Multilocus Enzyme Electrophoresis Typing Research Articles

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

BackgroundNew foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.Methodology/Principal FindingsThe Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.ConclusionThe discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database ( http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing ( Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.

Molecular typing and virulence of enteroaggregative Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro city, Brazil

Enteroaggregative Escherichia coli (EAEC) strains have been implicated as emerging aetiological agents of diarrhoea worldwide. In the present study, 43 EAEC strains were serotyped and characterized according to random amplification of polymorphic DNA profiles, PFGE, multilocus enzyme electrophoresis (MLEE) and the presence of putative virulence genes (hly, aero, kps, fim, aggA, aafA, aggR, astA, she, aap, shf and pet). The EAEC strains consisted of a diversity of serotypes including eight O-non-typable and 35 O-typable strains arranged into 21 O : H combinations. Amplification of specific genes revealed that all strains carried at least two of the virulence sequences investigated. fim, aggR and aap were the most frequent genes in both groups studied. hly, aero and aggA sequences were more prevalent in the diarrhoeal group. kps occurred exclusively in strains isolated from symptomatic children and showed strong association with diarrhoeal disease. The molecular approaches used to investigate the relatedness among EAEC strains revealed a high degree of polymorphism, suggesting that these micro-organisms have a non-clonal origin. A closer relationship was observed among EAEC strains sharing O : H types. No significant clustering could be identified related to the virulence traits investigated; however, the she locus showed clonal distribution by MLEE typing. These results are in accordance with previous findings in revealing the conservation of particular EAEC factors, despite the high degree of diversity related to both genotypic and phenotypic markers.

Typing of four genetic loci discriminates among closely related species of New World Leishmania

All New World Leishmania species can cause cutaneous lesions, while only Leishmania ( Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.

Use of Variable-Number Tandem Repeats To Examine Genetic Diversity of Neisseria meningitidis

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.

Multilocus enzyme electrophoresis typing of Candida albicans populations isolated from healthy children according to socioeconomic background

The aim of this research was to evaluate the genetic diversity within and between C. albicans populations isolated from the oral cavity of healthy Brazilian children classified into five socioeconomic categories (A to E). Multilocus Enzyme Electrophoresis (MLEE) analysis was the method used to assess genetic diversity. High genetic diversity was observed in all populations that showed predominance of some C. albicans subtypes (Electrophoretic Types - ETs). However, no correlation was observed between a specific ET and a specific population of children. Clustering analysis showed one or more highly related ET clusters, suggesting the existence of indirect and direct propagation routes of C. albicans among healthy children. Microevolutionary changes were observed in some C. albicans populations isolated from children with the same or very similar socioeconomic condition. Furthermore, low transition of C. albicans subtypes can be occurring among certain populations of children coming from high and medium/high, or high and medium/low, or medium/high and medium/low socioeconomic categories, which can also be explained by their own socioeconomic and cultural characteristics.

Pulsed-Field Gel Electrophoresis Study of Mycobacterium abscessus Isolates Previously Affected by DNA Degradation

DNA degradation (which results in a smear pattern) occurs with almost 50% of Mycobacterium abscessus strains during pulsed-field gel electrophoresis (PFGE). We assessed the potential benefit of using thiourea-containing buffer with M. abscessus by studying 69 isolates not previously typeable by PFGE (i.e., those with a smear pattern). Random (epidemiologically unrelated) isolates that were typeable (no smear pattern) were included as controls. Genomic DNA was digested with DraI, XbaI, and AseI. PFGE gels were run in regular gel buffer with and without 100 muM thiourea. All 69 isolates that generated smear patterns had clear band profiles when the thiourea buffer was used. These isolates were divided into only 30 patterns with DraI, 20 patterns with XbaI, and 20 patterns with AseI. The molecular profiles were all closely or possibly related, and the differences between the isolates ranged from zero to six bands. By multilocus enzyme electrophoresis (MEE), 45 of 53 smear isolates (85%) belonged to two closely related electrophoretic types. These isolates contained at least one enzyme allele seen almost exclusively in this group. Isolates without smear patterns were unaffected by thiourea and produced unrelated PFGE profiles, as well as multiple MEE types. The hsp65 and 16S rRNA gene sequences of the isolates with smear patterns were identical to those of M. abscessus type strain ATCC 19977, which had a nonsmear pattern, suggesting that this clone is a subgroup within M. abscessus. This demonstrates that the inability to type M. abscessus by PFGE is associated with a single clone of organisms.

Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States

Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61 % of cases, but meningitis, epiglottitis and osteoarthritis were also seen. Recent Danish isolates were compared to recent American isolates, historical Hif strains and non-Hif invasive strains. Results of conventional serotyping were confirmed by PCR detection of the serotype-f-specific cap and bexA gene sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized.

Comparison of repetitive element sequence-based PCR with multilocus enzyme electrophoresis and pulsed field gel electrophoresis for typing Listeria monocytogenes food isolates

Repetitive element sequence-based PCR (rep-PCR) was compared with multilocus enzyme electrophoresis (MEE) and pulsed field gel electrophoresis (PFGE) for typing Listeria monocytogenes food isolates. All 45 L. monocytogenes food isolates were typable by rep-PCR using two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2), with good reproducibility though with less discriminatory power than MEE or PFGE. MEE and rep-PCR types were more stable than PFGE types over a given time period. All three typing methods usefully differentiated between closely related L. monocytogenes strains. Results obtained using rep-PCR concurred broadly with those obtained using MEE, while the former method proved to be more reproducibly and conveniently performed. The greater discriminatory power of PFGE enabled subtyping of rep-PCR and MEE types. When examining L. monocytogenes isolates collected over an extended period, the slightly less discriminatory but more stable electrophoretic types (ETs) distinguished by MEE are useful for confirmation and interpretation of PFGE data. However MEE is a cumbersome technique requiring a high degree of technical expertise in order to obtain reproducible results. In the present study rep-PCR conveniently and reliably produced typing data broadly congruent with that obtained using MEE which should prove useful in aiding interpretation of PFGE data for L. monocytogenes isolates obtained over extended time periods.

Multilocus enzyme electrophoresis typing of clinical campylobacters from outbreak and sporadic sources

Multilocus enzyme electrophoresis typing of clinical campylobacters from outbreak and sporadic sources

Characterization of urease-positive thermophilic Campylobacter subspecies by multilocus enzyme electrophoresis typing.

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three C. lari isolates examined, and no ETs were shared by UPTC and C. lari isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C. lari cluster.

Phenotypic and genotypic relationship between Campylobacter spp isolated from humans and chickens in Northern Ireland – a comparison of three phenotyping and two genotyping schemes

Human campylobacteriosis is currently the most common cause of acute bacterial gastroenteritis on the island of Ireland, accounting for over 3,000 laboratory reports per year, where circa 2,000 reports originate from the Republic of Ireland and circa 1,000 reports from Northern Ireland. Elsewhere, consumption of contaminated poultry has been associated with the zoonotic transmission of disease, therefore it was the aim of this study to examine the phenotypic and genotypic relatedness of campylobacters isolated from chickens and humans locally. Sixty isolates were subtyped using phenotyping techniques (biotyping, phage-typing), as well as genotyping techniques (multilocus enzyme electrophoresis (MEE), ribotyping) and the data compared. The frequency of shared phenotypes and genotypes between poultry and humans varied depending on the typing technique employed ranging from 98.2% of human isolates sharing a similar resistotyping (MAST) disc type with poultry strains to 20% similarity with MEE typing. Overall, this small study is the first report on phenotypic and genotypic relatedness between human and poultry campylobacters in Northern Ireland, isolated under controlled conditions. The study demonstrated an association between chicken and human sub-species types, taken from a relatively contained epidemiological environment. Further work is required with larger numbers of isolates coupled with typing schemes, which are able to reliably cluster strains from chicken and humans, which share high degrees of clonality, before local poultry can be conclusively proven to be a significant source of human campylobacteriosis.

Subspecies characterization of porcine Campylobacter coli and Campylobacter jejuni by multilocus enzyme electrophoresis typing.

Enteropathogenic Campylobacterjejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated foodstuffs. C jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and L-phenylalanyl-L-leucine peptidase giving 5. 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C jejuni isolates and 19 unique ETs were detected for the 23 C coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C jejuni and C coli. respectively. Alleles were shared by C jejuni and C coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C coli in porcine livers. In certain cases, up to four phenotypically different strains of C coli were isolated from one liver, indicating multiple infections.

Multicentric genetic study of Candida albicans isolates from non-neutropenic patients using MLEE typing: population structure and mode of reproduction.

A mycological survey was conducted on non-neutropenic patients in three distinct intensive care units in two hospitals in Marseille (France) from November 1993 to November 1995. Candida albicans positive cultures from 62 patients were included in this study. Every first isolate of each patient was typed by multilocus enzyme electrophoresis (MLEE). The enzyme profiles obtained from 15 polymorphic loci were then compared. This analysis demonstrated a strong population differentiation of C. albicans infective strains within and between the different care units and confirmed the probable preponderant clonal mode of reproduction of this yeast.

Epidemic serogroup B meningococcal disease in Oregon: the evolving epidemiology of the ET-5 strain.

In 1993, Oregon's incidence of serogroup B meningococcal disease began to rise because of a highly clonal group of strains designated enzyme type 5 (ET-5), the first such increase observed in the United States. To evaluate the impact that the ET-5 strain has had on the epidemiology of meningococcal disease in Oregon. Epidemiologic analysis of surveillance data on Oregon meningococcal disease cases from 1987 through 1996 and multilocus enzyme electrophoresis typing of serogroup B isolates from June 1993 through April 1995 and from April through June 1996. A total of 836 persons with invasive meningococcal disease. Disease frequency and clonality of strains. Serogroup B disease incidence rates more than doubled from the preepidemic period in 1987-1992 (1.0 case per 100000 population) to the recent epidemic period in 1995-1996 (2.2 cases per 100000). The age-specific incidence rate of serogroup B disease among those 15 through 19 years old increased 13-fold between the preepidemic period (0.5 case per 100000) and 1995-1996 (6.4 cases per 100000). However, the proportion of cases with meningococcemia and the case-fatality rate did not change. Of 99 Neisseria meningitidis isolates obtained from 1993-1995, 88 (89%) belonged to the ET-5 complex. Of these, 69 (78%) were a single clone, designated 301. Of 20 serogroup B isolates from 1996, 18 (90%) belonged to the ET-5 complex; 17 (94%) were the 301 clone. Serogroup B meningococcal disease incidence continues at high levels in Oregon with increasing predominance of the ET-5 clonal strains.

A study of the use of multilocus enzyme electrophoresis as a typing tool in fowl cholera outbreaks

Multilocus enzyme electrophoresis (MLEE) was compared with restriction endonuclease analysis (REA) and ribotyping as a method for differentiating isolates of Pasteurella multocida in a retrospective study of eight fowl cholera outbreaks on seven turkey farms. MLEE typing matched the results previously obtained by REA and ribotyping, except that the two outbreaks linked by REA and ribotyping were not linked by MLEE typing. MLEE appears to be a useful technique for investigations of fowl cholera outbreaks.

Serogroup Y meningococcal disease in Chicago, 1991-1997.

In 1994, surveillance by the Chicago Department of Public Health detected a growing trend in the proportion of invasive meningococcal infections caused by serogroup Y. To examine the emergence of serogroup Y meningococcal disease and compare its clinical characteristics with those of other meningococcal serogroups. Population-based retrospective review of surveillance records; medical record review and cohort analysis of serogroup Y vs non-serogroup Y case patients. Chicago, III. City residents with Neisseria meningitidis isolated from a normally sterile site from January 1, 1991, through December 31, 1997; cohort analysis included those identified through March 31, 1996. Serogroup-specific incidence, demographics, and clinical outcomes. We identified 214 case patients; 53 (25%) had serogroup Y. The attack rate of serogroup Y meningococcal disease increased from 0.04 cases per 100000 in 1991 to a peak of 0.82 cases per 100000 in 1995 and subsequently decreased to 0.26 cases per 100000 and 0.34 cases per 100000 in 1996 and 1997, respectively. Compared with patients infected by other serogroups, patients with serogroup Y were older (median age, 16 years vs 1 year; P = .001) and more likely to have a chronic underlying illness (prevalence ratio, 2.3; 95% confidence interval, 1.2-4.4). Outcome did not differ significantly between the 2 groups. Multilocus enzyme electrophoresis typing of isolates from 19 case patients identified 5 different types. We found no clustering among the enzyme types by age, race/ethnicity, community area, or time. Serogroup Y emerged as the most frequent cause of meningococcal disease in Chicago in 1995 and accounted for a substantial proportion of cases in 1996 and 1997. Current data suggest that the magnitude of serogroup Y meningococcal disease is sufficient for vaccine developers to incorporate serogroup Y into new vaccines.

Multicentric epidemiological study of Aspergillus fumigatus isolates by multilocus enzyme electrophoresis

The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus.

Genetic diversity and population structure of Trypanosoma brucei: clonality versus sexuality

Genomic fingerprinting by arbitrarily primed PCR was used to analyze the genetic variability among 59 Trypanosoma brucei stocks representing the three T. brucei subspecies isolated from various hosts and different countries in Africa. 14 oligonucleotide primers revealed 355 polymorphic binary characters which were used for phenetic and phylogenetic analysis and to perform recombination tests exploring the linkage disequilibrium in the sample. These was good concordance between arbitrarily primed PCR polymorphisms and isoenzyme data previously collected for many of the same strains [1]. However, the arbitrarily primed PCR typing was more discerning than multilocus enzyme electrophoresis typing. Phenetic and phylogenetic analysis using arbitrarily primed PCR markers did not confirm T. brucei brucei and T. brucei rhodesiense as separate subspecies, but T. brucei gambiense group I was monophyletic, confirming this group as suitable for the subspecies status. With this exception, there were no clear lineages among the sample, other than clustering of East African stocks and clustering of West African stocks. Some features of the phylogenetic analysis suggested that the population structure was not strictly clonal though recombination tests showed linkage disequilibrium, even in the absence of repeated genotypes. While genotypes appear stable enough for tracking in applied studies, sexuality will impact at the evolutionary time scale, and may be more frequent under some ecological conditions. The arbitrarily primed PCR approach should be an effective and simple approach to follow epidemics and to quantify the role of sexuality in T. brucei populations.

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.