Comparison of repetitive-sequence-based polymerase chain reaction with random amplified polymorphic DNA analysis for rapid genotyping of nontuberculosis mycobacteria

Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit?- variable number of tandem repeat (MIRU-VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU-VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.     

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.     

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.     

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the 'acceptable' scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially 'false' scores. 'Acceptable' scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.     

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

碳青霉烯类耐药肺炎克雷伯菌基因型检测及同源性分析

目的了解福建医科大学附属第一医院耐碳青霉烯类肺炎克雷伯菌（KPC）的耐药基因分型及同源性分布情况。方法收集临床分离的29株KPC,改良Hodge试验检测碳青霉烯酶表型。PCR检测碳青霉烯酶blaKPC、blaIMP-1、blaVIM-1、blaOXA-48及blaNDM-基因;阳性结果进行DNA测序,并进行BLAST比对确定其基因型。采用肠杆菌科基因间重复序列（ERIC—PCR）对KPC进行同源性分析。结果29株KPC中Hodge试验阳性27株,阳性率为93．1％（27／29）。PCR扩增和DNA测序显示28株为blaKPC-2,株为blaIMP-1,未检出blaVIM-2、blaOXA-48和blaNDM-1基因。同源性分析发现KPC主要存在两种型别：28株A1和1株A2。结论blaKPC-2型碳青霉烯酶是该院KPC的主要型别,A1型已在该院流行,应加强院感监测和预防。ERIC—PCR是一种简便、快捷的基因分型技术,适合同源性的初步分型筛查。     

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.     

Evaluation of selected Listeria monocytogenes genotyping methods

Listeria monocytogenes infections are found in Poland and all world as sporadic episodes and large outbreaks of human illness as well, where bacteria-contaminated food is the source of infection. Genotyping of strains isolated from outbreak is one of many other stages of epidemiologic investigation. In case of L. monocytogenes pulse-field gel electrophoresis (PFGE) - "golden standard" of genotyping and also ERIC-PCR and rep-PCR could be used. The goal of this study was to evaluate the usefulness of methods: PFGE, ERIC-PCR and rep-PCR to Listeria monocytogenes strains genotyping. Moreover estimation of L. monocytogenes strains isolated in Poland during 2003 - 2006 years genetic diversity was performed. In performed studies, 44 strains of L. monocytogenes from PZH strains collection from 2003 - 2006 period were used. Standard strains of L. monocytogenes, L. ivanovii, L. welschimeri and L. innocua were used also. All those strains were genotyped using following methods: PFGE with ApaI and AscI restrictases and random amplification of polymorphic DNA fragments (ERIC-PCR, rep-PCR). Obtained results were analyzed using Tenover method and specialistic software - Gene Profiler (Scan Analytics, USA). In every analysis, clonal groups were obtained - using computer analysis as well as Tenover method. For PFGE with ApaI and AscI six clonal groups were obtained in each analysis. For ERIC-PCR and rep-PCR four clonal groups were obtained (every group was different except one, where four the same strains were included). Taking into account such factors, as efficiency of strains differentiation, work consumption and costs of procedure performing, for quick application, especially in local laboratories, the best genotyping technique is rep-PCR and ERIC-PCR a little less. On account of appreciable costs of PFGE analysis, this method should be used as reference technique.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.     

Application of PCR, rep-PCR and RAPD techniques for typing ofLactococcus lactis strains

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.     

Campylobacter spp. subtype analysis using gel-based repetitive extragenic palindromic-PCR discriminates in parallel fashion to flaA short variable region DNA sequence analysis

AIMS: The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology. METHODS AND RESULTS: Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. CONCLUSIONS: The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.     

Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups

REP- and ERIC-PCR genotyping were used to assess genetic heterogeneity among 81 strains of Yersinia enterocolitica biotype 1A isolated from India, Germany, France and the USA. Although both gave comparable results, ERIC fingerprints discriminated the strains better. The rep- (REP and ERIC) PCR genotyping showed that strains having different serotypes produced identical rep-profiles indicating their limited genetic diversity. The concatenated dendrogram of REP- and ERIC-PCR fingerprints clustered the biotype 1A strains into two major groups. In each group, majority of the Indian, European and American strains exhibited similarities ranging from 85% to >95%. Similarity of rep-PCR fingerprints amongst strains isolated from widely separated geographical regions revealed existence of a limited number of clonal groups of Y. enterocolitica biotype 1A. The present study failed to reveal unequivocal relationships between rep-PCR genotypes and the source of isolation. However, the clinical serotype O:6,30-6,31 strains formed a tight cluster and the aquatic O:6,30-6,31 strains formed a yet another tight cluster.     

Simple Tests to Detect Errors in High-Throughput Genotype Data in the Molecular Laboratory

With the advent of high-density DNA marker data sets for the mouse and other model systems, 100 or more genotypes are routinely generated from large groups of mice. Issues of the accuracy and reliability of the genotyping are extremely important but often not addressed until genetic analysis is conducted. Simple tests that rely on the robust predictions arising from Mendelian genetics can be made quickly in the molecular laboratory as the data are generated, and require only a spreadsheet program. In this report, genotype data from 392 mice tested at 96 marker sites were analyzed for errors that are typical when handling large volumes of data generated in a repetitive process. The testing consisted of: (1) repeating the genotyping of approximately 1% of the samples; (2) examining the deviation from the expected segregation ratio (1:2:1) on a marker-by-marker basis; and (3) testing the correlation of the genotype at one marker with that at neighboring genetic markers on a chromosome. These three steps allowed analysis at the level of the microtiter plate, where errors are most likely to occur. A set of 96 dinucleotide repeat markers that are polymorphic between the C57BL/6J and DBA/2J mouse strains and can be multiplexed is reported for use in other genotyping projects.     

Genetic Diversity of <Emphasis Type="Italic">Arcobacter</Emphasis> Species of Animal and Human Origin in Andhra Pradesh,India

Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.     

BOX-PCR is an Adequate Tool for Typing <Emphasis Type="Italic">Aeromonas</Emphasis> spp.

PCR-based methods of fingerprinting take advantage of the presence of repetitive sequences that are interspersed throughout the genome of diverse bacterial species. They include the repetitive extragenic palindromic (REP) sequence, the enterobacterial repetitive intergenic consensus sequence (ERIC) and the 154-bp BOX element. The combination of the three methods is used for fine discrimination of strains and is designated as rep-polymerase chain reaction (PCR). REP-PCR and ERIC-PCR have been shown to be useful for typing Aeromonas strains. To our knowledge, rep-PCR fingerprinting method using the BOXA1R primer has never been tested on aeromonads. In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of strains of some Aeromonas species. All strains were typeable and the majority showed unique banding patterns. Four strains from culture collections were used to investigate the reproducibility of the method. According to our results, BOX-PCR fingerprinting is applicable for typing of Aeromonas strains and can be considered as a useful complementary tool for epidemiological studies of members of this genus.     

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state‐of‐the‐art” equipment and well‐trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore‐concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN‐1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.     

Molecular Typing of Paenibacillus larvae Strains Isolated from Bulgarian Apiaries Based on Repetitive Element Polymerase Chain Reaction (Rep-PCR)

The aim of the present study was to perform molecular typing of Paenibacillus larvae (P. larvae) isolates from Bulgarian apiaries with repetitive element polymerase chain reaction (rep-PCR) using BOX A1R, MBO REP1, and ERIC primers. A total of 96 isolates collected from brood combs with clinical symptoms of American foulbrood originating from apiaries located in different geographical regions of Bulgaria, a reference strain P. larvae NBIMCC 8478 and 30 commercial honey samples with Bulgarian origin were included in the study. Rep-PCR fingerprinting analysis revealed two genotypes ab and AB of P. larvae isolates from brood combs and honey samples. A combination of genotypes ab/AB was detected in one apiary and honey sample. The prevailing genotype ab was found in 78.1 % of brood combs isolates as well as in the reference strain whereas genotype AB was determined in 21.9 % of isolates. The examination of honey samples confirmed the preponderance of ab genotype which was demonstrated in 20 of 30 samples analyzed. In conclusion, the genetic epidemiology of P. larvae revealed two genotypes—ab and AB for Bulgarian strains. Developed protocols for molecular typing of P. larvae are reliable and may be used to trace the source of infection.