Vol. 30, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1992, p. 2632-2637
0095-1137/92/102632-06$02.00/0
Outer Membrane Protein Profiles and Multilocus Enzyme Electrophoresis Analysis for Differentiation of Clinical Isolates of Proteus mirabilis and Proteus vulgaris TINA KAPPOS,' MICHAEL A. JOHN,2 ZAFAR HUSSAIN,2 AND MIGUEL A. VALVANO1* Department of Microbiology and Infection Control, Victoria Hospital,2 and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5CJ, Canada Received 1 April 1992/Accepted 14 July 1992 Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates ofP. mirabiis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.
Members of the genus Proteus are commonly present in the normal intestinal flora and become pathogenic only when they reach tissues outside the intestinal tract, particularly the urinary and biliary tracts, wounds and bums, and also the peritoneum, meninges, and lungs (18). Proteus species, especially P. mirabilis and less frequently P. vulgaris, are often isolated in cases of pyelonephritis (26). These infections are often acquired in a hospital and are observed in patients undergoing urologic manipulation or in patients with urinary tract obstruction (4, 31). Recurrent urinary tract infections with Proteus species either may be the result of reinfection with a new and different strain or may arise from a relapse because of failure to eliminate the original infecting strain. Highly specific typing schemes for Proteus species are needed to determine whether either of these possibilities explains recurrent infection in a specific patient and for epidemiological tracing of nosocomial strains. Several methods for typing Proteus strains have been reported in the literature. These involve the use of 0-specific typing (13, 19), biotyping and phage sensitivity (9, 21), proticine production and sensitivity (24), and Dienes typing (25). In one study, a combination of proticine production and sensitivity with 0-specific typing and Dienes typing has given the best results for discriminating among Proteus strains (27). Other investigators have found the use of bacteriophage typing useful in determining the epidemiology of infections caused by P. mirabilis, whereas the Dienes typing reaction was only useful at times (9). Unfortunately, all these typing schemes are restricted to centers that possess reference strains and panels of 0-specific antisera, bacteriophages, and/or proticine-producing strains. The study described here was designed to explore the use of outer membrane protein (MP) profiles derived by sodium dodecyl sulfate (SDS)-urea polyacrylamide gel electrophore*
Corresponding author.
sis (PAGE) and multilocus enzyme electrophoresis (MEE) analysis as means for differentiating among clinical isolates of Proteus spp. (10). MATERIALS AND METHODS
Bacterial strains, media, and chemicals. Forty-four clinical strains identified as P. mirabilis and 10 strains identified as P. vulgaris were kindly donated by D. Colby, University Hospital, London, Ontario, Canada. All the strains were from different patients, and they were identified to the species level by conventional biochemical tests as described previously (12, 18). Pure cultures were maintained on Luria agar slants at room temperature for working purposes, and stock cultures were stored in 20% glycerol at -110°C. All chemicals were purchased from Sigma Chemical Co., St. Louis, Mo. Preparation of outer membranes. Outer membranes were prepared by the method described by Achtman et al. (1), with some modifications. A loopful of culture taken from Luria agar slants was used to inoculate a 1-ml Luria broth tube; this was followed by overnight incubation at 37°C. A total of 500 ,ul of this culture was added to tubes containing 10 ml of Luria broth. Upon overnight incubation at 37°C, the cultures were centrifuged at 7,000 x g for 10 min. The sedimented cells were then suspended in 2.5 ml of 10 mM Tris-HCl (pH 7.0) and sonicated with an 80-s pulse by using a Sonifier Cell Disruptor 350 (Branson Ultrasonics Corp., Danbury, Conn.). After a brief centrifugation to pellet the unbroken cells, total membranes were pelleted by centrifugation at 40,000 x g for 30 min at 4°C. Pellets were carefully and thoroughly resuspended in 500 pl of 20 mM Tris-HCl with 1.5% Sarkosyl (N-laurylsarcosine sodium salt) and were incubated at room temperature for 20 min to solubilize linear MPs. Outer membranes were recovered by another cycle of centrifugation as described above, and the mem2632
VOL. 30, 1992
TYPING OF CLINICAL PROTEUS ISOLATES
TABLE 1. Enzyme electromorphs investigated in the Proteus strains
Enzyme
Abbreviation
No. of
electromorphs0
Aconitate hydratase 2 ACO Adenosine deaminase ADA 6 (11) Adenylate kinase AK 3 Alcohol dehydrogenase ADH 5 (7) Catalase CAT 3 Fumarate hydratase FUM 5 5 (1) Glucose 6-phosphate dehydrogenase G6P Glutamic oxaloacetic transaminase 5 GOT GPT 8 (6) Glutamic pyruvate transaminase 4 (4) G3P Glyceraldehyde 3-phosphate dehydrogenase HEX 4 Hexokinase IPO 6 Indophenyl oxidase 5 (13) Isocitrate dehydrogenase IDH Leucine aminopeptidase LAP 6 Nucleoside phosphorylase NSP 6 PGI 7 (1) Phosphoglucose isomerase a Includes null phenotypes. Values in parentheses are numbers of strains with a null phenotype.
brane pellets were washed with distilled water and stored at -200C. A rapid method of obtaining total proteins was carried out in a manner similar to that described by Senior and Voros (28). Briefly, 1.5-ml aliquots of an overnight culture were spun down, and cells were washed twice with phosphate saline buffer. After the last wash, cells were resuspended in 50 jtl of distilled water-50 ,ul of sample buffer and were used for gel electrophoresis (see below). PAGE. Polyacrylamide gels were cast by using acrylamide/bisacrylamide ratios of 30:0.8 and 4 M urea as described by Achtman et al. (1). Sample buffer consisted of 0.125 M Tris-HCl (pH 6.8), 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 10% (vol/vol) 2-mercaptoethanol, and 0.01% (wt/ vol) bromophenol blue. Prior to electrophoresis, membrane pellets were resuspended in 25 p,l of distilled water-25 p,l of sample buffer. Either outer membrane preparations or washed total cells were denatured by boiling for 5 min prior to loading onto the gels. Electrophoresis was carried out at 20 mA of constant current under previously described conditions (29, 30), and slabs were stained with Coomassie blue. MEE. Prior to the determination of enzymes, Proteus isolates were grown overnight on Columbia agar containing 5% (vol/vol) washed horse erythrocytes, and the cells were suspended in 8 ml of 0.2 mM Tris buffer (pH 8.0). After lysis by sonication and centrifugation at 15,000 x g for 15 min, the supernatants were divided into aliquots in small screw-cap vials and were stored frozen at -70°C. Electrophoresis was carried out by using horizontal starch gels by a previously described method (23). Specimens were loaded onto the gel, with Whatman no. 3 filter paper inserted in a continuous slit cut into the gel. Eighteen lysates were run on each gel, with GSB (sample buffer containing bromophenol blue) on either side of the gel to show migration of the front of the buffer line. During electrophoresis, a constant voltage of 125 V was maintained and the gel was cooled with a pan of ice. Following electrophoresis, three of four horizontal slices (2 mm thick) were cut from the gel with a thin wire and incubated individually at 37°C in various enzyme-staining solutions. The enzymes were stained as described previously (8, 23) and are listed in Table 1. Comparisons of the
am
b A B C D E F G HI I A B C D E m
kDa -97 -66
kDa 976645-
4ft
U
-
40. *.
31-
21-
2633
-Aw
mv 40
d* Amo-t
J~~:-45
a
0
-
..-1
1 4FIG. 1. MP patterns identified in P. mirabilis and P. vulganis isolates. MP profiles were determined by SDS-PAGE of partially purified outer MPs. (a) MP profiles of P. mirabilis MPml (lane A), MPm2 (lane B), MPm3 (lane C), MPm4 (lane D), MPm5 (lane E), MPm6 (lane F), MPm7 (lane G), MPm8 (lane H), and MPm9 (lane I). (b) MP profiles of P. vulganis MPv1 (lane A), MPv2 (lane B), MPv3 (lane C), MPv4 (lane D), MPv5 (lane E). m, molecular mass standards, as follows: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa).
relative mobilities of the enzymes were made visually by comparing them with each other on the gel slice. Alternative electrophoretic forms (electromorphs) of each enzyme were numbered in order of increasing relative anodal mobility (15). For strains that lacked the activity of a particular enzyme, a 0 (null) was assigned. Each strain was assigned an electrophoretic type (ET) on the basis of the combination of electromorphs for the enzyme tested, as described by Caugant et al. (7). Strains that expressed the same electromorphs for the 16 enzymes were included in the same ETs.
RESULTS
Outer membrane profile typing. The results of the SDSurea PAGE of outer MP preparations indicated the presence of different patterns among Proteus strains according to the number and relative mobilities of the major outer MPs which migrate in the region of the gel corresponding to 35 to 45 kDa (Fig. 1). Nine distinct MP patterns were identified among the clinical isolates of P. mirabilis, whereas five MP profiles were found among clinical isolates of P. vulgaris (Fig. 1). Most of the MP patterns were easily distinguishable. In some cases, such as in patterns 5 and 6, the major outer MPs displayed very similar migration rates (Fig. la, lanes E and F), but examination of the other minor proteins indicated that these MP patterns were different. Repeated protein preparations examined within a period of about 6 months revealed identical results, indicating that the MP patterns remain stable for at least that amount of time. Since the cell fractionation steps for obtaining enriched outer membrane fractions are somewhat time-consuming, we sought to examine the membrane pattern obtained from total cell lysates, as described by Senior and Voros for Morganella morganii (28). Using this method, the major outer MPs from the Proteus isolates could still be identified from the background created by the rest of the cellular proteins, and the migration
2634
KAPPOS ET AL.
J. CLIN. MICROBIOL.
TABLE 2. Distribution of MP profiles and ETs in
P. mirabilis. and P. vulgaris Species
profile
MP
strains
No. of
No. of
ETs
ET no.
P. mirabilis
MPml
25
18
MPm2 MPm3 MPm4
4 2 4 5 1 1 1 1
4 2 3 5 1 1 1 1
2-4, 6-7, 15-17,19-21, 24-25, 27-30, 34 5, 9,11, 31 3, 22 10,18, 35 1, 8,14, 32, 33 13 26 23 12
4 2 1 2 1
4 2 1 2 1
MPm5 MPm6 MPm7 MPm8
MPm9 P. vulgaris
MPvl MPv2 MPv3 MPv4 MPv5
2v, 5v, 8v, lOv 4v, 9v 7v 3v, 6v
lv
patterns of the major outer membranes were found to be identical to those observed with the more purified outer MP preparations (data not shown). However, the presence of a large number of proteins sometimes made it difficult to clearly discern differences between strains with similar MP profiles. The rapid method of protein preparation by using boiled cells could be used as an alternative method of examining membrane patterns if facilities for preparing outer membranes are not available. It is interesting that the MP patterns of P. mirabilis and P. vulgaris strains were different (Fig. 1). Therefore, MPm and MPv were chosen as the standard nomenclature to identify MP patterns from P. mirabilis and P. vulgaris, respectively. The majority of the P. mirabilis isolates were grouped into five MP patterns (Table 2). MPml, the most common pattern among the isolates of P. mirabilis, was found in 25 strains (57%) (Table 2 and Fig. la). Another 15 P. mirabilis strains were assigned to MPm patterns 2, 3, 4, and 5 (Table 2 and Fig. la, lanes B to E), whereas the remaining 4 isolates had unique MP patterns (Fig. la, lanes F to I). In the case of P. vulganis, four strains were assigned to the pattern MPvl (Table 2 and Fig. lb, lane A). Patterns MPv2 and MPv4 comprised two strains each, and the remaining two P. vulgaris isolates had unique MP patterns (Table 2 and Fig. lb, lanes B to E). MEE. All the clinical isolates of Proteus were also analyzed by MEE. We sought to use MEE to obtain an independent confirmation of the validity of MP profiles for strain differentiation and also to be able to subclassify the strains of P. mirabilis and P. vulgaris belonging to the most MP profiles. The 16 enzymes chosen (Table 1)
common
are known to occur in Escherichia coli and Shigella species (15, 16) and were also expected to be expressed by the Proteus isolates since members of this genus belong to the family Enterobacteriaceae. The conditions for enzyme analysis were those shown to be optimal for E. coli enzyme electrophoresis (16). All the enzymes assayed were detected in the majority of the Proteus isolates examined. The number of electromorphs detected for each enzyme among the P. mirabilis and P. vulgaris isolates is indicated in Table 1. CAT, ACO, AK, HEX, GOT, FUM, LAP, NSP, and IPO occurred in all isolates (enzyme abbreviations are listed in Table 1). The remaining enzymes were not always detected in all of the
isolates. No enzyme was found to be characteristic of either P. mirabilis or P. vulgaris. IDH was not found in seven isolates of P. mirabilis and six isolates P. vulgaris. ADA was not found in eight and three isolates of P. mirabilis and P. vulgaris, respectively, and ADH was absent from seven strains (13%). It is not possible to conclude whether these enzymes are indeed absent from certain Proteus strains or, alternatively, whether they are expressed as electromorphs which cannot be resolved under the conditions used for the electrophoresis. ETs consisting of a 16-digit number were generated for each isolate by using the 16 enzymes assayed. The distributions of ETs in P. mirabilis and P. vulgaris strains are given in Tables 3 and 4, respectively. Thirty-five distinct ETs were observed among the 44 isolates of P. mirabilis screened (Table 3). Only a few P. mirabilis ETs were common to more than one isolate, such as ET2 (two isolates), ET3 (five isolates), ET4 (four isolates), and ET10 (two isolates), whereas the remaining P. mirabilis strains each corresponded to a distinct ET (Table 3). In the case of P. vulgaris, 10 strains examined had 10 unique ETs (Table 4). The ETs expressed by isolates with the same MP profiles in P. mirabilis and P. vulgaris are shown in Table 2. The strains with the MPml pattern expressed 18 ETs, indicating that this group is genetically heterogeneous. Altogether, 40 isolates from the four most common MP groups of P. mirabilis were subgrouped into 30 ETs. Strains with unique MP patterns were also found to have unique ETs, except in the case of ET3, which was reproducibly found in four isolates with the MPml pattern and one isolate with the MPm3 pattern (Table 2) (data not shown). All P. vulgaris isolates had unique ETs, even though eight strains were grouped into three MPv patterns (Table 2).
DISCUSSION MP profile typing is a relatively simple and reliable typing technique which does not require any sophisticated equipment. The results reported here suggest that MP patterns can be used for the differentiation of clinical isolates of Proteus. Most of the P. mirabilis isolates (90%) fell into five MP patterns, and 8 of 10 P. vulgaris isolates were grouped into three MP patterns. The remaining P. mirabilis and P. vulgaris isolates had unique MP patterns. At present, we do not know whether the natural environment faced by these microorganisms could play some role in the selection of strains with specific MP patterns, the significance of which would require further investigation. However, it is likely that the distribution of MP patterns reflects local epidemiological factors. Previous studies by other investigators have shown that outer MP patterns correlate well with other indicators of clonal descendence (1-3, 5, 14, 15, 20, 30). MP patterns have been used for typing clinical strains of E. coli (1, 2), Shigella spp. (29), Haemophilus influenzae (3), Haemophilus pleuropulmoniae (14), Neisseria meningitidis (17), and Borrelia burgdorferi (5). Also, a protein typing system similar to that described in this report has recently been reported for clinical strains of M. morganii (28). Although the isolates examined in our study were obtained from different patients, we initiated a prospective study with sequential isolates from the same patients with urinary tract infections. Preliminary evidence shows that successive isolates of P. mirabilis obtained from two patients exhibited the same patterns, suggesting that in each of those cases reinfection with the same strain of P. mirabilis occurred (11). We
VOL. VTYPING OF CLINICAL PROTEUS ISOLATES 30, 1992
2635
TABLE 3. ETs identified in 44 isolates of P. mirabilis ET no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 a
Electromorph (isoenzyme)4
No. of
No.aof stramns
CAT
ACO
AK
G3P
G6P
HEX
IDH
ADH
GOT
FIM
LAP
NSP
ADA
IPO
PGI
GPT
1 2 5 4 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 3 2 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 0 0 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 0 1 1 1 1 1 1
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 4 1 1 1 3 0 1 1 1 2 0 1 0 1 0 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 3 4 1 1 1 1 1 1 1 0 1 1 1 3 1 0 1 1 1 0
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4 4 4 4 4 4 4 4 4 2 4 4 4 4 4 4
1 1 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 3 1 1 2 1 1 2
1 1 1 1 1 1 2 2 6 6 1 1 2 2 6 1 1 1 1 1 2 6 1 1 1 1 1 1 4 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 3 6 1 1 1 1 1 1
0 0 1 1 1 0 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 5 3 1 0 0 0 1 0
1 1 1 1 3 5 1 1 1 1 1 3 1 1 3 1 4 1 1 1 1 1 3 1 1 1 3 1 6 3 1 1 1 4 1
1 2 1 2 2 2 1 2 1 2 1 2 1 3 6 2 2 1 1 1 1 1 1 1 1 1 1 5 6 2 1 1 2 2 2
5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 7 0 5 2 0 0 5 4
The abbreviation for each enzyme is listed in Table 1. Numerical assignment of electromorphs is described in the text.
thus propose that MP profiles may have clinical application in the typing of Proteus species for cases of recurrent urinary tract infections in patients with numerous complications and also for typing isolates of epidemiological significance such as outbreak-related strains. MEE has been used extensively to analyze genetic variations in natural populations of bacteria since it gives a representative measure of relatedness between individual
members of a given species (7, 23). Thus, strains with the same ETs are also closely related in other characteristics such as outer MP profiles, biotypes, and serotypes (6, 14, 15, 22). To our knowledge, this is the first report of an analysis of MEE patterns in Proteus strains. Our results suggest that both P. mirabilis and P. vulgaris exhibit high genetic variability since 35 distinct ETs were identified in 44 strains of P. mirabilis and 10 ETs were found in 10 strains of P. vulgans.
TABLE 4. ETs identified in 10 isolates of P. ET no.'
lv 2v 3v 4v 5v 6v 7v 8v
9v lOv
No. of No.ofn sris CAT ACO
1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 2 1 1
1 1 1 2 1 2 2 2 2 2
vulgaris
~~~~~~~~~~~Electromorph (isoenzyme)b AK
G3P
G6P
HEX
IDH
ADH
GOT
RIM
LAP
NSP
ADA
IPO
PGI
GPT
1 2 2 1 1 2 2 2 2 2
1 1 1 0 2 1 1 2 0 1
4 1 1 1 3 1 1 3 1 1
2 1 1 2 2 1 2 1 2 1
0 0
0 0 0 1 1 1 1 3 0 1
4 1 1 3 2 5 2 1 2 1
1 1 1 1 5 1 4 1 1 4
2 2 3 2 1 5 2 3 2 2
1 2
1 0
1 2
1 1
3 0 2 4 3 3 0 3
2 1 2 5 1 2 1 2
3 1 1 6 1 4 5 3 0 1
0 3 6 0 4 6 3 3 0 2
vulgaris
0 0 3 1 0 0 0 0
5 1 1 4 1 2
v indicates the ET number for P. strains. b The abbreviation for each enzyme is listed in Table 1. Numerical assignment of electromorphs is described in the text.
a
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KAPPOS ET AL.
The somewhat higher genetic variability found in isolates of P. vulgaris could be related to the fact that P. vulgaris isolates are less frequently found in urinary tract infections caused by Proteus species (26). This could contribute to an apparently more pronounced variability in the ETs. The analysis of genetic variability in P. vulgaris strains requires a larger number of isolates than we used in this study. The observation that the same ET was found in strains with two distinct MP patterns may be interpreted as a case of convergent evolution or, alternatively, infectious transmission of outer MP-determining genes. Similar observations were reported to occur in genetically different strains of H. influenzae that expressed the same capsular antigens (20). The MP patterns taken alone may not be good indicators of genetic variability, since several different ETs were distinguished in strains classified as having the same MP patterns. Results of this and previous studies (2, 3, 17, 20) have shown that MP patterns can, however, be used for clinical applications in an attempt to ascertain whether a particular bacterial clone already identified is responsible for continued or recurrent infections in single patients or in outbreaks. The large number of ETs identified among the P. mirabilis and P. vulgaris strains offers a more precise means of differentiating Proteus strains of clinical interest. However, it may be impractical to perform MEE in many clinical laboratories because of the complexity of the procedure, which is labor intensive and time-consuming and which requires a considerable amount of standardization. Therefore, we propose that upon routine biochemical differentiation of specimens as P. mirabilis or P. vulgaris, clinically important strains can initially be distinguished by the use of MP patterns, and if more refined differentiation is required, MEE analysis can be applied only to the strains with identical MP profiles. At the moment we do not know how many different clonal groups are to be expected in clinical strains of Proteus spp. Additional typing systems such as 0-specific and proticine typing may be useful for comparing our results with those obtained by other methods used at other centers. Correlation of the prevalence of the most frequent patterns with antibiotic resistance and potential virulence factors in Proteus strains awaits further studies. ACKNOWLEDGMENTS
This work was supported in part by a Vicepresident (Research) Award from the University of Western Ontario (to M.A.V.). T.K. was supported by the Medical Students Summer Training Program,
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30. Valvano, M. A., R. P. Silver, and J. H. Crosa. 1986. Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Eschenichia coli Kl. Infect. Immun. 52:192-199. 31. Warren, J. W., D. Damron, J. H. Tenney, J. M. Hoopes, B. Deforge, and H. L. Muncie. 1987. Fever, bacteremia, and death as complications of bacteriuria in women with long-term urethral catheters. J. Infect. Dis. 155:1151-1158.
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1992, p. 2632-2637
0095-1137/92/102632-06$02.00/0
Outer Membrane Protein Profiles and Multilocus Enzyme Electrophoresis Analysis for Differentiation of Clinical Isolates of Proteus mirabilis and Proteus vulgaris TINA KAPPOS,' MICHAEL A. JOHN,2 ZAFAR HUSSAIN,2 AND MIGUEL A. VALVANO1* Department of Microbiology and Infection Control, Victoria Hospital,2 and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5CJ, Canada Received 1 April 1992/Accepted 14 July 1992 Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates ofP. mirabiis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.
Members of the genus Proteus are commonly present in the normal intestinal flora and become pathogenic only when they reach tissues outside the intestinal tract, particularly the urinary and biliary tracts, wounds and bums, and also the peritoneum, meninges, and lungs (18). Proteus species, especially P. mirabilis and less frequently P. vulgaris, are often isolated in cases of pyelonephritis (26). These infections are often acquired in a hospital and are observed in patients undergoing urologic manipulation or in patients with urinary tract obstruction (4, 31). Recurrent urinary tract infections with Proteus species either may be the result of reinfection with a new and different strain or may arise from a relapse because of failure to eliminate the original infecting strain. Highly specific typing schemes for Proteus species are needed to determine whether either of these possibilities explains recurrent infection in a specific patient and for epidemiological tracing of nosocomial strains. Several methods for typing Proteus strains have been reported in the literature. These involve the use of 0-specific typing (13, 19), biotyping and phage sensitivity (9, 21), proticine production and sensitivity (24), and Dienes typing (25). In one study, a combination of proticine production and sensitivity with 0-specific typing and Dienes typing has given the best results for discriminating among Proteus strains (27). Other investigators have found the use of bacteriophage typing useful in determining the epidemiology of infections caused by P. mirabilis, whereas the Dienes typing reaction was only useful at times (9). Unfortunately, all these typing schemes are restricted to centers that possess reference strains and panels of 0-specific antisera, bacteriophages, and/or proticine-producing strains. The study described here was designed to explore the use of outer membrane protein (MP) profiles derived by sodium dodecyl sulfate (SDS)-urea polyacrylamide gel electrophore*
Corresponding author.
sis (PAGE) and multilocus enzyme electrophoresis (MEE) analysis as means for differentiating among clinical isolates of Proteus spp. (10). MATERIALS AND METHODS
Bacterial strains, media, and chemicals. Forty-four clinical strains identified as P. mirabilis and 10 strains identified as P. vulgaris were kindly donated by D. Colby, University Hospital, London, Ontario, Canada. All the strains were from different patients, and they were identified to the species level by conventional biochemical tests as described previously (12, 18). Pure cultures were maintained on Luria agar slants at room temperature for working purposes, and stock cultures were stored in 20% glycerol at -110°C. All chemicals were purchased from Sigma Chemical Co., St. Louis, Mo. Preparation of outer membranes. Outer membranes were prepared by the method described by Achtman et al. (1), with some modifications. A loopful of culture taken from Luria agar slants was used to inoculate a 1-ml Luria broth tube; this was followed by overnight incubation at 37°C. A total of 500 ,ul of this culture was added to tubes containing 10 ml of Luria broth. Upon overnight incubation at 37°C, the cultures were centrifuged at 7,000 x g for 10 min. The sedimented cells were then suspended in 2.5 ml of 10 mM Tris-HCl (pH 7.0) and sonicated with an 80-s pulse by using a Sonifier Cell Disruptor 350 (Branson Ultrasonics Corp., Danbury, Conn.). After a brief centrifugation to pellet the unbroken cells, total membranes were pelleted by centrifugation at 40,000 x g for 30 min at 4°C. Pellets were carefully and thoroughly resuspended in 500 pl of 20 mM Tris-HCl with 1.5% Sarkosyl (N-laurylsarcosine sodium salt) and were incubated at room temperature for 20 min to solubilize linear MPs. Outer membranes were recovered by another cycle of centrifugation as described above, and the mem2632
VOL. 30, 1992
TYPING OF CLINICAL PROTEUS ISOLATES
TABLE 1. Enzyme electromorphs investigated in the Proteus strains
Enzyme
Abbreviation
No. of
electromorphs0
Aconitate hydratase 2 ACO Adenosine deaminase ADA 6 (11) Adenylate kinase AK 3 Alcohol dehydrogenase ADH 5 (7) Catalase CAT 3 Fumarate hydratase FUM 5 5 (1) Glucose 6-phosphate dehydrogenase G6P Glutamic oxaloacetic transaminase 5 GOT GPT 8 (6) Glutamic pyruvate transaminase 4 (4) G3P Glyceraldehyde 3-phosphate dehydrogenase HEX 4 Hexokinase IPO 6 Indophenyl oxidase 5 (13) Isocitrate dehydrogenase IDH Leucine aminopeptidase LAP 6 Nucleoside phosphorylase NSP 6 PGI 7 (1) Phosphoglucose isomerase a Includes null phenotypes. Values in parentheses are numbers of strains with a null phenotype.
brane pellets were washed with distilled water and stored at -200C. A rapid method of obtaining total proteins was carried out in a manner similar to that described by Senior and Voros (28). Briefly, 1.5-ml aliquots of an overnight culture were spun down, and cells were washed twice with phosphate saline buffer. After the last wash, cells were resuspended in 50 jtl of distilled water-50 ,ul of sample buffer and were used for gel electrophoresis (see below). PAGE. Polyacrylamide gels were cast by using acrylamide/bisacrylamide ratios of 30:0.8 and 4 M urea as described by Achtman et al. (1). Sample buffer consisted of 0.125 M Tris-HCl (pH 6.8), 4% (wt/vol) SDS, 20% (vol/vol) glycerol, 10% (vol/vol) 2-mercaptoethanol, and 0.01% (wt/ vol) bromophenol blue. Prior to electrophoresis, membrane pellets were resuspended in 25 p,l of distilled water-25 p,l of sample buffer. Either outer membrane preparations or washed total cells were denatured by boiling for 5 min prior to loading onto the gels. Electrophoresis was carried out at 20 mA of constant current under previously described conditions (29, 30), and slabs were stained with Coomassie blue. MEE. Prior to the determination of enzymes, Proteus isolates were grown overnight on Columbia agar containing 5% (vol/vol) washed horse erythrocytes, and the cells were suspended in 8 ml of 0.2 mM Tris buffer (pH 8.0). After lysis by sonication and centrifugation at 15,000 x g for 15 min, the supernatants were divided into aliquots in small screw-cap vials and were stored frozen at -70°C. Electrophoresis was carried out by using horizontal starch gels by a previously described method (23). Specimens were loaded onto the gel, with Whatman no. 3 filter paper inserted in a continuous slit cut into the gel. Eighteen lysates were run on each gel, with GSB (sample buffer containing bromophenol blue) on either side of the gel to show migration of the front of the buffer line. During electrophoresis, a constant voltage of 125 V was maintained and the gel was cooled with a pan of ice. Following electrophoresis, three of four horizontal slices (2 mm thick) were cut from the gel with a thin wire and incubated individually at 37°C in various enzyme-staining solutions. The enzymes were stained as described previously (8, 23) and are listed in Table 1. Comparisons of the
am
b A B C D E F G HI I A B C D E m
kDa -97 -66
kDa 976645-
4ft
U
-
40. *.
31-
21-
2633
-Aw
mv 40
d* Amo-t
J~~:-45
a
0
-
..-1
1 4FIG. 1. MP patterns identified in P. mirabilis and P. vulganis isolates. MP profiles were determined by SDS-PAGE of partially purified outer MPs. (a) MP profiles of P. mirabilis MPml (lane A), MPm2 (lane B), MPm3 (lane C), MPm4 (lane D), MPm5 (lane E), MPm6 (lane F), MPm7 (lane G), MPm8 (lane H), and MPm9 (lane I). (b) MP profiles of P. vulganis MPv1 (lane A), MPv2 (lane B), MPv3 (lane C), MPv4 (lane D), MPv5 (lane E). m, molecular mass standards, as follows: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa).
relative mobilities of the enzymes were made visually by comparing them with each other on the gel slice. Alternative electrophoretic forms (electromorphs) of each enzyme were numbered in order of increasing relative anodal mobility (15). For strains that lacked the activity of a particular enzyme, a 0 (null) was assigned. Each strain was assigned an electrophoretic type (ET) on the basis of the combination of electromorphs for the enzyme tested, as described by Caugant et al. (7). Strains that expressed the same electromorphs for the 16 enzymes were included in the same ETs.
RESULTS
Outer membrane profile typing. The results of the SDSurea PAGE of outer MP preparations indicated the presence of different patterns among Proteus strains according to the number and relative mobilities of the major outer MPs which migrate in the region of the gel corresponding to 35 to 45 kDa (Fig. 1). Nine distinct MP patterns were identified among the clinical isolates of P. mirabilis, whereas five MP profiles were found among clinical isolates of P. vulgaris (Fig. 1). Most of the MP patterns were easily distinguishable. In some cases, such as in patterns 5 and 6, the major outer MPs displayed very similar migration rates (Fig. la, lanes E and F), but examination of the other minor proteins indicated that these MP patterns were different. Repeated protein preparations examined within a period of about 6 months revealed identical results, indicating that the MP patterns remain stable for at least that amount of time. Since the cell fractionation steps for obtaining enriched outer membrane fractions are somewhat time-consuming, we sought to examine the membrane pattern obtained from total cell lysates, as described by Senior and Voros for Morganella morganii (28). Using this method, the major outer MPs from the Proteus isolates could still be identified from the background created by the rest of the cellular proteins, and the migration
2634
KAPPOS ET AL.
J. CLIN. MICROBIOL.
TABLE 2. Distribution of MP profiles and ETs in
P. mirabilis. and P. vulgaris Species
profile
MP
strains
No. of
No. of
ETs
ET no.
P. mirabilis
MPml
25
18
MPm2 MPm3 MPm4
4 2 4 5 1 1 1 1
4 2 3 5 1 1 1 1
2-4, 6-7, 15-17,19-21, 24-25, 27-30, 34 5, 9,11, 31 3, 22 10,18, 35 1, 8,14, 32, 33 13 26 23 12
4 2 1 2 1
4 2 1 2 1
MPm5 MPm6 MPm7 MPm8
MPm9 P. vulgaris
MPvl MPv2 MPv3 MPv4 MPv5
2v, 5v, 8v, lOv 4v, 9v 7v 3v, 6v
lv
patterns of the major outer membranes were found to be identical to those observed with the more purified outer MP preparations (data not shown). However, the presence of a large number of proteins sometimes made it difficult to clearly discern differences between strains with similar MP profiles. The rapid method of protein preparation by using boiled cells could be used as an alternative method of examining membrane patterns if facilities for preparing outer membranes are not available. It is interesting that the MP patterns of P. mirabilis and P. vulgaris strains were different (Fig. 1). Therefore, MPm and MPv were chosen as the standard nomenclature to identify MP patterns from P. mirabilis and P. vulgaris, respectively. The majority of the P. mirabilis isolates were grouped into five MP patterns (Table 2). MPml, the most common pattern among the isolates of P. mirabilis, was found in 25 strains (57%) (Table 2 and Fig. la). Another 15 P. mirabilis strains were assigned to MPm patterns 2, 3, 4, and 5 (Table 2 and Fig. la, lanes B to E), whereas the remaining 4 isolates had unique MP patterns (Fig. la, lanes F to I). In the case of P. vulganis, four strains were assigned to the pattern MPvl (Table 2 and Fig. lb, lane A). Patterns MPv2 and MPv4 comprised two strains each, and the remaining two P. vulgaris isolates had unique MP patterns (Table 2 and Fig. lb, lanes B to E). MEE. All the clinical isolates of Proteus were also analyzed by MEE. We sought to use MEE to obtain an independent confirmation of the validity of MP profiles for strain differentiation and also to be able to subclassify the strains of P. mirabilis and P. vulgaris belonging to the most MP profiles. The 16 enzymes chosen (Table 1)
common
are known to occur in Escherichia coli and Shigella species (15, 16) and were also expected to be expressed by the Proteus isolates since members of this genus belong to the family Enterobacteriaceae. The conditions for enzyme analysis were those shown to be optimal for E. coli enzyme electrophoresis (16). All the enzymes assayed were detected in the majority of the Proteus isolates examined. The number of electromorphs detected for each enzyme among the P. mirabilis and P. vulgaris isolates is indicated in Table 1. CAT, ACO, AK, HEX, GOT, FUM, LAP, NSP, and IPO occurred in all isolates (enzyme abbreviations are listed in Table 1). The remaining enzymes were not always detected in all of the
isolates. No enzyme was found to be characteristic of either P. mirabilis or P. vulgaris. IDH was not found in seven isolates of P. mirabilis and six isolates P. vulgaris. ADA was not found in eight and three isolates of P. mirabilis and P. vulgaris, respectively, and ADH was absent from seven strains (13%). It is not possible to conclude whether these enzymes are indeed absent from certain Proteus strains or, alternatively, whether they are expressed as electromorphs which cannot be resolved under the conditions used for the electrophoresis. ETs consisting of a 16-digit number were generated for each isolate by using the 16 enzymes assayed. The distributions of ETs in P. mirabilis and P. vulgaris strains are given in Tables 3 and 4, respectively. Thirty-five distinct ETs were observed among the 44 isolates of P. mirabilis screened (Table 3). Only a few P. mirabilis ETs were common to more than one isolate, such as ET2 (two isolates), ET3 (five isolates), ET4 (four isolates), and ET10 (two isolates), whereas the remaining P. mirabilis strains each corresponded to a distinct ET (Table 3). In the case of P. vulgaris, 10 strains examined had 10 unique ETs (Table 4). The ETs expressed by isolates with the same MP profiles in P. mirabilis and P. vulgaris are shown in Table 2. The strains with the MPml pattern expressed 18 ETs, indicating that this group is genetically heterogeneous. Altogether, 40 isolates from the four most common MP groups of P. mirabilis were subgrouped into 30 ETs. Strains with unique MP patterns were also found to have unique ETs, except in the case of ET3, which was reproducibly found in four isolates with the MPml pattern and one isolate with the MPm3 pattern (Table 2) (data not shown). All P. vulgaris isolates had unique ETs, even though eight strains were grouped into three MPv patterns (Table 2).
DISCUSSION MP profile typing is a relatively simple and reliable typing technique which does not require any sophisticated equipment. The results reported here suggest that MP patterns can be used for the differentiation of clinical isolates of Proteus. Most of the P. mirabilis isolates (90%) fell into five MP patterns, and 8 of 10 P. vulgaris isolates were grouped into three MP patterns. The remaining P. mirabilis and P. vulgaris isolates had unique MP patterns. At present, we do not know whether the natural environment faced by these microorganisms could play some role in the selection of strains with specific MP patterns, the significance of which would require further investigation. However, it is likely that the distribution of MP patterns reflects local epidemiological factors. Previous studies by other investigators have shown that outer MP patterns correlate well with other indicators of clonal descendence (1-3, 5, 14, 15, 20, 30). MP patterns have been used for typing clinical strains of E. coli (1, 2), Shigella spp. (29), Haemophilus influenzae (3), Haemophilus pleuropulmoniae (14), Neisseria meningitidis (17), and Borrelia burgdorferi (5). Also, a protein typing system similar to that described in this report has recently been reported for clinical strains of M. morganii (28). Although the isolates examined in our study were obtained from different patients, we initiated a prospective study with sequential isolates from the same patients with urinary tract infections. Preliminary evidence shows that successive isolates of P. mirabilis obtained from two patients exhibited the same patterns, suggesting that in each of those cases reinfection with the same strain of P. mirabilis occurred (11). We
VOL. VTYPING OF CLINICAL PROTEUS ISOLATES 30, 1992
2635
TABLE 3. ETs identified in 44 isolates of P. mirabilis ET no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 a
Electromorph (isoenzyme)4
No. of
No.aof stramns
CAT
ACO
AK
G3P
G6P
HEX
IDH
ADH
GOT
FIM
LAP
NSP
ADA
IPO
PGI
GPT
1 2 5 4 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 3 2 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 0 0 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 0 1 1 1 1 1 1
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 4 1 1 1 3 0 1 1 1 2 0 1 0 1 0 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 3 4 1 1 1 1 1 1 1 0 1 1 1 3 1 0 1 1 1 0
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 4 4 4 4 4 4 4 4 4 4 4 4 4 2 4 4 4 4 4 4
1 1 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 3 1 1 2 1 1 2
1 1 1 1 1 1 2 2 6 6 1 1 2 2 6 1 1 1 1 1 2 6 1 1 1 1 1 1 4 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 3 6 1 1 1 1 1 1
0 0 1 1 1 0 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 5 3 1 0 0 0 1 0
1 1 1 1 3 5 1 1 1 1 1 3 1 1 3 1 4 1 1 1 1 1 3 1 1 1 3 1 6 3 1 1 1 4 1
1 2 1 2 2 2 1 2 1 2 1 2 1 3 6 2 2 1 1 1 1 1 1 1 1 1 1 5 6 2 1 1 2 2 2
5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 7 0 5 2 0 0 5 4
The abbreviation for each enzyme is listed in Table 1. Numerical assignment of electromorphs is described in the text.
thus propose that MP profiles may have clinical application in the typing of Proteus species for cases of recurrent urinary tract infections in patients with numerous complications and also for typing isolates of epidemiological significance such as outbreak-related strains. MEE has been used extensively to analyze genetic variations in natural populations of bacteria since it gives a representative measure of relatedness between individual
members of a given species (7, 23). Thus, strains with the same ETs are also closely related in other characteristics such as outer MP profiles, biotypes, and serotypes (6, 14, 15, 22). To our knowledge, this is the first report of an analysis of MEE patterns in Proteus strains. Our results suggest that both P. mirabilis and P. vulgaris exhibit high genetic variability since 35 distinct ETs were identified in 44 strains of P. mirabilis and 10 ETs were found in 10 strains of P. vulgans.
TABLE 4. ETs identified in 10 isolates of P. ET no.'
lv 2v 3v 4v 5v 6v 7v 8v
9v lOv
No. of No.ofn sris CAT ACO
1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 2 1 1
1 1 1 2 1 2 2 2 2 2
vulgaris
~~~~~~~~~~~Electromorph (isoenzyme)b AK
G3P
G6P
HEX
IDH
ADH
GOT
RIM
LAP
NSP
ADA
IPO
PGI
GPT
1 2 2 1 1 2 2 2 2 2
1 1 1 0 2 1 1 2 0 1
4 1 1 1 3 1 1 3 1 1
2 1 1 2 2 1 2 1 2 1
0 0
0 0 0 1 1 1 1 3 0 1
4 1 1 3 2 5 2 1 2 1
1 1 1 1 5 1 4 1 1 4
2 2 3 2 1 5 2 3 2 2
1 2
1 0
1 2
1 1
3 0 2 4 3 3 0 3
2 1 2 5 1 2 1 2
3 1 1 6 1 4 5 3 0 1
0 3 6 0 4 6 3 3 0 2
vulgaris
0 0 3 1 0 0 0 0
5 1 1 4 1 2
v indicates the ET number for P. strains. b The abbreviation for each enzyme is listed in Table 1. Numerical assignment of electromorphs is described in the text.
a
2636
KAPPOS ET AL.
The somewhat higher genetic variability found in isolates of P. vulgaris could be related to the fact that P. vulgaris isolates are less frequently found in urinary tract infections caused by Proteus species (26). This could contribute to an apparently more pronounced variability in the ETs. The analysis of genetic variability in P. vulgaris strains requires a larger number of isolates than we used in this study. The observation that the same ET was found in strains with two distinct MP patterns may be interpreted as a case of convergent evolution or, alternatively, infectious transmission of outer MP-determining genes. Similar observations were reported to occur in genetically different strains of H. influenzae that expressed the same capsular antigens (20). The MP patterns taken alone may not be good indicators of genetic variability, since several different ETs were distinguished in strains classified as having the same MP patterns. Results of this and previous studies (2, 3, 17, 20) have shown that MP patterns can, however, be used for clinical applications in an attempt to ascertain whether a particular bacterial clone already identified is responsible for continued or recurrent infections in single patients or in outbreaks. The large number of ETs identified among the P. mirabilis and P. vulgaris strains offers a more precise means of differentiating Proteus strains of clinical interest. However, it may be impractical to perform MEE in many clinical laboratories because of the complexity of the procedure, which is labor intensive and time-consuming and which requires a considerable amount of standardization. Therefore, we propose that upon routine biochemical differentiation of specimens as P. mirabilis or P. vulgaris, clinically important strains can initially be distinguished by the use of MP patterns, and if more refined differentiation is required, MEE analysis can be applied only to the strains with identical MP profiles. At the moment we do not know how many different clonal groups are to be expected in clinical strains of Proteus spp. Additional typing systems such as 0-specific and proticine typing may be useful for comparing our results with those obtained by other methods used at other centers. Correlation of the prevalence of the most frequent patterns with antibiotic resistance and potential virulence factors in Proteus strains awaits further studies. ACKNOWLEDGMENTS
This work was supported in part by a Vicepresident (Research) Award from the University of Western Ontario (to M.A.V.). T.K. was supported by the Medical Students Summer Training Program,
University of Western Ontario. We express our gratitude to J. Welsh, L. Dafoe, and D. Moyles for technical assistance. REFERENCES 1. Achtman, M., A. Mercer, B. Kusecek, A. Pohl, M. Heuzenroeder, W. Aaronson, A. Sutton, and R. P. Silver. 1983. Six widespread bacterial clones among Escherichia coli Kl isolates. Infect. Immun. 39:315-335. 2. Achtman, M., and G. Pluschke. 1986. Clonal analysis of descent and virulence among selected Escherichia coli. Annu. Rev. Microbiol. 40:185-210. 3. Barenkamp, S. J., R. S. Munson, and D. M. Granoff. 1981. Subtyping isolates of Haemophilus influenzae type b by outer membrane protein profiles. J. Infect. Dis. 143:668-676. 4. Bergeron, M. G. 1985. Urinary tract infection, p. 253-256. In L. A. Mandell and E. D. Ralph (ed.), Essentials of infectious diseases. Blackwell Scientific Publications, Boston. 5. Bundoc, V. G., and A. G. Barbour. 1989. Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorfeni. Infect.
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multityping scheme for Proteus mirabilis and Proteus vulgaris. J. Med. Microbiol. 16:193-202. 28. Senior, B. W., and S. Voros. 1990. Protein profile typing. A new method for typing Morganella morganii strains. J. Med. Microbiol. 33:259-264. 29. Valvano, M. A., and C. L. Marolda. 1991. Relatedness of 0-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli 07:K1. Infect. Immun. 59:3917-3923.
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