Aug 1, 1995 - Flavimonas oryzihabitans has emerged as a potential nosocomial pathogen in recent years. The typing method for characterization of this ...
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 68–70 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 1
Epidemiological Typing of Flavimonas oryzihabitans by PCR and Pulsed-Field Gel Electrophoresis PETER YUK-FONG LIU,* ZHI-YUAN SHI, YEU-JUN LAU, BOR-SHEN HU, JAINN-MING SHYR, WEN-SHIH TSAI, YU-HUI LIN, AND CHING-YU TSENG Section of Infectious Diseases, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China Received 13 June 1995/Returned for modification 1 August 1995/Accepted 12 October 1995
Flavimonas oryzihabitans has emerged as a potential nosocomial pathogen in recent years. The typing method for characterization of this species has never been reported before. Pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-based PCR were used to generate DNA fingerprints for 14 F. oryzihabitans isolates obtained from eight episodes of nosocomial infections during a 2-year period. Both techniques successfully classified these clinical isolates into eight distinct genotypes, thus indicating that all of these episodes of infections were independent. In contrast, repeated isolates from the same patient were assigned to identical genotypes. The reproducibility of both techniques was good. Therefore, we conclude that both PFGE and ERIC-PCR have comparable reproducible and discriminatory powers for the typing of F. oryzihabitans and may be useful for clarifying the epidemiology of this species; however, ERIC-PCR has the advantages of both speed and simplicity. lase production, esculin hydrolysis, tyrosine hydrolysis, and b-galactosidase production and by failure to grow at 428C (8). All isolates were maintained at 2708C in Trypticase soy broth with 10% glycerol for further analysis. The type strain of the species, ATCC 43272, was also included in this study for comparison. PFGE. Genome DNA was prepared as described previously (12). Chromosomal DNA plugs were incubated with XbaI, DraI, or SpeI (GIBCO-BRL Life Technologies, Gaithersburg, Md.). Restriction fragments were separated by PFGE with a CHEF-DRII apparatus (Bio-Rad Laboratories, Richmond, Calif.) through 1.2% SeaKem GTG agarose (FMC Bioproducts, Rockland, Maine) at a field strength of 6 V/cm for 22 h at 148C, while the pulse time being increased from 5 to 40 s. A lambda ladder (Bio-Rad Laboratories) was used as the molecular weight marker. PFGE chromosomal fingerprints were compared with the criteria described by Prevost et al. (17). Reproducibility of the PFGE profiles was examined by repeated testing of the same isolate on separate occasions. ERIC-PCR. ERIC-PCR was performed as previously described (11). A bacterial growth with the size of a rice grain was taken from nutrient agar and dispersed in 100 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). The cells were lysed with 500 ml of GES reagent (5 M guanidium thiocyanate [Sigma Chemical Co., St. Louis, Mo.], 0.1 M EDTA, 0.5% [wt/vol] sarcosyl [Sigma]). After addition of 250 ml of 7.5 M ammonium acetate, the extract was held on ice for 10 min. For deproteinization, 500 ml of chloroform-isoamyl alcohol (24:1) was added, and the extract was centrifuged at 13,000 3 g for 10 min. DNA was precipitated from the aqueous phase with ethanol at 2208C for 1 h. This extracted DNA (0.1 mg) was used as template for amplification. The primers were ERIC-1 (59-GTGAATCCCCAGGAGCTTACAT-39) and ERIC-2 (59-AAGTA
Flavimonas oryzihabitans, which was known previously as Pseudomonas oryzihabitans and which is a member of the Centers for Disease Control group Ve-2, is a gram-negative organism that was rarely implicated as a human pathogen before the 1980s. It has been recovered from many environmental sources such as rice paddies and is a saprophyte in human beings and several warm-blooded animals (2, 9). However, in recent years F. oryzihabitans has emerged as a potential nosocomial pathogen. The most common infections due to this organism include bacteremia, wound infections, prosthetic valve endocarditis, peritonitis in patients undergoing continuous ambulatory peritoneal dialysis, and meningitis in patients following neurosurgery (1, 3–7, 9, 10, 13–16, 18, 19). The presence of foreign material, including indwelling intravascular catheters and artificial grafts, appears to predispose patients to infection with F. oryzihabitans. From May 1993 to May 1994, we noticed an apparent increase in the rate of isolation of F. oryzihabitans from the Pediatric Unit of Taichung Veterans General Hospital, compared with the rates in other units in this hospital. To investigate the clonal relationship of these isolates, we evaluated two epidemiological typing techniques, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-based PCR (20), in the present study.
TABLE 1. Characteristics of the F. oryzihabitans clinical isolates studied Patient no.
MATERIALS AND METHODS Bacterial isolates. Between 1993 and 1995, 24 bacterial isolates collected from infected patients of Taichung Veterans General Hospital were identified as F. oryzihabitans in our laboratory by Vitek AutoMicrobic System (Vitek AMS; BioMerieux Vitek, Inc., Hazelwood, Mo.). Ten isolates were excluded because of the absence of typical yellow wrinkled colonies after 48 h of incubation, and their identification probability was low (,90%) when rechecked with API 20NE system (API-BioMerieux, La Balme les Grottes, France). The remaining 14 isolates, which were obtained from eight different episodes of nosocomial infections (Table 1), were confirmed to be oxidase negative. They were differentiated from Chryseomonas luteola by the negative results of the tests for arginine dihydro-
1 2 3 4 5 6 7
* Corresponding author. Mailing address: Section of Infectious Diseases, Taichung Veterans General Hospital, 160 Taichung Harbour Rd., Section 3, Taichung, Taiwan, Republic of China. Phone and Fax: 886-4-4611523.
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Isolate
Unit
Source
Date of isolation (mo/day/yr)
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14
Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Neurology Orthopedic Neurosurgery Neurosurgery Gynecology
Blood Blood Blood Blood Blood Blood Blood Blood Spinal fluid Spinal fluid Bone Blood Blood Wound
12/29/93 12/31/93 5/11/94 5/11/94 5/15/94 5/19/93 5/20/93 5/21/93 5/20/93 7/28/93 6/4/93 2/20/95 2/27/95 8/26/93
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TYPING OF F. ORYZIHABITANS BY PCR AND PFGE
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RESULTS AND DISCUSSION
FIG. 1. PFGE of SpeI-digested genomic DNA from F. oryzihabitans isolates. Lane M, a lambda ladder (Bio-Rad) which served as molecular marker; lane 1, DNA digest of type strain ATCC 43272; lanes 2 to 15, DNA digests of isolates S1 to S14, respectively (see Table 1 for the origins of the isolates).
AGTGACTGGGGTG AGCG-39). Amplification reactions were performed in a 100-ml final volume with 1 U of Taq polymerase (Super Taq; HT Biotechnology Ltd., Cambridge, England)–10 mM Tris (pH 8.3)–50 mM KCl–2.5 mM MgCl2– 0.01% (wt/vol) gelatin–250 mM (each) deoxynucleoside triphosphates–1 mM of single primer. Amplification was performed in a PHC-3 thermal cycler (Techne, Princeton, N.J.) with temperatures ramped as follows: 958C for 5 min to denature the template; four low-stringency cycles of 948C for 1 min, 268C for 1 min, and 728C for 2 min; 40 cycles of 948C for 30 s, 408C for 30 s, and 728C for 1 min; and finally, 728C for 10 min. A negative control was run with each experiment. Amplified products (10 ml) were analyzed by electrophoresis in a 1.6% agarose gel containing ethidium bromide (1 mg/ml) at 30 V for 6 h and were detected by UV transillumination. The PCR patterns were considered identical on the basis of similar numbers and matching positions of all major bands. Small differences in the intensities of faint bands were ignored. Day-to-day reproducibility was examined by comparing patterns obtained on 3 different days.
DNA analysis by PFGE after digestion with endonuclease SpeI revealed nine different restriction patterns (Fig. 1), among which were 14 isolates of F. oryzihabitans (Table 1) and the type strain of the species. Many patients (patients 1, 2, 3, and 7) had F. oryzihabitans isolated on more than one occasion. All isolates from each patient were identical by restriction enzyme digestion pattern. However, the restrictive patterns of strains isolated from different patients were distinct. Thus, it was shown that the eight episodes of F. oryzihabitans infections were unrelated. DNA restriction with the enzymes XbaI and DraI gave different patterns (not shown) but the same conclusion. The SpeI enzyme was chosen throughout this study because the patterns it generated are more easily compared. Repeated testing of the same isolate on separate occasions gave identical PFGE profiles, and the reproducibility of this technique for typing F. oryzihabitans was established. Amplification of genomic DNAs from F. oryzihabitans isolates with the ERIC-1 or ERIC-2 primer resulted in patterns consisting of three to eight distinct DNA fragments ranging from approximately 0.3 to 2.0 kbp (Fig. 2). With either primer, nine different PCR patterns were generated among the 14 clinical isolates and the type strain of the species. The results were completely concordant with those of PFGE. Day-to-day reproducibility was examined by comparing patterns obtained on 3 different days (Fig. 3). Good reproducibility was obtained with either primer. Although sometimes the bands produced were less intense, their overall positions and presence or absence were highly consistent. Stability was also confirmed by the identical patterns produced when the isolates had undergone multiple passages. To our knowledge, epidemiological typing for F. oryzihabitans has never been reported before. Traditional typing techniques, such as biotyping and antibiogram profiling, always have some limitations in characterizing bacterial strains. During the last decade, approaches at the molecular level such as PFGE and ribotyping have been used to assess the relatedness of bacterial isolates. Despite the broad applicability of these techniques, their use in clinical microbiology laboratories has been limited because of being both time-consuming and labor-
FIG. 2. ERIC-PCR products (A) and ERIC-2 PCR products (B) of F. oryzihabitans analyzed by 1.6% agarose gel electrophoresis. Lane M, 1-kb molecular weight marker (GIBCO-BRL); lane 1, product of type strain ATCC 43272; lanes 2 to 15, products of isolates S1 to S14, respectively (see Table 1 for the origins of the isolates); lane 16, negative control.
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FIG. 3. Day-to-day reproducibility of F. oryzihabitans fingerprints. (A) Three epidemiologically unrelated isolates, I, II, and III, amplified on 3 different days (d1, d2, and d3) with primer ERIC-1; (B) three epidemiologically unrelated isolates, I, II, and III, amplified on 3 different days (d1, d2, and d3) with primer ERIC-2. Lanes M, 1-kb molecular weight marker (GIBCO-BRL).
intensive. In this study, we have proved the usefulness of PFGE for the subtyping of F. oryzihabitans; however, because it is difficult to emulsify the colonies of this species into homogeneous suspensions, the bands generated by PFGE are sometimes distorted (Fig. 1), and repeated testing is necessary before the patterns can be compared with confidence. To circumvent these problems, a novel DNA fingerprinting strategy based on PCR amplification of variable-length chromosomal sequences with a variety of primers was developed. ERICbased PCR, which utilizes oligonucleotide primers matching palindromic repeated sequences described for enterobacteria to generate DNA fingerprints that discriminate bacterial strains (20), is one of these approaches. Our results show that such repetitive sequences thus exist in F. oryzihabitans, as in other enterobacteria, possibly with some minor variations. Under the four low-stringency cycles (with low annealing temperature) of amplification, the ERIC primers can anneal to the sequences that match less more easily and with more stability, thus allowing the amplification to proceed. The discriminatory power of ERIC-PCR for the F. oryzihabitans isolates examined to date was good, and it provided a result which agreed with those given by the PFGE technique yet was less skill-demanding and less time-consuming. The preliminary results of the present study show a wide diversity in F. oryzihabitans when it is defined by PCR-based fingerprints. However, the discriminatory power of this technique needs further evaluation by a study of a large population of F. oryzihabitans isolates. In conclusion, both molecular typing techniques, PFGE and ERIC-PCR, are useful for the characterization of F. oryzihabitans isolates, and the application of these techniques may help to clarify the epidemiology of F. oryzihabitans infections. ERICPCR is especially useful because of its speed and simplicity. ACKNOWLEDGMENTS We thank Meei-Fang Liu for her technical assistance. We also thank Hsin-Jyur Rehn, Meei-Rurng Liu, and Yu-Mei Huang for their help in outbreak investigation. REFERENCES 1. Bending, J. W. A., P. J. Mayes, D. E. Eyers, B. Holmes, and T. T. L. Chin. 1989. Flavimonas oryzihabitans (Pseudomonas oryzihabitans; CDC group Ve2): an emerging pathogen in peritonitis related to continuous ambulatory peritoneal dialysis? J. Clin. Microbiol. 27:217–218. 2. Chaudhry, H. J., P. E. Schoch, and B. A. Cunha. 1992. Flavimonas oryzihabitans (CDC Group Ve-2). Infect. Control Hosp. Epidemiol. 13:485–488. 3. Conlu, A., J. Rothman, H. Staszewski, P. E. Schoch, P. Domenico, S. M.
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