Département de Microbiologie Médicale et Moléculaire, Unité de Bactériologie, CHU Bretonneau, 37044 Tours Cedex 1, France1
Institute of Molecular Evolutionary Genetics, Pennsylvania State University Park, PA 16801, USA2
Centre Héliomarin, 19 Boulevard Félix Faure, 17370 Saint Trojan-les-Bains, France3
Author for correspondence: Roland Quentin. Tel: +33 2 47 47 80 56. Fax: +33 2 47 47 38 12. e-mail: quentin{at}pop.med.univ-tours.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: multilocus enzyme electrophoresis, MLEE, electrophoretic types, cystic fibrosis, P. aeruginosa
Abbreviations: CF, cystic fibrosis; ET, electrophoretic type; FIGE, field-inversion gel electrophoresis; MLEE, multilocus enzyme electrophoresis; RAPD, random amplified polymorphic DNA
a Present address: Laboratoire de Bactériologie-Virologie-Hygiène, Unité de Bactériologie, Centre Hospitalier Universitaire Dupuytren, 87042 Limoges Cedex, France.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ability of P. aeruginosa to occupy a wide range of habitats is due at least in part to its genetic versatility. The lungs of CF patients constitute a particular biotope and it is plausible that this particular niche is generally occupied by a group of adapted P. aeruginosa strains.
A first step to elucidating the relationships between a bacterial population and its biotope is to study the genetic diversity of the population. The genetics of P. aeruginosa has not been extensively studied. However, there are data indicating high genetic diversity of populations of P. aeruginosa strains responsible for various infections of, for example, surgical wounds, the ear, the pulmonary tract and burns, or isolates recovered from the environment (Blanc et al., 1993 ; Griffith et al., 1989
; Grüner et al., 1993
; Picard et al., 1994
).
The genetic diversity of populations of P. aeruginosa strains isolated from patients suffering from CF has been investigated in various ways. A large number of studies have used molecular methods including simple restriction endonuclease analysis (Maher et al., 1993 ), Southern blotting analysis using DNA probes encoding exotoxin A (Wolz et al., 1989
) or pilin (Speert et al., 1989
), rDNA RFLP (Denamur et al., 1991
; Martin et al., 1995
) and DNA fingerprinting by pulsed-field electrophoresis (Grothues et al., 1988
). Various patterns of chronic pulmonary colonization of CF patients by P. aeruginosa have been observed, particularly multiple resident strains (Struelens et al., 1993
), a single strain or a periodically replaced dominant strain (Martin et al., 1995
; Wolz et al., 1989
). Most of the tools used for these studies are powerful typing methods for investigating nosocomial and epidemic infections but are not necessarily suitable for phylogenetic and population genetic studies.
Metabolic enzymes are central to the adaptation of a bacterium to its environment (Young et al., 1989 ). The analysis of the diversity of these enzymes by multilocus enzyme electrophoresis (MLEE) is a valuable and well-established way of studying the ecology of bacteria (Selander et al., 1986
, 1987
; Selander & Musser, 1990
). Here, we assess the polymorphism of 18 metabolic enzymes in a population of 314 P. aeruginosa strains isolated from French CF patients to determine the degree of variability of these strains, all living in the particular conditions of the pulmonary tract of CF patients. We also examined the association, if any, between ETs and patient characteristics and the properties of the bacteria.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients were examined periodically to monitor their clinical status, at which time one or more sputum samples were obtained. The samples were cultured and strains representative of all morphotypes were saved. For 18 of the patients, multiple isolates were obtained at various intervals for 1218 months.
Clinical evaluation of patients.
A modification (Doershuk et al., 1965 ) of the SchwachmannKulcyzckiKhaw scoring system (Schwachmann et al., 1965
) was used to evaluate patient status. A best possible score of 25 is assigned for each of four categories: chest X-ray, general activity, nutrition and physical condition. There is thus a maximum total score of 100. The patients were assigned to five clinical condition groups: A, excellent (score, 86100); B, good (7185); C, moderate (5670); D, poor (4155); and E, weak (<40).
Culture and storage of isolates.
Respiratory specimens were cultured on trypticase-soy with 5% horse blood, chocolate (bioMérieux) and selective cetrimide (Diagnostics Pasteur) agars for 4 d at 37 °C. After examining the primary culture carefully, each different colony type was subcultured on a nutrient agar no. 1 plate (Unipath). Thus, 314 isolates were obtained from 229 sputa. Each strain was identified as P. aeruginosa on the basis of colony form, oxidase reaction, growth at 42 °C, characteristic pigmentation on King A agar, and the appropriate response pattern in the API 20NE system (API, bioMérieux). Each of the 314 isolates was stored at -80 °C in vitamin K3 broth (bioMérieux) containing 20% (v/v) glycerol.
Determination of colony form.
At primary isolation, P. aeruginosa strains were classified into four morphological types after growth on nutrient agar no. 1 (Unipath) (Véron & Berche, 1976 ). Smooth colonies were classified as small (sm), rough colonies as large (la), viscous colonies as mucoid (M), and the very small mucoid colonies as dwarf (d).
Serotyping.
Serotypes were determined by slide agglutination with commercial O-antisera (Diagnostics Pasteur), according to the international typing scheme of Habs (1957 ) and the subtypes described by Véron (1961
).
MLEE.
Strains were grown overnight at 37 °C in 150 ml trypticase-soy broth, harvested by centrifugation, suspended in 1·5 ml 50 mM Tris/EDTA buffer (pH 7·5), and sonicated (Branson Sonifer Cell Disruptor, model 250, with microtip) for 30 s at 50% pulse, with ice-water cooling. Clear lysates were obtained by centrifugation at 16000 g for 20 min at 4 °C and stored at -70 °C.
Horizontal starch-gel electrophoresis and specific enzyme staining methods were used (Selander et al., 1986 ). Variation in electrophoretic mobility of the following 18 enzymes was assayed: alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), NADP-dependent glucose dehydrogenase (GLU), glucose-6-phosphate dehydrogenase (G6P), ß-hydroxybutyrate dehydrogenase (HBD), isocitrate dehydrogenase (IDH), NAD-dependent malate dehydrogenase (MDH), mannitol-1-phosphate dehydrogenase (M1P), shikimate dehydrogenase (SKD), threonine dehydrogenase (THR), xanthine dehydrogenase (XDH), acid phosphatase (ACP), glutamic oxaloacetic transaminase (GOT), indophenol oxidase (superoxide dismutase) (IPO), mannose phosphate dehydrogenase (MPI), nucleoside phosphorylase (NSP), phosphoglucose isomerase (PGI) and phosphoglucomutase (PGM). For each enzyme, mobility variants (electromorphs), numbered in decreasing order of rate of anodal migration, were equated with alleles at the corresponding structural gene locus; the absence of enzyme activity was attributed to a null allele. Each isolate was characterized by its combination of alleles at the 18 enzyme loci. Distinctive combinations of alleles, corresponding to multilocus genotypes, were designated as electrophoretic types (ETs) (Selander et al., 1986
).
Statistical analysis.
Genetic diversity (h) among ETs at an enzyme locus was calculated from allele frequencies using the formula h=n(1- xi2)/(n-1), where xi is the frequency of the ith allele and n is the number of ETs (Nei, 1977
; Selander et al., 1986
). Mean genetic diversity (H) was calculated as the arithmetic mean of the h values for all 18 loci.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic diversity of the total population
In the total sample of 314 isolates, 9 of the 18 enzyme loci investigated were polymorphic, with 24 alleles. The other 9 loci were monomorphic (Table 1). The mean number of alleles per locus was 2·0.
|
|
Association of ET1 and ET2 with colony morphology and with serotype class
Of the 314 clinical isolates, 163 were small (sm), 60 large (la), 81 mucoid (M) and 10 dwarf (d). The distribution of the colony types into ET1 and ET2 was significantly different (Table 3): 78% of the mucoid strains were in ET2 whereas the other colony types were identically distributed in ET1 and ET2.
|
Of the 314 isolates, 139 were serologically non-typable, 128 agglutinated with polyvalent antisera (PAG), and 47 could be typed as O1, O3, O6 or O13. The distributions of strains of ET1 and ET2 among these three serotype classes were significantly different (Table 3). Disproportionately more isolates of ET1 were PAG and more isolates of ET2 were non-typable.
Geographical variation in ET representation and absence of association between ET and sex, age or clinical condition of patients
To assess the difference in ET representation between patients attending the principal care centres participating, the first isolate collected from each patient was selected to represent each of the 87 patients and was analysed. There was a significant difference between centres, mainly due to a preponderance of ET2 at Necker and of ET1 at Tours (Table 4, P<0·01).
|
|
|
For the 12 other patients, temporal change was observed. Patients T, Q, K, F, O, J, S and M (Table 6) had two types of strains in some sputa. Patients G, P, K, X, W, F, O, J and S (Table 6
) had strains belonging to different ETs at different times. For all these patients, it is possible that each type of strain could have been present throughout the study period but that not all were isolated from each sample. This may be the case, especially if one type was predominant or if colonies on the primary plate had the same morphology. Nevertheless, in most of the patients, strains of ET1 and/or of ET2 were preponderant.
Three pairs of sibling (N and G; Q and R; and J and S) were investigated. ET representation was very similar within each pair (Table 6). The only divergence was the appearance of ET3 in patient S, but not in sibling J, at the end of the study period.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results indicate that there is little genetic diversity at 18 loci for metabolic enzymes in a population of French P. aeruginosa CF strains. Three hundred and fourteen French CF isolates were studied and only 50% of the loci were polymorphic. Although 17 ETs were distinguished, H was only 0·138 and 92% of the strains clustered in two ETs. These findings contrast with those of an MLEE analysis of isolates recovered in an oncology ward in the Michael Reese Hospital, in Chicago, Illinois (Griffith et al., 1989 ). For the 172 isolates examined, 83% of the loci were polymorphic, 64 ETs were distinguished, and H for the ETs was 0·229. Other studies have also reported genetic heterogeneity of clinical isolates of P. aeruginosa strains (Levin et al., 1984
; Picard et al., 1994
).
There are many possible explanations of why French CF isolates of P. aeruginosa belong to two major groups of strains. One is that the CF patients were repeatedly hospitalized in the same centres and may have been cross-colonized. The correlation between ET distribution (ET1 and ET2) and geographical origin of the strains argues for this hypothesis. However, the two most prevalent ETs differed at only the SKD locus. And in addition, it is difficult to imagine that all 87 patients, who were treated at 20 different hospitals scattered across 29 of the 95 departments of France, did not encounter genetically diverse P. aeruginosa strains in their environment.
Another possible explanation of the existence of the two major clusters of strains is that particular groups of P. aeruginosa have been selected for their ability to adapt to the bronchopulmonary environment and to survive on the surface of the respiratory tract epithelium. The existence of strains specifically adapted to CF airways does not exclude the possibility that they are transmitted by cross-colonization and are responsible for other illnesses.
The relative homogeneity and stability of strains repeatedly recovered from sputa collected from 18 patients over periods of from 12 to 28 months (Table 6), and the absence of associations between ET distribution and the clinical status of patients, indicate that deterioration of the clinical status was not due to superinfection or to the emergence of particular virulent clones. Probably, the expression of virulence factors or the host reaction are more important to lung degradation than superinfections or changes in strain population.
Some studies exploring other parts of the genome of P. aeruginosa isolates from CF airways argue for heterogeneity and variability of isolates in the pulmonary flora of chronically infected CF patients. For example, DNA fingerprinting using arbitrarily primed polymerase chain reactions (AP-PCR) (Renders et al., 1997 ) or random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR) (Hoogkamp-Korstanje et al., 1995
) give a large variety of patterns. Macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) also detected the presence of multiple genetic differences between the genomes of different P. aeruginosa isolates (Römling et al., 1994
; Struelens et al., 1993
). RFLP of the rRNA gene region is consistent with heterogeneity of the rRNA operon (Bennekov et al., 1996
). In a previous study (Martin et al., 1995
), we characterized the genetic heterogeneity of rRNA operons of 87 of the P. aeruginosa strains included in this work using four restriction enzymes (BamHI, ClaI, EcoRI and PstI). Each isolate was from a different CF patient. Ribotyping and MLEE analysis give different views of the structure of the population of strains studied (Table 7
). There were many more ribotypes (31 ribotypes) than ETs (15 ETs, with 92% of the strains in two ETs) in this group of strains, and strains in one ET were of different ribotypes. The inconsistencies between the results of different studies may indicate a high gene flow rate within P. aeruginosa isolates as previously suggested (Denamur et al., 1993
).
|
In conclusion, our results suggest that limited groups of P. aeruginosa strains are able to colonize CF patients. To assess the position of CF P. aeruginosa strains within the far-flung world of P. aeruginosa species, isolates from other sources and from other geographical origins should be examined. In addition, a better understanding of the way in which different parts of the genome of CF P. aeruginosa isolates vary may help elucidate the particular relationship that exists between CF patients and this bacterium.
![]() |
ACKNOWLEDGEMENTS |
---|
This research was supported by grants from the association Anjou Mucoviscidose (to R.Q.), and in part by grant AI22144 from the national Institutes of Health (to R.K.S.).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blanc, D. S., Siegrist, H. H., Sahli, R. & Francioli, P. (1993). Ribotyping of Pseudomonas aeruginosa: discriminatory power and usefulness as a tool for epidemiological studies. J Clin Microbiol 31, 71-77.[Abstract]
Chatellier, S., Huet, H., Kenzi, S., Rosenau, A., Geslin, P. & Quentin, R. (1996). Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis. J Clin Microbiol 34, 2741-2747.[Abstract]
Denamur, E., Picard, B., Goullet, P., Bingen, E., Lambert, N. & Elion, J. (1991). Complexity of Pseudomonas aeruginosa infection in cystic fibrosis: combined results from esterase electrophoresis and rDNA restriction fragment length polymorphism analysis. Epidemiol Infect 106, 531-539.[Medline]
Denamur, E., Picard, B., Decoux, G., Denis, J. B. & Elion, J. (1993). The absence of correlation between alloenzyme and rrn analysis indicates a high gene flow rate within human clinical Pseudomonas aeruginosa isolates. FEMS Microbiol Lett 110, 275-280.[Medline]
Doershuk, C. F., Matthews, L. W., Tucker, A. S. & Spector, S. (1965). Evaluation of a prophylactic and therapeutic program for patients with cystic fibrosis. Pediatrics 36, 675-688.[Abstract]
Griffith, S. J., Nathan, C., Selander, R. K., Chamberlin, W., Gordon, S., Kabins, S. & Weinstein, R. A. (1989). The epidemiology of Pseudomonas aeruginosa in oncology patients in a general hospital. J Infect Dis 160, 1030-1036.[Medline]
Grothues, D., Koopmann, U., von der Hardt, H. & Tümmler, B. (1988). Genome fingerprinting of Pseudomonas aeruginosa indicates colonization of cystic fibrosis siblings with closely related strains. J Clin Microbiol 26, 1973-1977.[Medline]
Grüner, E., Kropec, A., Hubner, J., Altwegg, M. & Daschner, F. (1993). Ribotyping of Pseudomonas aeruginosa strains isolated from surgical intensive care patients. J Infect Dis 167, 1216-1220.[Medline]
Habs, I. (1957). Untersuchungen über die O-Antigene von Pseudomonas aeruginosa. Z Hyg Infektionskr 144, 218-228.[Medline]
Hoogkamp-Korstanje, J. A. A., Meis, J. F. G. M., Kissing, J., van der Laag, J. & Melchers, W. J. G. (1995). Risk of cross-colonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients. J Clin Microbiol 33, 572-575.[Abstract]
Levin, M. H., Weinstein, R. A., Nathan, C., Selander, R. K., Ochman, H. & Kabins, S. A. (1984). Association of infection caused by Pseudomonas aeruginosa serotype O11 with intravenous abuse of pentazocine mixed with tripelennamine. J Clin Microbiol 20, 758-762.[Medline]
Maher, W. E., Kobe, M. & Fass, R. J. (1993). Restriction endonuclease analysis of clinical Pseudomonas aeruginosa strains: useful epidemiologic data from a simple and rapid method. J Clin Microbiol 31, 1426-1429.[Abstract]
Martin, C., Aït Ichou, M., Massicot, P., Goudeau, A. & Quentin, R. (1995). Genetic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis revealed by restriction fragment length polymorphism of the ribosomal RNA gene region. J Clin Microbiol 33, 1461-1466.[Abstract]
Musser, J. M., Kroll, J. S., Moxon, E. R. & Selander, R. K. (1988). Evolutionary genetics of the encapsulated strains of Haemophilus influenzae. Proc Natl Acad Sci USA 85, 7758-7762.[Abstract]
Musser, J. M., Mattingly, S. J., Quentin, R., Goudeau, A. & Selander, R. K. (1989). Identification of a high-virulence clone of type III Streptococcus agalactiae (group B Streptococcus) causing invasive neonatal disease. Proc Natl Acad Sci USA 86, 4731-4735.[Abstract]
Musser, J. M., Hauser, A. R., Kim, M. H., Schlievert, P. M., Nelson, K. & Selander, R. K. (1991). Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA 88, 2668-2672.[Abstract]
Nei, M. (1977). F-statistics and analysis of gene diversity in subdivided populations. Ann Hum Genet 41, 225-233.[Medline]
Nelson, K. & Selander, R. K. (1992). Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli. J Bacteriol 174, 6686-6695.
Picard, B., Denamur, E., Barakat, A., Elion, J. & Goullet, P. (1994). Genetic heterogeneity of Pseudomonas aeruginosa clinical isolates revealed by esterase electrophoretic polymorphism and restriction fragment length of the ribosomal RNA gene region. J Med Microbiol 40, 313-322.[Abstract]
Quentin, R., Martin, C., Musser, J. M., Pasquier-Picard, N. & Goudeau, A. (1993). Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections. J Clin Microbiol 31, 1111-1116.[Abstract]
Quentin, R., Huet, H., Wang, F.-S., Geslin, P., Goudeau, A. & Selander, R. K. (1995). Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease. J Clin Microbiol 33, 2576-2581.[Abstract]
Quentin, R., Ruimy, R., Rosenau, A., Musser, J. M. & Christen, R. (1996). Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA. J Clin Microbiol 34, 1380-1385.[Abstract]
Renders, N. H. M., Sijmons, M. A. F., van Belkum, A., Overbeek, S. E., Mouton, J. W. & Verbrugh, H. A. (1997). Exchange of Pseudomonas aeruginosa strains among cystic fibrosis patients. Res Microbiol 148, 447-454.[Medline]
Römling, U., Fiedler, B., Boßhammer, J., Grothues, D., Greipel, J., von der Hardt, H. & Tümmler, B. (1994). Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis. J Infect Dis 170, 1616-1621.[Medline]
Schwachmann, H., Kulczycki, L. L. & Khaw, K. T. (1965). Studies in cystic fibrosis: a report on sixty-five patients over 17 years of age. Pediatrics 36, 689-699.[Medline]
Selander, R. K. & Musser, J. M. (1990). Population genetics of bacterial pathogenesis. In Molecular Basis of Bacterial Pathogenesis, pp. 11-36. Edited by B. H. Iglewski & V. L. Clark. San Diego: Academic Press.
Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M., Gilmour, M. N. & Whittman, T. S. (1986). Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 51, 873-884.[Medline]
Selander, R. K., Musser, J. M., Caugant, D. A., Gilmour, M. N. & Whittman, T. S. (1987). Population genetics of pathogenic bacteria. Microb Pathog 3, 1-7.[Medline]
Selander, R. K., Beltran, P., Smith, N. H., Barker, R. M., Crichton, P. B., Old, D. C., Musser, J. M. & Whittman, T. S. (1990). Genetic population structure, clonal phylogeny, and pathogenicity of Salmonella paratyphi B. Infect Immun 58, 1891-1901.[Medline]
Smith, N. H. & Selander, R. K. (1990). Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (flicC) among strains of Salmonella typhimurium. J Bacteriol 172, 603-609.[Medline]
Speert, D. P., Campbell, M. E., Farmer, S. W., Volpel, K., Joffe, A. M. & Paranchych, W. (1989). Use of a pilin gene probe to study molecular epidemiology of Pseudomonas aeruginosa. J Clin Microbiol 27, 2589-2593.[Medline]
Struelens, M. J., Schwam, V., Deplano, A. & Baran, D. (1993). Genome macrorestriction analysis of diversity and variability of Pseudomonas aeruginosa strains infecting cystic fibrosis. J Clin Microbiol 31, 2320-2326.[Abstract]
Véron, M. (1961). Sur lagglutination de Pseudomonas aeruginosa: subdivision des groupes antigéniques O:2 et O:5. Ann Inst Pasteur 101, 456-460.
Véron, M. & Berche, P. (1976). Virulence et antigènes de Pseudomonas aeruginosa. Bull Inst Pasteur 74, 295-337.
Wolz, C., Kiosz, G., Ogle, J. W., Vasil, M. L., Schaad, U., Botzenhart, K. & Döring, G. (1989). Pseudomonas aeruginosa cross-colonization and persistence in patients with cystic fibrosis. Use of a DNA probe. Epidemiol Infect 102, 205-214.[Medline]
Young, J. P. (1989). The population genetics of bacteria. In Genetics of Bacterial Diversity, pp. 417-438. Edited by D. A. Hopwood & K. E. Chater. London: Academic Press.
Received 3 November 1998;
revised 28 April 1999;
accepted 13 May 1999.