Restriction Fragment Length Polymorphism Typing Research Articles

Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen affecting the global swine industry. The first open reading frame 5 (ORF5)-based genetic lineage classification system describing global PRRSV-2 genetic diversity was introduced over a decade ago. Although refinements have been proposed for the predominant lineage in the USA (lineage 1), PRRSV-2 phylogenetic classification system at international levels has not been thoroughly evaluated and updated since 2010. In this study, based on analysis of 82,237 global ORF5 sequences reported during 1989-2021, we classified PRRSV-2 into 11 genetic lineages (L1‒L11) and 21 sublineages (L1A‒L1F, L1H‒L1J, L5A‒L5B, L8A‒L8E, and L9A‒L9E). The proposed classification system is flexible for growth if additional lineages, sublineages, or more granular classifications are needed. For example, for more fine-scale epidemiological investigation, L1C was further divided into five groups (L1C.1‒L1C.5), with L1C.5 corresponding to the recently emerged L1C variant. Comparison between restriction fragment length polymorphism (RFLP) typing and phylogenetic classification revealed the inaccuracy of using RFLP to determine PRRSV-2 genetic relatedness in most scenarios. Genetic homology of six commercial PRRSV-2 vaccines to each lineage/sublineage and detection frequency of vaccine-like viruses were determined. Global geographic distribution of each lineage/sublineage was presented. Temporal dynamic changes of PRRSV-2 in the USA during 1989-2021 were investigated by analyzing 73,092 ORF5 sequences. In summary, this study refined PRRSV-2 ORF5-based phylogenetic classification and investigated the geographic distribution and temporal changes of PRRSV-2 at the lineage/sublineage levels. The refined classification system and molecular epidemiology data in this study will be invaluable for future characterization of PRRSV-2.IMPORTANCEIn this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

Molecular typing and antifungal susceptibility of clinical isolates of Cryptococcus neoformans and Cryptococcus gattii species complexes from the National Invasive Fungal Surveillance Network of Uruguay.

Cryptococcus neoformans and C. gattii species complexes (phylum: Basidiomycota) are environmental yeasts and are the main cause of human cryptococcosis worldwide. The most recent molecular typing studies in Latin America have focused on the intertropical region. Thus, this study aimed to update the knowledge of human cryptococcosis in the South American temperate region. We obtained and analyzed 116 C. neoformans/C. gattii species complexes isolates from the Public Health Surveillance Laboratory between 2008-2013 and 2017-2021 (C. gattii species complex=1 and C. neoformans species complex=115). The average patient age was 45 years, with an overall male:female ratio of 3.1:1. The proportion of HIV-negative patients was significantly higher in the second study period. Restriction fragment length polymorphism typing of URA5 gene revealed that the C. neoformans species complex comprised 75.7% VNI, 2.6% VNII, 0.9% VNIII, 1.7% VNIV, 17.4% VNII/VNIV hybrids, and one C. neoformans isolate (0.9%) misidentified as VGI; the C. gattii species complex isolates comprised one VGII. The overall case fatality rate was 49.5%, with no differences in lethality between VNI and hybrid isolates. Of the four isolates responsible for episodes of reoccurrence, only one had a genotype different from the first episode. Antifungal susceptibility testing revealed that most isolates fell below the local epidemiological cut-off value. This study provides additional information for the analysis of C. neoformans/C. gattii species complexes dynamics in the South American temperate region.

Campylobacter jejuni and other emerging Campylobacteraceae in retail beef liver - an underestimated potential source?

Bovine by-products, such as liver, could be an underestimated source of Campylobacter jejuni. Therefore, our aims were to evaluate the occurrence of C. jejuni and other Campylobacteraceae in retail beef liver and characterize their antibiotic resistance (ciprofloxacin, tetracycline, erythromycin and gentamicin) and potential genetic relationship by flagellin gene restriction fragment length polymorphism (flaA-RFLP) and multilocus sequence typing with clinical strains. Seventy-six out of 206 samples (36·9%) were positive for Campylobacter and related organisms. Arcobacter butzleri was the most frequently isolated species (21·8%), followed by C. jejuni (9·7%), C. fetus (7·8%) and C. coli (1%). The C. jejuni strains showed resistance to tetracycline (17·2%) or ciprofloxacin (6·9%), with only one strain resistant to both antibiotics. Meanwhile, 8·3% of ciprofloxacin resistance was observed in C. fetus. The other species showed no resistance. Most of the clonal complexes (CC) in which the C. jejuni genotypes were grouped (CC-21, 42, 48 and 52), coincided with genotypes of clinical strains previously reported in Chile. As such, this study provides evidence that beef liver could be an underestimated route for resistant C. jejuni to humans. Further studies should assess whether this food could play a role in the transmission of other emerging Campylobacteraceae such as those reported here.

DNA in forensic odontology: An overview

Human remains are tremendously damaged and degraded in forensic cases and the identification becomes very difficult, in those cases teeth and bones are often the only reliable sources of DNA for identification. Advances in DNA extraction techniques and short-amplicon DNA typing have greatly increased our potential to identify human remains which were previously considered to be too compromised for genetic analysis. As the teeth are largely protected within the jawbones and remains protected from the environmental conditions and prevent these tissues from postmortem decomposition and DNA decay. DNA profile tests which are performed nowadays are totally reliable and give details about an individual’s physical characteristics, ethnicity, place of origin and sex. These tests are also accepted as legal proofs in courts. These tests are: Restriction Fragment Length Polymorphism Typing, STRs Typing, Mitochondrial DNA Analysis, Y-Chromosome Analysis, X-Chromosome STR, Single Nucleotide Polymorphism. The aim of this review is to provide a better understanding and knowledge about the latest techniques in DNA for human identification in the field of forensic odontology.

PRRSV2 genetic diversity defined by RFLP patterns in the United States from 2007 to 2019.

The genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) increases over time. In 1998, restriction-fragment length polymorphism (RFLP) pattern analysis was introduced to differentiate PRRSV wild-type strains from VR2332, a reference strain from which a commercial vaccine (Ingelvac PRRS MLV) was derived. We have characterized here the PRRSV genetic diversity within selected RFLP families over time and U.S. geographic space, using available ISU-VDL data from 2007 to 2019. The 40,454 ORF5 sequences recovered corresponded to 228 distinct RFLPs. Four RFLPs [2-5-2 (21.2%), 1-7-4 (15.6%), 1-4-4 (11.8%), and 1-8-4 (9.9%)] represented 58.5% of all ORF5 sequences and were used for cluster analysis. Over time, there was increased detection of RFLPs 2-5-2, 1-7-4, 1-3-4, 1-3-2, and 1-12-4; decreased detection of 1-4-2, 1-18-4, 1-18-2, and 1-2-2; and different detection trends for 1-8-4, 1-4-4, 1-26-1, 1-22-2, and 1-2-4. An over-time cluster analysis revealed a single cluster for RFLP 2-5-2, supporting that sequences within RFLP 2-5-2 are still relatively conserved. For 1-7-4, 1-4-4, and 1-8-4, there were multiple clusters. State-wise cluster analysis demonstrated 4 main clusters for RFLP 1-7-4 and 1-8-4, and 6 for RFLP 1-4-4. For the other RFLPs, there was a significant genetic difference within them, particularly between states. RFLP typing is limited in its ability to discriminate among different strains of PRRSV. Understanding the magnitude of genetic divergence within RFLPs helps develop PRRSV regional control programs, placement, herd immunization strategies, and design of appropriate animal movements across borders to minimize the risk of PRRSV transmission.

Recent acute reduction in macrolide-resistant Mycoplasma pneumoniae infections among Japanese children

IntroductionMycoplasma pneumoniae contributes to numerous pneumonia cases among children and young adults. Therefore, this study aimed to investigate the prevalence of M. pneumoniae infections among Japanese children, occurring since 2008. MethodsNasopharyngeal swab specimens were obtained from all cases, following which real-time PCR was performed to identify M. pneumoniae. Further, the p1 genotypes of isolates were determined using the PCR restriction fragment length polymorphism typing method. ResultsThe annual rate of macrolide-resistant M. pneumoniae (MRMP) infections peaked at 81.8% in 2012 and decreased annually until 2015. Although the infection rate increased to 65.3% in 2016, it decreased again to 14.3% in 2018. Although >90% of isolates harbored the type 1 genotype until 2012, this rate decreased, and approximately 80% harbored p1 genotypes other than type 1 in 2018. Furthermore, the occurrence rate of MRMP among the type 1 isolates was very high (82.4%), whereas that among p1 genotypes other than type 1 was very low (6.5%). ConclusionsMRMP occurrence potentially decreased owing to changes in not only antibiotic usage but also in the distribution of p1 genotype among isolates.

Characterization of antibiotic resistance integrons harbored by Romanian Escherichia coli uropathogenic strains

Abstract Because little is known about the integrons which constitute an important means of spreading resistance in bacteria circulating in Romania, this study aimed to detect antibiotic resistance gene cassettes embedded in integrons in a convenient collection of 60 ciprofloxacin-resistant Escherichia coli isolates of various phylogroups, associated with community-acquired urinary tract infections. Characterization of the integrons was accomplished by PCR, restriction fragment length polymorphism typing, and DNA sequencing of each identified type. More than half of the tested E. coli strains were positive for integrons of class 1 (31 strains) or 2 (1 strain). These strains derived more frequently from phylogenetic groups A (15 of 21 strains), B1 (10 of 14 strains), and F (3 of 4 strains), respectively. While 20 strains carried class 1 integrons which could be assigned to nine types, eleven strains carried integrons that lacked the 3’-end conserved segment. The attempts made to characterize the gene cassettes located within the variable region of the various integrons identified in this study revealed the presence of genes encoding resistance to trimethoprim, aminoglycosides, beta-lactams or chloramphenicol. The evidence of transferable resistance determinants already established in the autochthonous E. coli strains highlights the need for improved control of resistance-carrying bacteria.

Isolation and molecular characterization of a variant of Chinese gC-genotype II pseudorabies virus from a hunting dog infected by biting a wild boar in Japan and its pathogenicity in a mouse model.

We isolated a variant of Chinese pseudorabies virus from a hunting dog with symptoms similar to Aujeszky's disease and designated the isolate MY-1 strain. The dog developed symptoms 6days after hunting and biting a wild boar and died the day after onset. The Bam HI restriction profile of MY-1 DNA was different from those of the Japanese reference strain Yamagata-S81 and two vaccine strains, Bartha and Begonia, and resembled Bam HI-RFLP (restriction fragment length polymorphism) type IV. Complete nucleotide sequences were determined, and phylogenetic analyses revealed that MY-1 belonged to the same cluster of old Chinese strains and variant strains isolated recently in China, but most of the open reading frames of MY-1 were located on a different branch from those of these Chinese strains. Based on a gC phylogenetic analysis, MY-1 belonged to gC-genotype II composed of those Chinese strains. In mice, the 50% lethal dose (LD50) of MY-1 (103.0 TCID50) was almost the same as those of Yamagata-S81 and Bartha. The LD50 value of Begonia was 10≥4.5 TCID50. The mean survival periods of mice after infection with 104 TCID50 of MY-1, Yamagata-S81 and Bartha were 3.9days, 2.3days, and 8.0days, respectively. The results suggested that the variant of Chinese PRV with slightly weaker pathogenicity than that of wild virulent viruses might be maintained in wild boars in Japan. Furthermore, we would like to propose that old Chinese strains, recent Chinese variant strains, and MY-1 should be grouped as an Asian type PRV.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV) 1-7-4-type strains in Peru.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. While PRRSV has been endemic in North America since 1989, it was not until 1999 that the virus was first described in South America. Notably, recently an increased number of PRRSV outbreaks have been reported in South American countries. However, epidemiological information related to these outbreaks is limited and the genetic characteristics of the PRRSV strains circulating in the region are poorly understood. In this study, we describe the genetic analyses of PRRSV strains associated with severe PRRS outbreaks in Peru. Samples originating from 14 farms located in two Departments in Peru (Lima and Arequipa), were subjected to RT-PCR amplification of the PRRSV ORF5 gene and sequencing followed by restriction fragment length polymorphism (RFLP) analysis. Results demonstrated the circulation of PRRSV-2 in Peru. Notably ORF5 RFLP typing revealed that 15 (75%) of the PRRSV strains detected in this study belong to the RFLP 1-7-4 type. Phylogenetic analysis showed that the Peruvian strains are closely related to the highly virulent PRRSV 1-7-4 strains that emerged in the US in 2013-2014. Results here indicate the presence of highly virulent PRRSV 1-7-4 strains in Peru and provide important information on the geographical distribution of PRRSV, confirming the recent geographical expansion of this important swine pathogen towards South America.

Genetic Diversity of the Cryptococcus gattii Species Complex in Mato Grosso State, Brazil.

Cryptococcosis is caused by fungi of the genus Cryptococcus. Owing to its importance, this study aimed to analyze the genetic diversity of C. gattii isolates from animals, humans, and the environment in Mato Grosso State (MT), Brazil, during November 2010-December 2017. All isolates of the C. gattii species complex were subjected to molecular genotyping via Restriction Fragment Length Polymorphism (PCR-RFLP) and Multi-locus Sequence Typing (MLST). PCR-RFLP analysis revealed that 21 isolates presented the genotype VGII, which is considered the most common and virulent genotype globally among. MLST analysis revealed the presence of 14 sequence types (STs), of which 5 are considered new genotypes. Clonal Complex (CC) CC182 (n = 5; 23,80%) and CC309 (n = 3; 14,28%) were the most frequent. CC distribution in relation to origin revealed that three CCs were found in animals with a predominance of CC182 (66,66%), while nine were found in humans, and two CCs were found in the environment. Extensive genetic variability was observed among the isolates in the State of Mato Grosso. STs belonging to the already described clonal complexes (CC) indicate the global expansion and adaptation of isolates in several other countries. Therefore, detection of clonal complexes and STs already described in other regions and the occurrence of new STs in the present study help further the current understanding of the geographic dispersion and genetic origin of the C. gattii species complex.

Relation between broiler and human Campylobacter jejuni strains isolated in Belgium from 2011 to 2013.

This study inquires the relationship between Campylobacter jejuni isolated from broiler meat carcasses (n=97) and human clinical samples (n=72) in Belgium, from 2011 to 2013. The evaluation of the relation was based on the characteristics determined using multilocus sequence typing (MLST) alone and combined with flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, antibiotic microbiological resistance profiling (AMRp), lipooligosaccharide class typing or virulence gene profiling (Vp). Clusters containing both human and broiler meat strains were more common when MLST was used alone, followed by MLST/flaA-RFLP and then by MLST/AMRp. More logical chronologically relations broiler-human were obtained for MLST/flaA-RFLP, then for MLST, and finally for MLST/AMRp: i.e. the isolates would first be detected in the broiler meat and at the same time or later in humans. In several cases, the C. jejuni strains isolated from the consumed broiler meat and from the campylobacteriosis case had the same profile, according to the used typing methods. The circulating Campylobacter strains appear to have remained the same from 2011 till 2013 in Belgium. This study corroborates previously published data from Belgium that suggest a strong correlation between C. jejuni strains isolated from broiler meat and from campylobacteriosis patients.

Molecular epidemiology and antimicrobial resistance mechanisms of Campylobacter coli from diarrhoeal patients and broiler carcasses in Belgium.

The aim of this study was to better understand the molecular epidemiology of Campylobacter coli isolated from multiple sources in Belgium, by studying the genotypic diversity and antimicrobial resistance phenotypes and resistance mechanisms of 59 C.coli isolates. Isolates from broiler carcasses and human cases were genotyped using multilocus sequence typing (MLST), porA typing, flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, and by PCR binary typing (P-BIT). Thirty-two MLST sequence types, 24 flaA types, 31 porA alleles, and 29 P-BIT types were identified among the screened isolates. Some types and alleles were shared among strains recovered from both broiler carcasses and diarrhoeal patients. Both porA typing and MLST revealed a similar discriminatory power (0.969), which was the highest discriminatory power when compared to other methods. Minimum inhibitory concentrations against seven different antibiotics (ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, tetracycline, gentamicin, and erythromycin) were analysed. Strains were most frequently resistant to tetracycline (81.4%), followed by: ciprofloxacin and nalidixic acid (76.3%); streptomycin (33.9%); erythromycin (27.1%); and chloramphenicol (3.4%). All isolates were sensitive to gentamicin. Multidrug resistance was observed in 24 of 59 C.coli isolates (40.7%). Molecular screening of antimicrobial resistance mechanisms revealed the predominance of the gyrA T86I substitution among ciprofloxacin- and nalidixic acid-resistant isolates, the tet(O) gene among tetracycline-resistant isolates and the 23S rRNA A2075G mutation among erythromycin- resistant isolates. Furthermore, some erythromycin-resistant isolates harboured a diverse array of resistance mechanisms, including the presence of ermB, 23S rRNA A2074G mutation, and point mutations the rplD and rplV ribosomal genes, and the cmeABC multidrug efflux pump genes.

Polymorphism of ompH gene of Pasteurella multocida serotype A strains isolated in Iran

One of the most frequent causes of respiratory infection and death in sheep and goats is Pasteurella multocida. In humans, it has been associated with diseases of the respiratory tracts, arthritis, osteomyelitis and meningitis. Outer membrane protein H (OmpH) has a role in immunogenicity and pathogenicity of P. multocida. The aim of this study was to characterize the genetic diversity of ompH gene of a panel of P. multocida serotype A strains isolated in sheep. Forty P. multocida serotype A strains isolated in previous study were selected and analyzed by restriction fragment length polymorphism (RFLP) of a species-specific PCR assay. RFLP amplified fragment produced five different cleavage patterns. On the basis of combinations resulting from ompH gene digestion, the 40 P. multocida isolates were classified in six RFLP type. It seems that isolates with variants genetic profile represent different pathogenecity. New vaccine formulation should consider multivariants of P. multocida in order to confer a wider protection.

Molecular and physiological analysis of Anopheles funestus swarms in Nchelenge, Zambia

BackgroundAnopheles funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm.MethodsAnopheles funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males.ResultsA total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. funestus s.s. MW-type, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation.ConclusionsThis is the first time that An. funestus swarms have been molecularly identified to species level. Anopheles funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.

Occurrence and Nature of Double Alleles in Variable-Number Tandem-Repeat Patterns of More than 8,000 Mycobacterium tuberculosis Complex Isolates in The Netherlands

ABSTRACTSince 2004, variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates has been applied on a structural basis in The Netherlands to study the epidemiology of tuberculosis (TB). Although this technique is faster and technically less demanding than the previously used restriction fragment length polymorphism (RFLP) typing, reproducibility remains a concern. In the period from 2004 to 2015, 8,532 isolates were subjected to VNTR typing in The Netherlands, with 186 (2.2%) of these exhibiting double alleles at one locus. Double alleles were most common in loci 4052 and 2163b. The variables significantly associated with double alleles were urban living (odds ratio [OR], 1.503; 95% confidence interval [CI], 1.084 to 2.084; P = 0.014) and pulmonary TB (OR, 1.703; 95% CI, 1.216 to 2.386; P = 0.002). Single-colony cultures of double-allele strains were produced and revealed single-allele profiles; a maximum of five single nucleotide polymorphisms (SNPs) was observed between the single- and double-allele isolates from the same patient when whole-genome sequencing (WGS) was applied. This indicates the presence of two bacterial populations with slightly different VNTR profiles in the parental population, related to genetic drift. This observation is confirmed by the fact that secondary cases from TB source cases with double-allele isolates sometimes display only one of the two alleles present in the source case. Double alleles occur at a frequency of 2.2% in VNTR patterns in The Netherlands. They are caused by biological variation rather than by technical aberrations and can be transmitted either as single- or double-allele variants.

Restriction Fragment Length Polymorphism (RFLP) typing of Mycobacterium tuberculosis isolates from Nepal

Restriction Fragment Length Polymorphism (RFLP) typing of Mycobacterium tuberculosis isolates from Nepal

Comparison of the characteristics of Mycobacterium tuberculosis isolates from sputum and lung lesions in chronic tuberculosis patients.

Mycobacterium tuberculosis (Mtb) in sputum originates from lung cavities in tuberculosis (TB) patients. But drug susceptibility testing (DST) of sputum Mtb can not be conducted the same as in the lung because mutagenesis of bacilli may be happening in the lung during treatment and result in the possibility of the presence of heterogeneous drug-resistant subpopulations in the different lung lesions. This could be one of the reasons for low cure rates for multi-drug resistant (MDR)-TB. We studied the resected lungs of nine surgery patients with chronic TB. The isolates isolated from the sputum and different lung lesions of each patient were tested for phenotypic DST and genotyped using restriction fragment length polymorphism (RFLP) typing method. Genetic analysis to resistance to first and second line drugs was also performed. Five of nine patients were MDR-TB and three XDR-TB. DST results for ten anti-TB drugs were in accordance among different lung lesions in eight patients. However, only three of these eight patients showed the concordance of DST with sputum. Even though the isolates were heteroresistant, genotyping them by RFLP showed the clonal population in each individual patient. Six of eight followed-up patients achieved successful cure. In conclusion, the heteroresistance between sputum and lung lesions and a clonal population without mixed infection might provide useful information in establishing treatment regimen and surgery decision for MDR- and XDR-TB.

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.

Mitochondrial DNA Analysis of Mazahua and Otomi Indigenous Populations from Estado de México Suggests a Distant Common Ancestry.

The indigenous Mazahua and Otomi have inhabited the same localities in Estado de México since pre-Columbian times. Their languages, Mazahua and Otomi, belong to the Oto-Manguean linguistic family, and although they share cultural traditions and a regional history that suggest close genetic relationships and common ancestry, the historical records concerning their origin are confusing. To understand the biological relationships between Mazahua and Otomi, we analyzed mitochondrial DNA (mtDNA) genetic variation. We identified the mtDNA haplogroups by restriction fragment length polymorphism typing and sequenced hypervariable region 1 of the mtDNA control region in 141 Mazahua and 100 Otomi. These results showed that Otomi exhibit a higher frequency of haplogroup A than B, whereas Mazahua exhibit the opposite pattern. In the Otomi EM population the most frequent subhaplogroups are, in order of frequency, A2, B2, and C1, whereas in the Mazahua 1 population they are B2, D1, and A2. The most frequent haplotypes (Ht) of haplogroups A and B are Ht2 (A) and Ht58 (B2g1) in Mazahua 1 and Ht8 (A2), Ht22 (A2ao1), and Ht53 (B2c2b) in Otomi EM. The genetic differences between the Mazahua 1 and Otomi EM suggest a distant shared ancestry and a moderate degree of maternal admixture that has not obscured the difference of their mtDNA patterns. These unexpected results suggest the Mazahua and Otomi probably descend from the same group but separated very early and admixed with other Mesoamerican populations before their arrival in Central Mexico. The historical evidence of conflicting relations between the Mazahua and Otomi and the almost nonexistence of marriage between them could be responsible for maintaining only a moderate degree of maternal admixture.

Characterization of Extended-Spectrum β-Lactamase-Carrying Plasmids in Clinical Isolates ofKlebsiella pneumoniaefrom Taiwan

We analyzed the replicon types, sizes, and restriction fragment length polymorphism (RFLP) typing of plasmids carrying extended-spectrum β-lactamase (ESBL) genes in Klebsiella pneumoniae isolates from Taiwan. Fifty-one Escherichia coli transconjugant strains with plasmids from ESBL-producing K. pneumoniae from the Taiwan Surveillance of Antimicrobial Resistance III Program in 2002 were included. All the 51 plasmids carried a blaCTX-M gene, the majority of which were blaCTX-M-3 (28/51 [54.9%]). Plasmids ranged in size from 126 to 241 kb by S1 nuclease digestion and subsequent pulsed-field gel electrophoresis, and the most common plasmid size (37.3%) was 161-170 kb. The most common replicon type of plasmids was incompatibility group (Inc)A/C (60.8%). The IncA/C plasmids all carried blaCTX-M (blaCTX-M-3, -14, -15), and some also carried blaSHV (blaSHV-5, -12) genes. All 51 plasmids could be typed with PstI, and 27 (52.9%) belonged to 10 clusters. Thirty-eight of the 51 plasmids were typable with BamHI, and 21 plasmids (55.3%) fell into 7 clusters. Plasmids in the same cluster belonged to the same incompatibility group, with the exception of cluster C6. In conclusion, IncA/C plasmids are the main plasmid type responsible for the dissemination of ESBL genes of K. pneumoniae from Taiwan. RFLP with PstI possessed better discriminatory power than that with BamHI and PCR-based replicon typing for ESBL-carrying plasmids in K. pneumoniae in this study. Greater than 50% of plasmids fell into clusters, and >60% of cluster-classified plasmids were present in clonally unrelated isolates, indicating that horizontal transfer of plasmids plays an important role in the spread of ESBL genes.