INTRODUCTION

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Pseudomonas aeruginosa is a major cause of serious nosocomial infections, particularly in immunocompromised patients, and is the most common pathogen associated with cystic fibrosis (4), in which chronic lung colonization is a major factor in morbidity and mortality. P. aeruginosa has also been isolated from a number of environmental sources, including water (5, 12, 15), soil (19), plants, such as barley roots (7) and stored onions (21), and a gasoline-contaminated aquifer (3). However, the main habitat of P. aeruginosa remains controversial. Although P. aeruginosa is widely thought to be ubiquitous, the frequency of recovery of P. aeruginosa isolates is often very low (5, 15). It is also not clear whether environmental and clinical isolates of P. aeruginosa are different (12, 16). This fact has significant consequences for the proposed environmental release of P. aeruginosa as a plant growth promoter (6) and for bioremediation, including bioremediation that involves stimulation of natural Pseudomonas populations (23).

Recently, molecular typing methods have become useful for separating strains belonging to the same species. For example, the bacterial flagellin gene provides a new way to differentiate or subtype strains (27). In P. aeruginosa, two types of flagellin protein have been identified. These two types have been designated type a flagellin and type b flagellin, which can be distinguished on the basis of molecular size and reactions with type-specific polyclonal and monoclonal antibodies (1). Unlike Salmonella flagellins, the type a and b flagellins of P. aeruginosa do not exhibit phase variation; a single strain produces a single type of flagellin, and no switching between types a and b has been observed. Oligonucleotide primers for PCR amplification of the flagellin genes of P. aeruginosa have been developed. Restriction fragment length polymorphism (RFLP) analysis of PCR products has been used to separate 64 P. aeruginosa clinical isolates into 13 groups (29), and the usefulness of this method as a tool for strain separation was demonstrated when it was employed to provide evidence that a -lactam-resistant strain had spread in a cystic fibrosis clinic (2).

In this paper we describe development of a method for isolating P. aeruginosa and characterization of strains obtained from a new experimental mushroom-growing unit and other sources in which a detailed analysis of flagellin gene sequences was performed. In addition, we discuss ways that flagellin gene variation may have occurred in P. aeruginosa.

	MATERIALS AND METHODS

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Bacterial strains and culture. The isolates used in this study and their sources are listed in Table 1. All of the clinical isolates were identified as P. aeruginosa by using the API-NE test (29). The following additional tests were performed on strains: ability to grow on nutrient agar at 42°C and production of soluble pigments on King's B medium (10). To confirm the identity of strains, the exotoxin A (ETA) gene was amplified from a selection of strains by using primers ETA1 and ETA2 as described by Khan and Cerniglia (9). All of the strains were cultured on nutrient agar plates at 30°C for 24 h and were stored at 4°C. Liquid cultures were prepared by using Luria broth inoculated with strains and grown at 30°C and 150 rpm for 24 h.

Isolation of P. aeruginosa from environmental samples. Samples were obtained from the mushroom-growing unit at Horticulture Research International, Wellesbourne, Warwickshire, United Kingdom, which opened in 1995, during August 1996 and were examined to determine whether P. aeruginosa was present. Samples of individual mushrooms (button stage), casing (a mushroom-growing medium), straw, and floor water were collected within the mushroom unit, and samples of straw were collected outside the unit, as were samples of the tap water supplied to the unit, unused casing, pasteurized compost, and chicken manure. Chicken manure and straw are components of the pasteurized compost that is used in the unit to grow mushrooms. Additional samples of casing and mushrooms were collected within the unit and straw samples were collected outside the unit in November 1996. Various soil samples from locations surrounding Horticulture Research International (Wellesbourne), including soil samples WQ, WQ1, and B1, were also analyzed. Water samples were obtained from the Coventry Canal, the River Avon (at Stratford upon Avon), and a pond site at Horticulture Research International (Wellesbourne).

PCR amplification of the flagellin gene. The central region of the P. aeruginosa flagellin gene was amplified by the method of Winstanley et al. (29). The conserved primers which were used, CW46 and CW45, bind at positions 146 and 2024 on the Pseudomonas putida Paw8 flagellin gene (28). After the PCR, aliquots (5 µl) of each reaction mixture were subjected to electrophoresis on a standard 0.7% (wt/vol) agarose gel containing 0.1 µg of ethidium bromide per ml to confirm the presence of an amplified product. Samples (5 µl) of the flagellin gene amplified products were digested in 15-µl (final volume) reaction mixtures with restriction enzymes MspI, HaeIII, CfoI, MboI, RsaI, and SalI. Digestion was performed by using the conditions recommended by the supplier (Life Technologies), and the digests were subjected to electrophoresis on 3% (wt/vol) MetaPhor agarose (Flowgen). The digested amplified products were electrophoresed alongside a PCR size marker (fragment sizes, 50, 150, 300, 500, 750, 1,000, 1,500, and 2,000 bp; R & D Systems).

Computer analysis of RFLP groups. Levels of pairwise similarity between isolates were calculated with a metric that assigned equal weight to each fragment length for which at least one of the isolates produced a fragment; a score of 1 was assigned when both isolates produced the fragment, and a score of 0 was assigned when only one isolate produced the fragment. A hierarchical cluster analysis was performed by using the unweighted pair group with mathematical average algorithm of the GENSTAT 5 package, and a dendrogram was constructed.

DNA sequencing. PCR products were purified by using a QIAquick PCR purification kit (Qiagen, Dorking, United Kingdom) according to the manufacturer's instructions. Cycle sequencing was performed by using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) with 0.2 µg of DNA per 200 bp of product and 10 ng of primer per reaction mixture. Samples were analyzed with an ABI automated sequencer (Applied Biosystems). The initial sequencing was performed by using primers CW45 and CW46. Internal primers were designed to determine the DNA sequence at intervals of 150 to 200 bp downstream from each previous reaction. The sequences obtained were compared to sequences obtained from databases and to each other by using the GAP, FASTA, and CLUSTALV packages of the Genetics Computer Group (University of Wisconsin). A DNA sequence composition and GCCG/CGGC motif analysis was performed by using Clone Manager for Windows 4.0 (S & E Software, State Line, Pa.).

Nucleotide sequence accession numbers. The GenBank accession numbers for the nucleotide sequences of the central regions of the flagellin genes determined in this study are X99098, X98280, X98281, X98461, X98462, X98463, X98464, and X98465.

	RESULTS AND Discussion

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Isolation of P. aeruginosa. During the first sampling period (August 1996), P. aeruginosa was isolated within the mushroom unit from casing, straw, floor water, and mushrooms and outside the unit from yard straw. No P. aeruginosa was detected in the tap water supplied to the unit or in the external samples of unused casing, pasteurized compost, or chicken manure. Strains were identified by PCR amplification of the ETA gene by using oligonucleotide primers ETA1 and ETA2, which gave a single 396-bp amplified product (ETA positive) for more than 150 isolates obtained from the mushroom-growing facility. Early in this work we found that using the selective medium alone was not sufficient to prevent isolation of many non-P. aeruginosa colonies. Although we found that a higher temperature could be used to select for P. aeruginosa, direct incubation at 42°C on a selective medium resulted in reduced levels of recovery. For example, the average number of CFU per plate obtained without a recovery period from the casing sample was 19, compared with an average of more than 200 CFU per plate when preincubation on King's B medium at 35°C for 4 h was included. These values may not represent a genuine increase in the level of recovery as the method of swabbing cells from one plate to another could also lead to an increase in the number of CFU. However, if detection is more important than enumeration, then inclusion of a recovery step can provide greater reliability. Certainly there is a need for further assessment and development of methods for isolation of P. aeruginosa, since the methods commonly used have been designed for use with clinical samples rather than for use with environmental samples.

Flagellin gene RFLP analysis. PCR amplification performed with oligonucleotide primers CW45 and CW46 gave a single amplified product with all of the ETA-positive strains of P. aeruginosa. Of the 45 mushroom unit ETA-positive strains tested, 43 had a 1.02-kb flagellin gene amplified product (type a flagellin) (29), while 2 had a 1.25-kb product (type b flagellin) (29). Both soil isolates had type b flagellins, whereas all of the aquifer isolates had type a flagellins. The results of the RFLP analysis of the flagellin gene products allowed us to place the two soil isolates, 45 mushroom unit isolates, 13 aquifer isolates, and five selected clinical isolates of P. aeruginosa into flagellin gene RFLP groups as described previously (29) (Table 1). Five of these groups, designated RFLP groups XIV, XV, XVI, XVII, and XVIII (Table 1), were not found in a previous study of clinical isolates of P. aeruginosa (29). Figure 1 shows all of the restriction patterns that have been observed to date when P. aeruginosa flagellin gene amplified products have been digested with restriction enzyme MboI. Although the majority of the new RFLP groups were the result of different combinations of previously described restriction patterns, one restriction fragment pattern that was not previously observed was obtained with MboI (Fig. 1, lane H) from the amplified flagellin genes of two clinical isolates (RFLP group XVII). A dendrogram showing the relationships among the 18 RFLP groups is shown in Fig. 2.

View larger version (83K): [in this window] [in a new window]   FIG. 1.   MboI-digested P. aeruginosa flagellin gene PCR products. The gel shows all of the patterns (patterns A through H) obtained with restriction enzyme MboI. Restriction pattern E is shown twice; it was derived once from a clinical isolate and once from a mushroom unit isolate. Lanes M contained a PCR marker (fragment sizes, 50, 150, 300, 500, 750, 1,000, 1,500, and 2,000 bp).

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Relationships among P. aeruginosa flagellin gene RFLP groups. The dendrogram shows the relationships among the 18 RFLP groups (groups I through XVIII). RFLP groups II, III, IV, VI, VIII, IX, X, XI, XII, and XIII were not observed in this study but have been identified previously (29).

Flagellin gene sequence variation. All seven strains used for flagellin gene sequencing were identified as ETA-positive P. aeruginosa strains. The estimated sizes of the PCR products representing most of the flagellin genes of seven P. aeruginosa strains were 1.02 kb (strains 590 and K701), 1.25 kb (strains 409, 593, C201, and E4193), and 2.0 kb (strain K979). The two type a sequences included in this study (the strain 590 and K701 sequences) aligned with more than 90% similarity with the previously published P. aeruginosa PAK sequence (22). The four type b flagellins were much larger, and their sequences aligned with the previously published type b sequences (20). The final strain, K979, produced an abnormally large central flagellin region that was 2,100 bp long. This sequence exhibited similarity to other flagellin sequences only in the N- and C-terminal regions. The overall levels of similarity of the P. aeruginosa type a and b and K979 flagellin sequences to each other and to previously published flagellin sequences are shown in Table 2.

Flagellin gene sequence composition analysis. In an attempt to determine how the three different P. aeruginosa flagellin genes may have arisen, the G+C contents of the genes and the frequency of the P. aeruginosa GCCG/CGGC motif (25) were compared. The central regions of the type a and b flagellin genes had an average G+C content of 63%, which is equivalent to the G+C content of the P. aeruginosa chromosome (13, 14). The G+C contents determined for 50-bp segments of the type a and b flagellin genes were also found to be similar throughout the gene (Fig. 3). The P. aeruginosa K979 flagellin gene is more than twice as big as the type a gene. The average G+C content of this gene was 53%, but the G+C content along its length varied (Fig. 3). In the first 500 bp the G+C content was high and equivalent to typical values obtained for P. aeruginosa. After the first 500 bp the G+C content declined and was restored to the high value only from 1,800 bp to the end of the gene. In addition to a low G+C content (53%), this gene had a very low frequency (1 in 102) of the GCCG/CGGC motif. In the type a flagellin gene this motif occurred at a frequency of 1 in 32, which was equivalent to the frequency in the P. aeruginosa chromosomal genes in the GenBank database (25). This frequency was only slightly lower in type b genes, 1 in 42 bases. If the GCCG/CGGC motif occurred at random in a sequence, then it would be apparent once in every 254 bases, yet in P. aeruginosa it occurs once in every 32 bases. The reason for the frequent occurrence of this motif in the P. aeruginosa chromosome is not known, but its presence does allow it to be used as an indicator for determining P. aeruginosa sequences within DNA. Both the low G+C content of the K979 flagellin gene and the low frequency of the GCCG/CGGC motif indicate that this gene is not typical of the P. aeruginosa chromosome and differs considerably from the type a and b flagellin genes. This suggests that the K979 flagellin gene has acquired an additional section of DNA from outside the P. aeruginosa chromosome, and the low G+C content of the region between 500 and 1,800 bp may indicate that this region is the most likely area where recombination has occurred (Fig. 3). The predicted amino acid sequence revealed 25-amino-acid repeats at positions 172 to 197 and 268 to 293. This corresponds to base pair positions starting at positions 516 and 804, which may also indicate that a recombination event occurred. Recombination of large segments of DNA within and between bacterial strains offers the most likely explanation for P. aeruginosa flagellin gene variation. This scenario is similar to what is believed to occur in the genus Salmonella, in which the flagellin gene (fliC) is highly polymorphic, especially among Salmonella enterica strains with genes containing segments of DNA from diverse origins (18).

View larger version (23K): [in this window] [in a new window]   FIG. 3.   G+C contents of strain 590, which has a type a flagellin (a), strain 409, which has a type b flagellin (b), and strain K979 (c). G+C contents were calculated for 50-bp intervals and were plotted along the length of the PCR product. The average G+C content of P. aeruginosa is indicated by the dotted lines.