 gene. Plasmid-mediated quinolone resistance was present in 10 strains (10/32). The data obtained in this study reveal the pathogenic potential of this pathogen and highlight the need for surveillance and monitoring.

Genotyping of Rotavirus by Using RT-PCR Methods

There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994) and Gentsch et al. (1992). There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990) and Simmond et al. (2008) for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses. Key words: rotavirus, G typing, P typing

Analysis of clinical and environmental Candida parapsilosis isolates by microsatellite genotyping—a tool for hospital infection surveillance

Candida parapsilosis emerged as an important opportunistic pathogen, causing candidaemia worldwide. Nosocomial outbreaks triggered by this species have been frequently described, particularly in cancer patients. For a better understanding of its epidemiology, several typing methods are used and microsatellite analysis has been reported as highly discriminant. The main objective of this work was to study C. parapsilosis isolates by application of microsatellite genotyping to distinguish epidemiologically related strains, compare clinical and environmental isolates and determine possible routes of dispersion of the isolates in the hospital setting. A total of 129 C. parapsilosis isolates from different origins, including hospital environment and hands of healthcare workers, were genotyped using four microsatellite markers. The isolates were recovered from different health institutions. Analysis of C. parapsilosis isolates from hospital environment showed great genotypic diversity; however, the same or very similar genotypes were also found. The same multilocus genotype was shared by isolates recovered from the hand of a healthcare worker, from the hospital environment and from patients of the same healthcare institution, suggesting that these could be possible routes of transmission and that infections due to C. parapsilosis may be mainly related with exogenous transmission to the patient. Examination of sequential isolates from the same patients showed that colonizing and bloodstream isolates had the same multilocus genotype in the majority of cases. We demonstrate that this typing method is able to distinguish clonal clusters from genetically unrelated genotypes and can be a valuable tool to support epidemiologic investigations in the hospital setting.

Norovirus genotype diversity associated with gastroenteritis outbreaks in aged-care facilities.

Noroviruses are a major cause of gastroenteritis. Vaccine strategies against norovirus are currently under consideration but depend on a detailed knowledge of the capsid genotypes. This study examined the incidence of norovirus outbreaks in residential aged-care facilities in Victoria, Australia over one year (2013) and documented the (capsid) norovirus genotypes associated with these outbreaks. It was found that 65·0% of 206 outbreaks tested were associated with norovirus infection, thereby showing norovirus to be the major cause of viral gastroenteritis in residential aged-care facilities. Fifteen capsid (open reading frame 2) genotypes were identified as follows: GI.2 (0·9%), GI.3 (1·8%), GI.4 (3·7%), GI.6 (0·9%), GI.7 (0·9%), GI.8 (0·9%), GII.1 (0·9%), GII.2 (0·9%), GII.3 (1·8%), GII.4 (2009-like) (0·9%), GII.4 (2012) (48·6%), GII.4 (2012-like) (16·5%), GII.4 (unknown) (9·2%), GII.5 (2·8%), GII.6 (0·9%), GII.7 (0·9%), GII.13 (6·4%) and an as yet unclassified GII genotype (0·9%). Although GII.4 was the most common norovirus capsid genotype detected, the great diversity of norovirus genotypes in the elderly indicates vaccination strategies for this demographic are not straightforward.

Fitness tradeoffs between spores and nonaggregating cells can explain the coexistence of diverse genotypes in cellular slime molds

Cellular slime molds, including the well-studied Dictyostelium discoideum, are amoebae whose life cycle includes both a single-cellular and a multicellular stage. To achieve the multicellular stage, individual amoebae aggregate upon starvation to form a fruiting body made of dead stalk cells and reproductive spores, a process that has been described in terms of cooperation and altruism. When amoebae aggregate they do not perfectly discriminate against nonkin, leading to chimeric fruiting bodies. Within chimeras, complex interactions among genotypes have been documented, which should theoretically reduce genetic diversity. This is however inconsistent with the great diversity of genotypes found in nature. Recent work has shown that a little-studied component of D. discoideum fitness--the loner cells that do not participate in the aggregation--can be selected for depending on environmental conditions and that, together with the spores, they could represent a bet-hedging strategy. We suggest that in all cellular slime molds the existence of loners could resolve the apparent diversity paradox in two ways. First, if loners are accounted for, then apparent genotypic skew in the spores of chimeras could simply be the result of different investments into spores versus loners. Second, in an ecosystem with multiple local environments differing in their food recovery characteristics and connected globally via weak-to-moderate dispersal, coexistence of multiple genotypes can occur. Finally, we argue that the loners make it impossible to define altruistic behavior, winners or losers, without a clear description of the ecology.

Genetic diversity of the Mycobacterium tuberculosis Complex in San Luis Potosí, México

BackgroundAlthough epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce.MethodsComplete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined.ResultsPulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities.ConclusionsIn San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage.

Prevalence and genotypic diversity of cervical human papillomavirus infection among women from an urban center in Brazil

Human papillomavirus (HPV) infection is a common viral sexually transmitted infection and the main cause of cervical cancer in women worldwide. Epidemiological data on the prevalence of HPV in a given population is essential for the establishment of effective prevention strategies. The aim of this study was to determine HPV prevalence in women who attended a public health service within an urban center in Brazil. Cervical samples were collected from 337 women recruited from a primary public health care clinic in the city of Cruz Alta located in Rio Grande do Sul, the southernmost State of Brazil. Samples were analyzed for HPV DNA and with Pap smear screening tests. HPV was detected in 114 (34%) women. HPV type analysis revealed that 95 (83.3%) of those represented infections with a single genotype, while 19 (16.7%) were mixed genotype infections. High- and low-risk HPV genotypes were detected in 83 (72.8%) and 48 (42.1%) samples, respectively. Furthermore, a great diversity of HPV genotypes was observed (18 high-risk, 12 low-risk, and 1 indeterminate). The most commonly identified low-risk types were candHPV62 (7.9%) and 61 (5.3%), while the most common high-risk types were 16 and 33 (8.8% each). Abnormal cytology was observed in 10 (3.0%) women, 9 of which were infected with HPV. Of the remaining 327 women with normal cytology results, 107 (32.7%) were positive for HPV DNA. HPV infection was correlated with younger age (less than 40 years), a first Pap smear, and other vaginal infections.

Diversity of ARSACS Mutations in French-Canadians

The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.

Distribution of rotavirus genotypes after vaccine introduction in the Triângulo Mineiro region of Brazil: 4-Year follow-up study

BackgroundRotaviruses are the major cause of diarrhea in children for which a monovalent G1P[8] vaccine has been provided free for all Brazilian infants since March 2006. ObjectivesTo investigate prevalence and genotypes of rotavirus strains causing diarrhea in children in Triângulo Mineiro, Minas Gerais, during 2007–2010, and to assess local vaccine impact. Study designFecal specimens were analyzed for rotavirus detection and characterization by PAGE, RT-PCR and PCR-genotyping assays. ResultsOverall, rotavirus was diagnosed in 12.1% (76/630) cases, accounting for 35.8% of the hospitalizations and 6.5% of outpatient attendance due to diarrhea. A trend in rotavirus disease reduction occurred in both cities (71.8% and 83.4% in Uberaba; 95.3% in Uberlândia) up to 2009, but it reversed in 2010 with increased rotavirus cases in Uberlândia. Short pattern G2P[4] strains were detected in all but three (96%) cases of mixed/P[NT] infections with long electropherotypes. ConclusionsThis 4-year follow-up study showed a reduction in rotavirus-related diarrhea and even skipped a rotavirus season, which is consistent with vaccine mediated protection. The 2007–2010 rotavirus epidemic curve reflected the natural cyclic fluctuation of the single G2P[4] genotype, with sharp reduction of cases in 2008 leading to lack of a rotavirus 2009 season (both cases and hospitalizations) followed by its come back in 2010. Diarrhea cases related to either vaccine serotype/genotype (G1 or P[8]) were not detected. Thus, a new scenario emerged with a single epidemic genotype replacing the cocirculation of great diversity of genotypes, thus far, a hallmark of the epidemiology of rotavirus in Brazil.