A comparison of probes for restriction fragment length polymorphism (RFLP) typing of Legionella pneumophila serogroup 1 strains.

The use of probes derived from rRNA sequences to detect restriction fragment length polymorphisms (RFLPs) associated with the ribosomal RNA genes for epidemiological typing (ribotyping) is a powerful and readily applicable tool. Different probes and enzymes for ribotyping were compared for a series of 73 unrelated Legionella pneumophila serogroup 1 strains. The probes compared were cDNAs, transcribed from L. pneumophila or Escherichia coli rRNA subunits, and a cloned L. pneumophila rRNA gene. The cloned rRNA gene probe gave the best discrimination and this probe was further compared with cloned probes comprised of randomly selected (non-rRNA) parts of the L. pneumophila chromosome. In this instance the greatest discrimination was achieved when one of the non-ribosomal RNA gene probes was employed. The overall discrimination of RFLP typing was enhanced by combining the data obtained with both rRNA and non-rRNA probes.     

An epidemiologically valuable typing method for Neisseria meningitidis by analysis of restriction fragment length polymorphisms.

A restriction fragment length polymorphism (RFLP) typing method was developed for Neisseria meningitidis. A cloned EcoRI fragment from a Neisseria meningitidis Group B serotype 15P1.16 sulphonamide-resistant strain was used to probe Southern blots of total chromosomal DNA restriction fragments (enzyme AvaI). A group of 75 apparently unrelated organisms gave rise to 26 different restriction fragment length patterns and two different groups of epidemiologically related strains had RFLP patterns that were distinct for each group. The technique was highly reproducible and discriminatory. The RFLP data were compared with the results of serotyping and subtyping and isoenzyme electrophoretotyping. The RFLP data were consistent with those from the alternative typing methods; clones defined by isoenzyme analysis were subdivided by this technique. The use of RFLP typing by cloned probes should be of considerable epidemiological value.     

HLA-DP typing by analysis of DNA restriction fragment length polymorphisms in the HLA-DPβ subregion

HLA class II antigens are encoded in the HLA-D region and are highly polymorphic. Southern hybridization technique was used to analyze restriction fragment length polymorphisms (RFLPs) in the DPß gene and an attempt was made to correlate these with DP haplotypes derived from primed lymphocyte typing (PLT) analysis. Digestion of DNA from 32 Epstein-Barr virus (EBV)-transformed cell lines (of haplotypes DPw2, DPw3, DPw4, DPw5, and Cp63) with three different restriction endonucleases. Southern transfer, and hybridization to the DPß cDNA probe revealed multiple fragments in all cell lines tested. The polymorphic patterns of these fragments were found to correlate with DP haplotypes, suggesting the possibility that the analysis of DNA RFLPs (DNA typing) in the HLA-DPß subregion can distinguish and identify HLA-DP haplotypes.     

Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis.

As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.     

Epidemiological typing of Yersinia enterocolitica by analysis of restriction fragment length polymorphisms with a cloned ribosomal RNA gene

Intra-species restriction fragment length polymorphisms (RFLPs) of Yersinia enterocolitica were detected in assays with a cloned DNA fragment from Legionella pneumophila that included the 16S and 23S rRNA genes. By use of this method it was possible to identify different RFLP types within biogroups/serogroups which were indistinguishable by other means. Thus the 37 biogroup IV/serogroup O3 strains isolated worldwide from pig carcasses, pork and human patients, were subdivided into five different RFLP-types. Typing based on RFLP analysis was sensitive and independent of phenotypic characters. The method will be of value for the identification and evaluation of possible reservoirs and routes of infection.     

Similar DNA restriction endonuclease profiles in strains of Legionella pneumophila from different serogroups

DNA of strains of Legionella pneumophila serogroups 1, 3, 4, and 6, isolated from patients and environmental sources, was examined by restriction endonuclease analysis (REA). Major differences in profiles enabled subtyping in many strains with the same serogroup antigen. However, a cluster of L. pneumophila strains, originating from all the examined serogroups, had similar restriction endonuclease profiles, sometimes with minor differences. This suggests that the genetic similarity between strains of L. pneumophila of different serogroups is sometimes closer than in strains with the same serogroup antigen. Seven environmental sources harbored two L. pneumophila strains with various serogroup antigens; six sources had similar restriction endonuclease profiles. The resolution of small differences in profiles is hampered in REA by the great magnitude of DNA fragments; even upon extensive analysis, these differences are not always readily visualized. Double digestions with the restriction enzymes HpaI and HpaII showed the best results and sometimes revealed differences not evident by digestions with a single endonuclease. REA has a great capacity for accurate epidemiological typing of L. pneumophila, in addition to classical serogrouping; it appeared that the results of the two techniques do not necessarily correlate. On the other hand, it should be stressed that small differences in profiles are not easily detected by REA.     

DNA probe specific for Legionella pneumophila.

A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila.     

Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies.

A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.     

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.     

Sequence-based typing of Legionella pneumophila serogroup 1 offers the potential for true portability in legionellosis outbreak investigation

Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.     

Subtyping of Legionella pneumophila serogroup 1 isolates by monoclonal antibody and plasmid techniques.

A group of environmental and clinical Legionella pneumophila serogroup 1 isolates was subtyped by monoclonal antibody dot immunoblotting and plasmid analysis. Monoclonal antibody analysis defined seven subtypes within three major groups. Plasmid analysis (including restriction endonuclease digestion) revealed 10 subtypes. By combining plasmid and monoclonal techniques, all 16 strains were shown to be distinct. Plasmid profiles and monoclonal antibody reactivities of selected strains were stable despite serial passage (greater than 100 times). No plasmid-associated antigen was defined by this panel of monoclonal antibodies. The observed dissociation of plasmid profiles and monoclonal antibody reactivity patterns suggests that accurate epidemiologic typing of L. pneumophila serogroup 1 strains will require use of both techniques.     

Distribution, expression, and long-range mapping of legiolysin gene (lly)-specific DNA sequences in legionellae.

The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal lly-specific DNA probe was used in Southern hybridizations for the detection of lly-specific DNA in the genomes of legionellae and other gram-negative pathogenic bacteria. Under conditions of high stringency, the lly DNA probe specifically reacted with DNA fragments from L. pneumophila isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bacteria of other genera. The lly gene was mapped by pulsed-field gel electrophoresis to the respective genomic NotI fragments of Legionella isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.     

Insertion sequences as highly resolutive genomic markers for sequence type 1 Legionella pneumophila Paris

The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.     

Theileria parva: genomic DNA studies reveal intra-specific sequence diversity

Theileria parva parva piroplasm DNA was purified from 11 different infections of cattle with 6 different East African isolates of the parasite. Total DNA was also prepared from bovine lymphoblastoid cells infected with schizonts of one of the isolates. Two of the infections were with cloned parasites. The DNA was of high molecular weight and free from protein and RNA, but some of the samples contained a proportion of bovine DNA. The 6 samples least contaminated with bovine DNA had a mean 'melting' temperature (Tm) of 84 degrees C and a mean GC content of 31.3%. Reassociation kinetics gave an estimate of 1.2 x 10(7) base pairs for the genome of T. parva. Repetitive restriction fragments were cloned from two samples, separated from the recombinant vectors and used as probes to demonstrate RFLPs between isolates. Discrimination into five groups was achieved. Schizont and piroplasm DNAs from the same isolate gave identical RFLPs, and one of the cloned parasites appeared to be a sub-population selected from a mixed-infection field isolate. Comparison of RFLPs with monoclonal antibody profiles suggested that neither technique yet provides discrimination between all the isolates which may comprise a strain. The importance of DNA probes for studying the epidemiology of theileriosis and for control programs is discussed.     

Paleoepidemiologic investigation of Legionnaires disease at Wadsworth Veterans Administration Hospital by using three typing methods for comparison of legionellae from clinical and environmental sources.

Multilocus enzyme electrophoresis, monoclonal antibody typing for Legionella pneumophila serogroup 1, and plasmid analysis were used to type 89 L. pneumophila strains isolated from nosocomial cases of Legionnaires disease at the Veterans Administration Wadsworth Medical Center (VAWMC) and from the hospital environment. Twelve L. pneumophila clinical isolates, obtained from patients at non-VAWMC hospitals, were also typed by the same methods to determine typing specificity. Seventy-nine percent of 33 VAWMC L. pneumophila serogroup 1 clinical isolates and 70% of 23 environmental isolates were found in only one of the five monoclonal subgroups. Similar clustering was found for the other two typing methods, with excellent correlation between all methods. Enzyme electrophoretic typing divided the isolates into the greatest number of distinct groups, resulting in the identification of 10 different L. pneumophila types and 5 types not belonging to L. pneumophila, which probably constitute an undescribed Legionella species; 7 clinical and 34 environmental VAWMC isolates and 2 non-VAWMC clinical isolates were found to be members of the new species. Twelve different plasmid patterns were found; 95% of VAWMC clinical isolates contained plasmids. Major VAWMC epidemic-bacterial types were common in the hospital potable-water distribution system and cooling towers. Strains of L. pneumophila which persisted after disinfection of contaminated environmental sites were of a different type from the prechlorination strains. All three typing methods were useful in the epidemiologic analysis of the VAWMC outbreak.     

Cloning and expression of a common Legionella outer membrane antigen in Escherichia coli

A genomic library of Legionella pneumophila was constructed by inserting L. pneumophila knoxville-1 strain (LPK-1) chromosome fragments into cosmid vector pHC79. Screening of the library with antibodies directed against a major outer membrane protein/lipopolysaccharide complex from LPK-1 resulted in the identification of six clones that reacted with the antiserum. Western blot analysis indicated that a 19,000 dalton (19 kDa) component was the reactive antigen in all of the clones. Western blot analysis of outer membranes from L. pneumophila serogroups and other Legionella species revealed that the cloned 19 kDa antigen was common to all serogroups and all but one of the five other Legionella species examined. One of the 19 kDa expressing clones was used as an immunoabsorbent to recover antibody to the 19 kDa antigen thus confirming the surface localization of this L. pneumophila antigen in E. coli.     

Cloning and expression of Legionella pneumophila antigens in Escherichia coli.

To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L. pneumophila serogroup 1 (strain 130b). L, pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtained by partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient were inserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells of strain HB101 were transformed with hybrid plasmids. To detect expression of antigens, 2,559 ampicillin-resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzyme immunoassay (EIA) with E. coli-absorbed rabbit anti-L. pneumophila sera. A total of 77 (3%) of the colonies were reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colony lysis. Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E. coli-absorbed antisera. Absorption of antisera with heat- and Formalin-killed L. pneumophila antigen eliminated or diminished the reactivity of the antigenic bands in representative clones. These studies confirm that several L. pneumophila antigens can be cloned and expressed in E. coli.     

Molecular typing of Legionella pneumophila serogroup 1 by pulsed-field gel electrophoresis with SfiI and comparison of this method with restriction fragment-length polymorphism analysis.

A panel of 131 well-characterised Legionella pneumophila serogroup 1 strains, previously used to evaluate a restriction fragment-length polymorphisms (RFLP) typing scheme, was examined by pulsed-field gel electrophoresis (PFGE) with the restriction endonuclease SfiI. The data obtained show that PFGE with SfiI is a highly discriminatory method yielding an index of discrimination (IOD) of 0.992 and 0.975, with 100% and 90% similarity thresholds respectively, compared with an IOD of 0.909 for the RFLP typing method. Reproducibility of PFGE profiles within gels was excellent and it was possible to compare the profiles visually. However, the reproducibility of the technique between gels was poor and visual comparison of the patterns was extremely difficult. Computer-aided analysis assisted the assessment of inter-gel reproducibility. Of 11 duplicates examined only four pairs showed 100% similarity, although 9 of 11 showed > or =90% similarity. In an attempt to determine if the PFGE banding patterns were sufficiently unambiguous to allow the method to be used as a definitive typing method, 20 coded strains were examined. At a 90% similarity level, 16 of these were placed in the correct PFGE type and four were not allocated to a type. Partial digestion of DNA by SfiI was noted despite careful control of DNA and enzyme concentrations, suggesting that an alternative enzyme might give more reproducible results.     

Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains

The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.     

Evaluation of arbitrarily primed polymerase chain reaction analysis for typing Legionella pneumophila

Objective: To evaluate the performance of arbitrarily primed polymerase chain reaction (AP-PCR) analysis in epidemiologic typing of Legionella pneumophila.
Methods: Sixty-two isolates of L. pneumophila of serogroups 1, 3, 6 and 10, including epidemiologically related and unrelated isolates, were analyzed by AP-PCR using the primer BG2. Twenty-six of the serogroup 1 isolates were typed by pulsed-field gel electrophoresis (PFGE).
Results: AP-PCR analysis showed 98% typeability and complete reproducibility. A majority of unrelated isolates of each serogroup could be distinguished (discrimination index: 92%). Clinical isolates showed AP-PCR patterns indistinguishable from those of the isolates of the related environmental source. PFGE and AP-PCR results were in agreement for 88% of isolates.
Conclusions: Single-primer AP-PCR analysis can be used as a simple and reproducible screening method for typing L. pneumophila strains of different serogroups.