Outbreak of Gut Colonization by Pseudomonas aeruginosa in ...

Vol. 29, No. 9

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 2068-2071

0095-1137/91/092068-04$02.00/0 Copyright © 1991, American Society for Microbiology

Outbreak of Gut Colonization by Pseudomonas aeruginosa in Immunocompromised Children Undergoing Total Digestive Decontamination: Analysis by Pulsed-Field Electrophoresis JALEL BOUKADIDA,1 MARIANE DE MONTALEMBERT,l JEAN-LOUIS GAILLARD,' JOVANA GOBIN,1 FRANCINE GRIMONT,2 DANIELE GIRAULT,3 MICHEL VERON,1 AND PATRICK BERCHEl* Laboratoire de Microbiologiel and Service d'Immuno-Hematologie,3 H6pital Necker-Enfants Malades, rue de Sevres 75015, and Centre National de Typage Moleculaire Ente'rique, Unite' des Ente'robacteries, Institut National de la Sante et de la Recherche U.199, Institut Pasteur,2 Paris, France Received 22 April 1991/Accepted 12 June 1991

We analyzed an outbreak of gut colonization by Pseudomonas aeruginosa occurring in an intensive care hematology unit by using conventional typing methods and pulsed-field electrophoresis. In October and November 1989, the feces of four immunocompromised children undergoing total digestive decontamination were colonized by P. aeruginosa. Ten isolates were obtained from the gut flora in pure culture. Retrospective investigations found that one P. aeruginosa isolate from stools of one of the patients was already present at high counts 6 months before the outbreak. This patient had been discharged from the unit in May 1989 and had been readmitted concomitantly with the outbreak. Only pulsed-field electrophoresis could demonstrate that a single epidemic strain was present in the fecal flora of the children. This strain had probably been brought into the unit by the patient with chronic fecal carriage.

nostics Pasteur, Marnes-La-Coquette, France). The presence of P. aeruginosa was associated in three cases with concomitant symptoms, including diarrhea, fever, or recurrent episodes of intestinal ileus (Table 1). Samples of stools and blood were regularly taken in the four patients for surveillance cultures. P. aeruginosa disappeared from the feces within 1 to 4 weeks following appropriate systemic antibiotherapy. No bacteremia due to this bacterial species was observed. Bacterial investigation in the intensive care unit failed to isolate P. aeruginosa from the close environment of the patients (tap water, sinks, antiseptic solutions, and enteral alimentation). Stool specimens were obtained from the other children on the same ward. They were cultured on blood agar and cetrimide agar (minimal detection limit, 102 CFU/g of feces). Fecal carriage of P. aeruginosa was detected in two patients who were not receiving pro-

Pseudomonas aeruginosa is a major cause of nosocomial infections, especially in immunocompromised patients (10). The epidemiology of these infections is usually studied by the analysis of phenotypic markers of P. aeruginosa, including serovars, pyocin types, phage types, and antibiotic susceptibility patterns (12). Other methods consist of analyzing the DNA polymorphism of isolates. Restriction fragment length polymorphism types can be determined by hybridizing electrophoresed chromosomal DNA with Pseudomonas gene probes (8, 11, 14, 17). More recently, pulsedfield electrophoresis has also been used. Indeed, this method allows the separation of large DNA fragments (50 to 9,000 kb) after digestion with low-frequency-cleavage restriction endonucleases (2, 3, 16). This approach has already been applied to the study of bacterial strains and has been proved to be useful for investigations of the molecular epidemiology of P. aeruginosa infections (1, 5). This report describes the analysis of an outbreak of gut colonization by P. aeruginosa occurring in an intensive care hematology unit by using conventional methods and pulsed-field electrophoresis. In October and November 1989, we observed the sudden emergence of P. aeruginosa in stools of children hospitalized in the Immunology and Hematology Unit of our hospital (Pediatric Department, HOpital Necker-Enfants Malades, Paris, France) (Table 1). During this period, the feces of four patients were colonized only by P. aeruginosa. Ten isolates were thus obtained in pure culture from the gut flora. This was surprising, since the four patients were all receiving prophylactic oral antibiotics (vancomycin [50 mg], tobramycin [50 mg], and colistin [500,000 IU], given every 8 h) at the time of the study. The stools of patients on this regimen are usually sterile as assessed by aerobic and anaerobic culture on blood agar. No. other P. aeruginosa isolate was recovered from the patients by culturing swabs from the nose, oropharynx, axilla, and prepuce or vulva on cetrimide agar (Diag*

phylactic oral antibiotics. Retrospective investigations found that one P. aeruginosa isolate had previously been found in pure culture 6 months before the outbreak from stools of patient 1. This patient had been discharged from the unit in May 1989 and had been readmitted in October 1989. All isolates from stools of patient 1 were found at high counts (106 to 107 CFU/g of feces). P. aeruginosa isolates were identified by colony morphology, growth at 42°C, and biochemical activities as determined in the API20 NE system (API System, La Balme les Grottes, France). The serovar, phage type, antibiotic susceptibility, and pulsed-field electrophoresis restriction pattern were each determined for each isolate, including the isolate obtained 6 months before from patient 1. Serotyping was performed by the slide agglutination technique, using commercially prepared antisera (Diagnostics Pasteur) to 16 somatic 0 antigens, according to the antigenic classification of Habs (6) with the subtypes of Veron (18). Strains that were agglutinated by more than one typing serum were designated polyagglutinable. A set of 17 reference phages, designated 7, 16, 21, 24, 31, 44, 68, 73, F7, F8, Flo, 109, 119x, 352, 1214, M4, and M6 was used for phage typing (9).

Corresponding author. 2068

VOL. 29, 1991

NOTES

2069

TABLE 1. Nosocomial outbreak due to P. aeruginosa P. aeruginosa isolation

Epidemiological markers

Patient no. and

symptom(s)l (age)

Isolate date

1. Cartilage hair hypoplasia May 10 (6 yr) Nov. 8 Nov. 9 2. SCID with BMT (2 yr) Oct. 27 Nov. 10 3. AIDS (2.5 yr) Oct. 31 Nov. 8 Nov. 17 Nov. 17 4. Adrenoleucodystrophy Oct. 31 with BMT (3 yr) 5. San Filipo A syndrome Nov. 7 with BMT (2 yr) Nov. 17 6. SCID with BMT (3 mo) Nov. 24

prophylactic

Associated

antibioticsb

symptoms

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

None Diarrhea Diarrhea None None Diarrhea and episodes of intestinal ileusg

Phage

Antibiotic

FIGE

Serovarc

typing'

susceptibility'

Fever (39.5'C)

0:11 0:11 0:11 PA PA PA PA PA PA PA

R 31 R 31 31 R 31 R 31 31

S to tic, caz, and ipm R to tic, S to caz and ipm R to tic, S to caz and ipm S to tic, caz, and ipm R to tic and caz, S to ipm R to tic and caz, S to ipm R to tic, S to caz and ipm S to tic and caz, R to ipm R to tic and caz, S to ipm R to tic, S to caz and ipm

1 1 1 1 1 1 1 1 1 1

No

None

0:11

R

S to tic, caz, and ipm

2

No No

None None

0:11 PA

R S to tic, caz, and ipm 16, 31, 44, F8, S to tic, caz, and ipm 109, 1214, M4

1 3

restriction

patternf

BMT, bone marrow transplantation; SCID, severe combined immunodeficiency. Vancomycin (50 mg), tobramycin (50 mg), and colistin (500,000 IU), given every 8 h. PA, polyagglutinable. d Phage(s) to which the strain is susceptible. R, resistance to the 17 phages tested. e tic, ticarcillin; caz, ceftazidime; ipm, imipenem; S, susceptible; R, resistant. f For explanation of restriction patterns, see legend to Fig. 1. g Associated symptoms in patient 3 apply to all four isolation times. a

b

c

Antibiotic susceptibility was tested by a disk diffusion technique (Diagnostics Pasteur). Pulsed-field electrophoresis restriction patterns were determined as follows, using a procedure modified from Grothues et al. (5). Bacteria grown in tryptone-water (Diagnostics Pasteur) to log phase were pelleted and adjusted to an optical density at 650 nm of 1.5 in phosphate-buffered saline. The bacterial suspension was mixed (vol/vol) with Incert agarose (FMC BioProducts, Rockland, Maine) and dispensed in a slot former. The resulting inserts were then incubated with a mixture of 0.5 M EDTA, 1% sarcosine (Sigma Chemical Co., St. Louis, Mo.), and proteinase K (1 mg/ml) (Appligene, Illkirch, France) for 48 h at 55°C. They were washed twice for 30 min at 45°C in TE buffer (0.1 M Tris, 0.01 M EDTA, pH 7.5)-0.01 M phenylmethylsulfonyl fluoride (Sigma), twice for 1 h at 45°C in TE buffer alone, and twice for 1 h at 4°C in 10 mM Tris-50 mM NaCl, pH 7.5. One insert of DNA was incubated overnight with 30 U of DraI or Xbal (Appligene). Restriction fragments were separated by field inversion gel electrophoresis (FIGE), using a Rotaphore 10035 apparatus (Biometra, Gottingen, Germany). Gels were run in 0.25x Tris-borateEDTA buffer (1 x Tris-borate-EDTA buffer is 0.089 M Trisborate, 0.089 boric acid, and 0.002 M EDTA, pH 8.0) for 96 h at 10°C, with a field strength of 5 V/cm and a forward-toreverse ratio of 3:1. Lambda concatemers (FMC BioProducts) were used as DNA size standards. Controls included DNA prepared from P. aeruginosa isolates of serovar 0:11, obtained from various departments of our hospital. Table 1 depicts the epidemiological markers of the P. aeruginosa isolates obtained from the six patients. Among the 13 strains, 5 belonged to serovar 0:11 and 8 were polyagglutinable. The phage types of the strains were as follows: six strains were susceptible to phage 31, six strains were resistant to all phages, and one strain was susceptible to 7 phages (patient 6). The same apparent heterogeneity was observed in the antibiotic susceptibility patterns to ticarcillin, ceftazidime, and imipenem. Five isolates were suscepti-

ble to the three 1-lactam compounds, four isolates were susceptible to ceftazidime and imipenem but resistant to ticarcillin, three isolates were only susceptible to imipenem, and one isolate was only resistant to imipenem. The patterns of susceptibility to aminoglycosides were more homogeneous: all isolates were resistant to gentamicin and tobramycin but susceptible to amikacin, except the isolate from patient 6, which was susceptible to all three aminoglycoside antibiotics. DNA analysis by FIGE after digestion with endonuclease DraI revealed three different restriction patterns (Table 1; Fig. 1). Eleven isolates obtained from five patients exhibited a common restriction pattern. All isolates from the patients receiving oral prophylactic antibiotics were related. Interestingly, the isolate taken from patient 1 6 months before the outbreak was identical to the epidemic strain. The two isolates obtained successively from patient 5 differed from one another, one of them being the epidemic strain. The only isolate found in patient 6 was unrelated to the other strains. DNA restriction with the enzyme XbaI gave different patterns (not shown) but the same conclusions. The results obtained with FIGE strongly suggest that a single epidemic strain was present in the fecal flora of five patients, including the four patients treated with oral prophylactic antibiotics. This confirms that genotyping of P. aeruginosa by FIGE is very useful for epidemiological investigations, since this method allows the discrimination between isolates that are indistinguishable or difficult to type by conventional techniques. In hematology-oncology patients, systemic infections due to P. aeruginosa are thought to result primarily from prior colonization of the gastrointestinal tract (15). In most cases, these infections are endemic and involve several different strains (4), leading to the assumption that gastrointestinal tract colonization usually starts from resident strains. Other mechanisms include transmission to patients from the hospital environment (4). Recently, it has been shown that colonized or infected patients may also be a possible reser-

2070

J. CLIN. MICROBIOL.

NOTES A It

C

1)

Fv C 11

aJ K 1I

\I

\N

0

()

It should be noted that the epidemic strain was resistant to tobramycin, which was given to the patients for digestive decontamination. Colistin, which was active against the epidemic strain, was unable to restrict P. aeruginosa growth. Although we have no evidence about the mode of transmission of P. aeruginosa, it can be speculated that bacteria were transmitted via transient carriage on the hands of hospital personnel. Finally, this study suggests that heavily colonized patients can be an important source of contamination in a proportion of outbreaks. It also illustrates

the potential 'dangers of total digestive decontamination. We thank Madame Rosine Fournier for typing the manuscript. This work was supported by a grant from the Association Frangaise de Lutte contre la Mucoviscidose.

1.

2. 3.

FIG. 1. FIGE analysis of P. aeruginosa DNA after digestion with endonuclease DraI. Patient numbers and isolation dates are as follows: patient 1, May 10 (lane A), November 8 (lane B), and November 9 (lane C); patient 2, October 27 (lane D) and November 10 (lane E); patient 3, October 31 (lane F), November 8 (lane G), November 17 (lane H), and November 17 (lane I); patient 4, October 31 (lane J); patient 5, November 7 (lane K) and November 17 (lane L); patient 6, November 24 (lane M). Lanes N to P, P. aeruginosa isolates of serovar 0:11, obtained from patients located in various departments of our hospital; lane Q, lambda concatemers used as DNA size standards. Three different restriction patterns in the pediatric hematology patients were observed: patterns 1 (lanes A to J and lane L), 2 (lane K), and 3 (lane M). Restriction patterns of control isolates (lanes N to P) were heterogeneous and different from that of the epidemic strain (lanes A to J and lane L). X molecular sizes (arrowheads, going from top to bottom): -1,020, 145, and 48 kb.

4.

5.

6. 7.

8.

REFERENCES Allardet-Servent, A., N. Bouziges, M. J. Carles-Nurit, G. Bourg, A. Gouby, and M. Ramuz. 1989. Use of low-frequency-cleavage restriction endonucleases for DNA analysis in epidemiological investigations of nosocomial bacterial infections. J. Clin. Microbiol. 27:2057-2061. Carle, G. F., M. Franck, and M. V. Olson. 1986. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232:65-68. Carle, G. F., and M. V. Olson. 1984. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12:5647-5664. Griffith, S. J., C. Nathan, R. K. Selander, W. Chamberlin, S. Gordon, S. Kabins, and R. A. Weinstein. 1989. The epidemiology of Pseudomonas aeruginosa in oncology patients in a general hospital. J. Infect. Dis. 160:1030-1036. Grothues, D., U. Koopmann, H. von der Hardt, and B. Tummler. 1988. Genome fingerprinting of Pseudomonas aeruginosa indicates colonization of cystic fibrosis siblings with closely related strains. J. Clin. Microbiol. 26:1973-1977. Habs, I. 1957. Untersuchungen uber die O-Antigene von Pseudomonas aeruginosa. Z. Hyg. Infectionskr. 144:218-228. Hentges, D. J., A. J. Stern, S. W. Casey, and J. U. Que. 1985. Protective role of intestinal flora against infection with Pseudomonas aeruginosa in mice: influence of antibiotics on colonization resistance. Infect. Immun. 47:118-122. Kern, W., C. Wolz, and G. Doring. 1990. Molecular epidemiological study of Pseudomonas aeruginosa isolates from patients with acute leukemia. Eur. J. Clin. Microbiol. Infect. Dis. 9:257-261.

voir for epidemic strains (13). In this study, the epidemic strain was shown to be genetically identical to the isolates obtained after inadequate long-term prophylactic antibiotherapy from a patient with chronic fecal carriage. This child had been discharged from the unit 6 months before the outbreak with high titers of P. aeruginosa in the stools and had been readmitted concomitantly with the outbreak with unchanged fecal flora. Together with the failure to isolate P. aeruginosa from the environment, this suggests that the epidemic strain was brought into the unit by. this patient at the time of his readmission. Two factors may have contributed to the dissemination of the epidemic strain in the unit. First, this strain was present at high counts (106 to 107 CFU/g of feces) in stools of the patient with chronic fecal carriage. Second, total digestive decontamination of patients with orally administered vancomycin, colistin, and tobramycin has probably favored P. aeruginosa colonization of the gastrointestinal tract by altering the barrier effect of the endogenous anaerobic flora. In experimental animal models, resistance to colonization by P. aeruginosa is notably modified by antibiotics, including aminoglycosides (7).

9. Lindberg, R. B., and R. L. Latta. 1974. Phage typing of Pseudomonas aeruginosa: clinical and epidemiologic consider-

ations. J. Infect. Dis. 130:S33-S42. 10. Morrison, A. J., and R. P. Wenzel. 1984. Epidemiology of infections due to Pseudomonas aeruginosa. Rev. Infect. Dis.

6:S627-S642. 11. Ogle, J. W., J. M. Janda, D. E. Woods, and M. L. Vasil. 1987. Characterization and use of a DNA probe as an epidemiological marker for Pseudomonas aeruginosa. J. Infect. Dis. 155:119-

126. 12. Pitt, T. L. 1988. Epidemiological typing of Pseudomonas aeruginosa. Eur. J. Clin. Microbiol. Infect. Dis. 7:238-247. 13. Richet, H., M. C. Escande, J. P. Marie, R. Zittoun, and P. H. Lagrange. 1989. Epidemic Pseudomonas aeruginosa serotype 016 bacteremia in hematology-oncology patients. J. Clin. Microbiol. 27:1992-1996. 14. Samadpour, M., S. L. Moseley, and S. Lory. 1988. Biotinylated DNA probes for exotoxin A and pilin genes in the differentiation of Pseudomonas aeruginosa strains. J. Clin. Microbiol. 26:

2319-2323. 15. Schimpff, S. C., J. Aisner, and P. H. Wiernik. 1979. Infection in acute non-lymphocytic leukemia: the alimentary canal as a major source of pathogens, p. 12-27. In D. Van der Waaij and J. Verhoef (ed.), New criteria for antimicrobial therapy: mainte-

VOL. 29, 1991 of digestive tract colonization resistance. Elsevier/NorthHolland Publishing Co., Amsterdam. 16. Schwartz, D. C., and C. R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67-75. 17. Speert, D. P., M. E. Campbell, S. W. Farmer, K. Volpel, A. M. nance

NOTES

2071

Joffe, and W. Paranchych. 1989. Use of a pilin gene probe to study molecular epidemiology of Pseudomonas aeruginosa. J. Clin. Microbiol. 27:2589-2593. 18. Veron, M. 1961. Sur l'agglutination de Pseudomonas aeruginosa: subdivision des groupes antigeniques 0:2 et 0:5. Ann. Inst. Pasteur (Paris) 101:456-460.