Application of pulsed-field gel electrophoresis for the discrimination of leptospiral isolates in Brazil

Aims:  Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars.
Methods and Results:  Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200.
Conclusions:  PFGE offers the advantages of simple, reliable and reproducible results.
Significance and Impact of the Study:  PFGE provides a convenient tool for the identification of clinical isolates.     

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in S?o Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.     

Leptospira strains kept at the National Centre for Leptospirosis in Rome, Italy

Since the National Centre for Leptospirosis (Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome) was established in 1956 by B. Babudieri, efforts have been devoted to identifying new Leptospira isolates and maintaining a collection of strains that today comprises 670 strains, 550 of which have been totally or partially classified, and 120 are still under study. This collection includes 23 serogroups and 156 serovars of pathogenic leptospires, and 32 serogroups and 54 serovars of saprophytic leptospires. The conventional serogroup and serovar identification, mainly based on antigenic relatedness, is tedious and time-consuming, requiring the maintenance of a comprehensive collection of serovar reference strains and the preparation of the corresponding rabbit antisera. Although considerable difficulties are encountered in the classification of leptospires at the serogroup and serovar level, this classification system is essential to obtain information on the epidemiology of leptospirosis in the different geographical areas. Serovar identification has become faster with the introduction of pulsed-field gel electrophoresis (PFGE) of large DNA fragments obtained after digestion of leptospiral DNAs with rare-cutting restriction enzymes. This technique has been successfully utilized to discriminate between closely related serovars of the Leptospira interrogans complex. We have recently used PFGE to characterize several Italian leptospiral isolates, confirming that PFGE analysis combined with microscopic agglutination test (MAT) with monoclonal and polyclonal antibodies can be used as an accurate and reliable method to compare and classify leptospires.     

An application of monoclonal antibodies to identification of leptospires of serogroup icterohaemorrhagiae found in China

For the purpose of improving the procedures of identification of leptospires, a set of 5 monoclonal antibodies with different serological reactivity against serovars of Leptospira interrogans Icterohaemorrhagiae serogroup isolated in China was developed. One hundred and eight strains isolated from epidemic fields in 5 provinces in southern China were distinctly identified into 4 serovars of Icterohaemorrhagiae serogroup by the monoclonal antibody procedure, i.e., 98 isolates were identified as serovar lai, 7 as icterohaemorrhagiae, 2 as copenhageni, and 1 as H2. Factor antiserum procedure was used at the same time as control for typing these strains and an identical result was obtained.     

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.     

Etiological structure of icterohemorrhagic leptospirosis in gray rats (Rattus norvegicus)

In 93 Leptospira strains isolated from Norwegian rats serovar determination was made. As a result, leptospires circulating among Norwegian rats were found to belong mainly to serovar copenhageni, group Icterohaemorrhagiae, while leptospires of serovar icterohaemorrhagiae, even if occurring, were found only in the animals inhabiting pigsties. Leptospirosis epizooty among rats, caused by L. icterohaemorrhagiae, took its course independently of leptospirosis epizooty among mice, caused by L. hebdomadis, and simultaneously with it.     

Analysis of Leptospira isolates from mainland Portugal and the Azores islands

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.     

Muskrats as carriers of pathogenic leptospires in The Netherlands

Leptospires were isolated from 24 of 327 (7%) muskrats (Ondatra zibethicus) caught in The Netherlands. All isolates were identified asLeptospira interrogans. One isolate was typed as serovarcopenhageni in the Icterohaemorrhagiae serogroup, one as serovarlora in the Australis serogroup. Twenty-one isolates showed a close relationship with serovarsgrippotyphosa, valbuzzi, muelleri andratnapura from the Grippotyphosa serogroup. One isolate was lost. Sera from 196 muskrats were examined by the microscopic agglutination test. Forty-five (23%) sera reacted positively (titers1: 160), 42 (21%) of these 45 sera to Grippotyphosa and 3 (2%) to Sejroe serogroup antigens. This is the first report of serological and cultural evidence of leptospira infection in muskrats in The Netherlands.Abbreviations CAAT cross agglutination absorption test - 5-FU 5-fluorouracil - MAT microscopic agglutination test - MCA monoclonal antibodies - PBS phosphate buffered saline - REA restriction endonuclease analysis - SDS sodium dodecyl sulphate     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.     

Leptospira-rat-human relationship in Luzon,Philippines

Leptospirosis is a zoonotic infection that is caused by the pathogenic species of Leptospira. Rats are the most important reservoirs of these organisms. Our study aimed to characterize Leptospira isolates from humans and rats and elucidate the Leptospira-rat-human relationship in Luzon, Philippines. Forty strains were isolated from humans and rats. The isolates were confirmed to be Leptospira and pathogenic through rrl- and flaB-PCR, respectively. Around 73% of the isolates were found to be lethal to hamsters. Serotyping showed that there were mainly three predominant leptospiral serogroups in the study areas namely Pyrogenes, Bataviae, and Grippotyphosa. Gyrase B gene sequence analysis showed that all the isolates belonged to Leptospira interrogans. Most had 100% similarity with serovar Manilae (15/40), serovar Losbanos (8/40), and serogroup Grippotyphosa (8/40). Strains from each group had highly identical pulsed-field gel electrophoresis patterns and were further grouped as A (Pyrogenes, 14), B (Bataviae, 8), and C (Grippotyphosa, 10). Results further revealed that similar serotypes were isolated from both humans and rats in the same areas. It is suggested that these three predominant groups with highly similar intra-group PFGE patterns may have been primarily transmitted by rats and persistently caused leptospirosis in humans particularly in the Luzon islands.     

PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci.     

Genome conservation in isolates of Leptospira interrogans.

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.     

Presence of a single genotype of the newly described species Mycobacterium immunogenum in industrial metalworking fluids associated with hypersensitivity pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates

Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.     

Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Pulsed-field gel electrophoretic identification of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H

Salmonella enterica subsp. enterica serovar Typhimurium ( Salm. Typhimurium) live vaccine strain Zoosaloral H was characterized by pulsed-field gel electrophoresis (PFGE). Each of the two suitable restriction enzymes, Xba I and Spe I, produced a unique restriction fragment pattern for this live vaccine strain which was not shared by field isolates of the same serovar. The characteristic fragment pattern proved to be stable during a 22 month observation period and was also not altered after animal passage of the vaccine strains. Thus PFGE analysis proved to be a helpful tool in the identification of Salm. Typhimurium live vaccine strain Zoosaloral H and its differentiation from wild-type isolates of the same serovar.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.