Epidemiological studies of nosocomial infections with ...

ORIGINAL ARTI C LE

Epidemiological studies of nosocomial infections with

Pseudomonas aeruginosa

using a DNA probe

A MARK JOFFE, MD, KATHY VOLPEL, PAMELA C KIBSEY. MD, WI LLI AM PARANC HYCH, PHD

AM J OFFE, K VOLPEL, PC KmSEY , W P ARANCHYCH. Epidemiological studies of nosocomial i nfections with Pseudomonas a eruginosa using a DNA probe . Can J Infect Dis 1992 ;3(6) :299-306 . A DNA probe encod ing th e Pseudomonas aerug inosa pili n gen e has bee n developed in th e a uth ors ? la bora to ry a nd has been shown to be a usefu l epidemiological tool. In th e p resen t s tudy this tec hn ology. toge th er wi Lh oth er typ ing met h ods. has bee n used to cle nn c re la ted ness a n d po si blc tra ns m ission routes of P aerug inosa strains isolated in seve ra l h ospita l wa rds. Clu s te rs o f P aerug inosa infec! ions . s u s pec ted to be the res ult of no. ocom ia l tra ns m ission . clcvclopccl in a ge ne ra l inte ns ive ca re un it (I CU) a nd a neu ros u rgica l wa rci / ICU. as we ll as in a b urn uni t. were tu d iccl us ing a ntibiogra m s . li popolysacc ha ri clc-s crotypin g. a nd ge ne p robe ana lysis. Res ults of these tucli cs d em onstra ted th a t eac h of the ge ne ra l a nd ne uros u rgical ICU isola tes were diffe rent. m a king nosoco mi a l tra ns m ission ve ry un likely . Howeve r . wiU1in th e burn uni t. pa ti ent isolates had iden tical p rofil es. s ugges ting tha t sp read be twee n pa ti e n ts was occ urring or tha t a com mon source of infection was p rese nt. Changes in infec ti on co nt ro l meas ures wi thin the un it were introd u ced and may have con tri b u ted to e ra dication of the ou tb rea k DNA probe st u d ies we re \?a lu ab le in cla rifyi ng cpiclemiological related ness of isolates that was not evide nt wil h the other typ ing s tra tegies a nd idc nlin ed a po s iblc bum -associated stra in .

Key Words: Epidemiology. Nosocom ia l ir ](ect ion. Pse ud om on as ac ru ginosa . Typ ing

a Etudes epidemiologiques sur les infections nosocomiales Pseudomonas a aeruginosa !'aide d'une sonde d 'ADN

RESUME: Un e so nd e d"AD N po ur l' cn cocl a(jc du ge n e de Pseudomonas aerug inosa a etc m ise a u point cl a ns cc Jaboralo ire et s?csl revclee un ou ti l cpicl cm iologiq uc u ti le. Dans Ia prcse n tc e lude . cc tle techn ologic. de memc que cl'autres methodes de lypage. ont etc uti lisces po u r clc nni r lcs inte rre la ti ons e t lcs voics de transm ission possibles de so uc hes de P. aerug inosa isolees cla ns d iffc rcntes u n ites h ospita lieres. Des

infec ti ons a P. aerug inosa. pres um em c n t a ttri b uab les a u nc trans mission nosoco mi a le, om de observees

da ns unc u nite de soins intc ns ifs gene raux. clans u nc un ite de soins imcnsifs ne uroch irurgica ux et dans

unc unite de brt:ll es. ou ellcs on t etc etuclices a !'a ide cl'a n libiogrammcs. de mCLhocles de typage se rologiqu e

pa r li popolysacc ha ri des ct cl'a na lyscs par sonde gc nc ti q u c. Lcs rcs ul tats d e ces e tu des on t dcm ontrc qu e chac u n des isolats p relevc d a ns les uni tes de soin s inle ns ifs ge nc ra u x ctnc u ro-c hirurgica u x Clail d iiTere n t. ce q ui a pe rm is d'cca rte r !' hypot hese de Ia tra ns miss ion nosocom ia lc. Ce pe nda nl. a u sein d e !' u nite des

brt:lles. les isolats etaient scmb lab les cl'un pa ti ent a !'a u tre . cc qui do nn e a pen se r qLd l y a cu co nta m ina tion entre lcs pa ti en ts ou qu'i l y a c u u nc so urce commune a !'i nfec ti o n . Des mod ifi cation s a u x mcs u rcs de lutLc contrc !'infec ti on onl etc a ppo rt ccs cla ns !'unite cl pc u vc n l avo ir conl ribu c a r climina lion d e l'cp iclemi c. Lcs

etudes par sonde ci"ADN on l etc uliles pour c la rifi er lcs liens cpiclem iologiq ues en tre lcs isolats. liens q ue ne pe rmcttaien t pas cl'iclcntin c r lcs m itres strategies de typage c t ont per m is Ia reconn a issa nce cl' un c so uche

possiblcmcnt lice a Ia b rt:ll urc .

Department of Medica l Microb iology wtd lr ]f'ectious Diseases . Facu lty of Med icine: Deparuneni qf M icrobiology. Facu lty of Science. University of A lberta. Ed monton . A lber ta

Correspondence: Dr A Mark Joffe. Depar tmen t q{ Microbiology a nd Immu nology . Slcu]jord Univers ity School qf Medicine. D3 12 Sherman Fairchild Science Building. Siaqford. Cal(fo mia 94305 -5402. US/\. Telephone (4 15) 723 -267 1. Fax (4 15) 725-7282

Received .for publication September 9. 199 1. 1\ccept ed November 15. 199 1

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JOFFE et Of

PSEUDOMONAS AERUCINOS/\ IS A MAJOR OPPORTUN ISTI C pathogen in immunocompromised individuals. and patients with cystic fibrosis and lhermal trauma. Though recognized as a prevalent organ ism in U1e hos pital environ m ent a nd a re latively frequent cause of nosocomial disease. Lhe reservo ir of U1e organ ism and U1e precise mode of transmission wilhin hospita ls are usually unclear.

Existing meti1ods for typ ing strains of P aeruginosa include plasmid analysis. bacteriocin (pyocin) typing. phage sensitivity. biotyping. colonial morpho logy. antibiotic sensitivity patterns and the use of DNA probes. Serotyping generally is accepted as the most practical and reliable for routine use in clinical microbiology laboratories and is based on an agglutination reaction between bacterial lipopolysaccharide (LPS) and type spec ific rabbit antisera (l ). While most clinica l isol ates may be typed using Lhis method. strains from cystic fibrosis patients are unique in that the majority po lyagglutinate or are nontypab le in 0 - typ ing sera (2) . In addition. il has been reported previously that LPS serotype may not be a stable ep idemiological marker and may change over lime (3.4).

A DNA probe encoding the P aeruginosa strain PAK pilin gene has been developed (5) and is used to probe restriction enzyme digested P aeruginosa chromosomal DNA yieldi ng strain-specilk restriction fragment lengti1 po lymorphism (RFLP) pallerns . It has been repo rted that serial P aeruginosa isolates from a patient with cystic fibrosis maintained a constant RFLP pattern and pilin gene sequence despite considerable phenotypic variability (4). In add ition. recent stud ies of over 300 P aeruginosa isolates from cystic fibros is patients and a variety of olher sou rces have demonstrated 21 RFLP patterns and have shown U1e p ilin gene probe to be a u efu l ep id emiological tool (6). The occurrence of clus ters of P aeruginosa infections in hospilalizecl patients that were closely related ep idem iological ly provided U1 e unique opportuni ty lo evalu ate U1e pil in gen e probe in the study of nosocomial ep id em iology. The goa ls of t.his study were to determine if nosocomial spread of P aeruginosa was occw-ring and to compare the pi! in gene probe wilh LPS-serotyping as ep id em iological typing techniques in U1is selling.

PATIENTS AND METHODS Infections with Pseudomonas aeruginosa: The Uni versity of Alberta Hosp itals is a 1250-becl tertiary ca re facility representing a ll areas of clinica l med icine. in cluding long term ca re fac ili ties and separate intensive care units (ICUs) for neonates. pediatrics. neurosurgery. coronary care. ca rdi ovascular surgery. burns and a genera l surgical-med ical !CU. Clu sters of infec tions \-vilh P aeruginosa were observed over a s hort pci-iod of Lime \viti1in ti1ree of the hosp ita r s ICUs (Table I). These three units- Lhe gene ral intens ive ca re unit (G ICU). neurosurgica l ward/ICU (NW ICU) and the burn

intens ive ca re unit (BICU) -are physically remote from each other within the hospital. Clusters included at least Lhree epidem iologically related patients s imul taneous ly or sequential ly hospitalized within one of U1e ICUs. from whom P aeruginosa was isolated from an infected s ite. Pneumon ia was defin ed as t h e presence of pu rulent sputum (grossly and microscopically) \vi.Lh or \vi.Lhoul fever associated wilh a chest x-ray infiltrate.

The GlCU is a 16-bed unit consisting of 10 open beds and six private rooms. Nosocomial P aeruginosa pneu monias developed in three mechanically ventilated patients over a 10-day period. with patient GICU-1 a lso having a sternal wound infection \vi.th this organism follo\.ving coronary artery bypass surgery (Table 1). These three patients occupied U1ree of four adjacent private rooms \vi.lh patients GICU - 1 and GICU-2 sideby -s ide and \vi.th a noncolonized patient in Lh e room separating patients GICU-2 and GlCU -3. Patient GICUX was transferred from anoti1er institution d uring Lhis outbreak and was colonized \viti1 P aeruginosa upon adm ission to t he GICU .

The NWICU consists of a 24-bed ward \vi.Lh a n attached lO -bed !CU. This outbreak involved one venli lated an d two unvenlilated patients. all of whom developed P aeruginosa nosocomial pneumonias over a period of one week.

The BICU is a lO-bed self-conta ined unit in which all necessary care of Lhe burn patient. including venUlalion and hydroU1erapy. is carried out. The nature of Lhis outbreak is described unde r 'Resu lts ?. Bacterial strains: P aeruginosa strains isolated from patients involved in clusters of nosocomial infections. as well as concurrent isolates from h ospitalized patients nol involved in outbreaks. were obtained from Lhe clini ca l microbiology laboratory of ti1e University of Alberta Hospitals. Clinical specim ens including sputum. trach eal secretions. and wound a nd burn swabs were p lated on sheep blood and MacConkey aga r plates. Blood cu ltures were innoc ul ated into broth a nd growth was detected by Lhe BACTEC NR660 system (Becton Dic kin so n. Maryland). Isolates were identified by con ventional methods including standard tests for oxidase. growlh at42?C and fluorescence under ultraviolet light. One iso late from each of patients BICU 1. 2 and 3 was inclu ded in ti1e ep idem ioloO"ical survey repo rted previously (6). Antibiograms: Sensitivi ty testing of a ll isol a tes was performed in ti1 e clini cal microbiology laboratory of th e University of Alberta Hospita ls u si ng U1e automated Vitek microbroth dilution technique (Vitek Systems Inc. Missouri) . Table 2 lists the antib iotic susceptibility profil es observed for isolates in Lhis study. Lipopolysaccharide 0-serotyping: All strains were se rotyped using ti1e International Antigenic Typing System (DIFCO. Michigan) in e iU1er the Provincial Laborat01y for Public Health of No rthern Alberta o r Lhe Department of Pediatrics. University of British Co lumbia. Isola tes

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Nosocomial infections with P aeruginosa

TABLE 1 Results of strain typing studies for Pseudomonas aeruginosa isolates from clusters of nosocomial infections

Patient

Date/ site of isolate

Antibiogram Serotype

Pi lin

Probe U

GeneraiiCU oubreak

GICU-1

January 11. 1988/sternum

1

Polytypeable

2

January 11. 1988/incision

2

Polytypeable

2

January 14. 1988/sputum

Polytypeable

2

February 1. 1988/sputum

Polytypeable

2

6.7/2.6

GICU-2

January 27. 1988/sputum

3

6

13

9.5/6 8

January 27. 1988/sputum

4

3

13

9.5/6 8

GICU-3

January 21. 1988/sputum

5

2

5

GICU-X'

January 25. 1988/sputum

6

4

4

Neurosurgical ward/ICU outb reak

NWICU-1

January 11. 1988/sputum

5

5

11

NWICU-2t

January 14. 1988/sputum

7

3

9

January 14. 1988/sputum

7

3

9

NWICU-3

January 15. 1988/sputum

8

2

5

Burn ICU outbreak

BICU-1

November 9. 1987/back

9

11

7

November 12. 1987/blood

9

11

7

BICU-2

February 10. 1988/back

10

11

7

February 11. 1988/abdominal

9

11

7

February 11 . 1988/left thigh

9

11

7

February 11. 1988/blood

9

11

7

4.3/9.0

BICU-3

February 20. 1988/right thigh

9

11

7

February 20. 1988/back

9

11

7

4.3/9.0

BICU-4

April18. 1988/arm

11

6

6

April 27. 1988/burn site

12

6

5.3:3.2/23

BICU-5

April22. 1988/graft site

11

11

9.5/3.0:16

April 22. 1988/foot

11

11

BICU-6

March 3. 1989/blood

13

3

9.5/55

March 3. 1989/burn biopsy

13

3

9.5/5.5

'Patient was colonized with Paeruginosa on transfer from another institution (see text): 1Different colony morphologies isolated from the same specimen:

BICU Burn mtensive care unit; GICU General intensive care unit: NWICU Neurosurgical ward intensive care unit: Pi/in Pi/in gene probe pattern (reference 6)

whjch agglu tinated in more than one typing serum were designated as polytypeable. DNA probe studies : Ch romosomal DNA was prepared from late-log phase bacteria usinO' a modWcation of U1e procedure of Coleman et al (7). Purified DNA (1 to 2 pg) was digested wi.th th e restriction endonucleases PsU and Hindiii (GIBCO Laboratories . New York). electrophoresed in 0. 7% agarose and transferred lo ni Lrocel lulose paper by the m e thod of Southern (8). A 1.2 kilobase Hindiii fragment. of PAK chromosomal DNA (5) was nick t.ranslaled and used to probe the nilrocellulose filters under high stri ngen cy conditions (9). washing a l 21 ?C rather than 65?C. Au toradiographs we re U1en exposed at - 70?C . Fragments hybridizing with U1e radiolabelled probe were s ized by comparison wi.lli m olecular weight standa rds run togeth er with U1e sarnp les in th e agarose gel. Twenty-one RFLP pallems were defined in screen ing large numbers of isolates ar1d were reported previously (6). Experience wiU1 lliis techniqu e has demonsLrated complete reproducibility ofRFLP patlems a long as ge nomic DNA is digested to completion and Southern lransfer is adequate (4.6). In lliis study. a techn ica lly satisfacto ry result was gene ra lly obtained

on the first. attempt. wiili occasional isolates requiting a second or confirmatory testing.

Selecled s lrains were also typed with a second DNA probe to clarify a pparent iliscrepancies in resulls belween antibiograrn . LPS serotype and pilin gene probe. Probe U. a 741 base pair fragm ent en coiling U1e region upslream of U1e P aeruginosa exotoxin A structural gene (10) . vvas isolated from a 5% acrylam id e gel (9) following digestion of plasmid pBRTox wiili Psll and Nrul (GIBCO Laboralories) . SouU1ern blots were prepared of Bgiii- and Xhol- (GIBCO) digested c hromosoma l DNA a nd were probed. washed and exposed to x-ray film as described a bove except U1al ilie 741 base pair probe was rarno la belled using U1e random primer lechnique (11) . Results a re expressed as U1e s ize of probe reactive fragments in ldlobases for ilie Bglll/Xhol diges tion s in Table l.

RESU LTS Table 1 s ummarizes the resu lts of ilie U1 ree clusters investigated in ili is s ludy. including the antibiotic sens ilivity profiles. serotypes and RFLP pallerns oblain ed in DNA probe slud ies .

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JOFFE eta/

TABLE 2 Antibiogram patterns

Pattern * 1 2 3 4

Amikacin Sensitive Resistant Sensitive Sensitive

Gentamicin Sensitive Resistant Sensitive Sensitive

Tobramycin Sensitive Sensitive Sensitive Sensitive

Ticarcillin Sensitive Sensitive Sensitive Sensitive

5

Sensitive

Sensitive

Sensitive

Sensitive

6

Sensitive

Sensitive

Sensitive

Sensitive

7

Moderately

Resistant

Sensitive

Sensitive

sensitive

8

Sensitive

Sensitive

Sensitive

Sensitive

9

Sensitive

Resistant

Resistant

Resistant

lO

Resistant

Resistant

Resistant

Resistant

11

Sensitive

Sensitive

Sensitive

Moderately

sensitive

12

Resistant

Resistant

Sensitive

Sensitive

13

Not applicable

Sensitive

Sensitive

Sensitive

' The follow1ng groups may not be mutally exclusive. 1 11 and 13. 2 and 12. 3 and 4

Ceftazidime Sensitive

Not applicable Resistant Resistant

Resistant

Sensitive

Sensitive

Piperacillin Sensitive

Not applicable Resistant

Moderately sensitive Sensitive

Sensitive

Sensitive

lmipenem Sensitive Not applicable Sensitive Sensitive

Moderately sensitive

Moderately sensitive Sensitive

Resistant

Sensitive Sensitive Sensitive

Moderately sensitive Resistant Resistant Sensitive

Resistant

Sensitive Sensitive Sensitive

Sensitive Sensitive

Sensitive Not applicable

Moderately sensitive

Sensitive

General ICU: DNA probe studies revealed U1at all four patients involved in this outbreak - including the three patients who sequentially became co lonized while in this unit as well as the patient who wa transferred from another institution - were co lonized w ith diffe rent

train of P aeruginosa. This cone! usion is supported by the antib iocrram patterns and LPS-serotyping.

Four isolates were taken over a three-week period and from two different sites from patient GlCU-1. All isolates were identical in DNA probe studies. suggesting that this patient remained colon ized with a single train of P aeruginosa througho u t. the study peri od . Slight. variab ili ty in U1e anlib i ogram pa tterns raised lhe pos -

ibilily of colonization wiU1 two different. str ain s while LPS- erotyping reveal ed the iso lates from palient. G lCU - 1 to be polytypeab le.

1\vo colon ies of P aeruginosa \Vith different colon i al morphologies were isolated from a single sputum specimen for patient GlCU -2. While LPS-serotyping suggested that Lhe patient was co lonized wiU1 two different strains. DNA probe tuclie with bolh the pilin gene and probe U demonstrated Lhat U1is was mo t p robably a single strain. Neurosurgical ward ICU: All three patients involved in Lhis cluster of infections were co lon ized by different. strain of P aeruginosa (indicated by each of the t hree

t.udy techniques). 1\vo colonies wiU1 differing morphologies were isolated from the sputum specimen of patient NWICU -2 and. in contrast. to patient GICU -2. LPS- erolyping and D A probe studies concurred that these two colony types represented a ingle strain. Burn unit: An outbreak of burn wound sepsis in Lhe burn unit was first recognized when palient BICU -2

devel oped septic shock from bacteremia with a r esistant str ain of P aer-uginosa: wiU1in a few days anoU1er patient in Lhe burn unit (patient BICU-3) had al so become co loni zed with a simil arly resistant str ain (Table 1). LPS -se rotyping and DNA probe studi es using bolh probes demonstrated that U1ese two patients were pr obab ly colonized wiU1 U1e identical slrain . In retrospect. there had been a patient in the bum unit three monU1s earlier infected with a resistant strai n of P aemginosa (patient BI CU-1). Stud i es of Lhis isolate revealed that. it. too. was U1e identical strain. Environ m enta l sampling was perfoi-med and ch anges were made in U1e routine care of patients in the burn unit to prevent further transmission (see D iscu ssion).

'[\vo other patients became colonized with P aeruginosa in the two monU1s subsequent to the recognition of lh is outbreak. Patient BICU-4 was colonized with a different strain as demonstrated by all three typing techniques. Patient Bl U-5 was colonized by a strain wilh the same serotype (0 11) as U1e ea rlier ou tbreak strain . However. DNA probe studies using boU1 probes in add ition to U1e antibiog r am patterns indicated U1at U1is represented a different and uniqu e str ain. Typing of P aeruginosa isolates from U1e first bacteremic patient following recognition of lliis outbreak (patient B ICU -6) also revealed a str ain which was different from the epidemic strain. Background strains: 1\vo sporadic clin ical isolates were available from P aer-uginosa-infected patients hospi talized during this period but unrelated to the identified outbreaks. One. an eye isolate from a patient. wiU1 conjunctivitis . was typed as RFLP pattern 6 with U1e pili n gene probe. while U1e second. a sputum isolate

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Nosocom ia l infections with P aeruginosa

from a mechanically ventilated patient in the cardiovasc ul ar !CU . could not be typed with lh is probe despite rep ated attempts. This was unusual as less than 5% of isolates have been fo u nd to be nontypeable in U1is system (6). It does. however. suggest Lhat this isolate was different from others studi.ed in this report. Isolates obtained from a GICU patient one year prior to U1e recognition of the cluster were also analyzed and found to be type 5. No environmental isolates obtained from hospital sources during this pe1iod were available for typing.

DI SCUSSION P aeruginosa is a ubiquitous organism found in soil. plants. water and food . Indeed. 5 to 29% of nonnal individuals are colonized in their gastrointestinal tracts (12). A recent study (13) found that 22.6% of patients were colonized with P aeruginosa on admission to a general hospital. with an additional 10.8% acquiring the organism during their hospitalization. Within U1e hospital. potential sources of paU1ogenic organisms are numerou and include U1e patient's own microflora. lhose in U1e hospital environment or canied by medical personnel. contaminated equipment and other patients. It generally is agreed that colonized and in fected palients are the major reservoir of resistant Gram-neo"alive organisms within hospitals (14.15). There is also considerable evidence U1at spread of such resistant strains between hospitalized patients occurs on U1e hands of health care personnel. 30 to 40% of whom carry Gran1-negative organisms on their hands (some of them continuously) (16-20). Gastrointestinal acqu is ition and carriage of Gram-negatives appears to be an important intermediate step in certain situations (14). while some patients are self-infected from their own gastro intestinal microflora (21). A gastrointestinal reservoir was fe lt to be important in one prospective study in which transfer of P aeruginosa between surgical patients in a hospital ward was demonstrated (22) . Previous studies of ICUs have demonstrated conflicting results related to cross-infection of P aeruginosa between patients. Large epidemics related to contaminated respiratory therapy equipment have been reported in U1e past (23). but are rarely a problem today. Cro s -infection of P aeruginosa between patients in one prospective ICU study was attributed to heaiU1 care personnel. as 24% of h and cultures and 30% of uniform cultures grew this organism (24). On lhe other hand. prospective studies in a spinal cord unit (25) and a surgical-medical ICU (26) concluded that cross-infection wilh P aeruginosa is not a major problem in these settings. Clearly. the validity of all such studies depends on U1e reliability of the strain typing systems employed. The present authors have investigated clusters of nosocomial infections wiU1 P aeruginosa in three reus in which transmission between patients was strongly

suspected on clinical grounds. The results demonstrated U1al the patients involved in U1e GTCU and NWlCU outbreaks vve re each infected with a different a nd unique strain. revea ling U1atlransmission between patients was not a fac tor in U1ese 1:\vo outbreaks. From an infection control standpoint it is important to ensure U1at pote ntially viru lent and multiply resistant Gramnegative organisms are not being spread between hospita lized patients. ll was not possible in this study to exam ine potential sources of the P aeruginosa colonizing the patients.

Of the currently available typing schemes for P aeruginosa. LPS -serotyping genera lly is considered to be U1e most practical. while colony morphology and antibiogram patterns are U1e least accurate (27.28). While reproducibility in testing serial isol ates has been questioned. antim icrobial susceptibil ity data assessment is likely lhe most readily available typing technique for most clinicians and was Ulerefore included in this investigation. In this study. multiple isolates from in dividual patients revealed s light variability in antibiogran1 s. malting interpretation confusing. As such. it is difficult to establish firmly epidem iological relatedness between strains on U1is basis. FurU1ermore. rapid changes in antibiogram patterns frequently are seen in s ubpopulalions of P aeruginosa in response to anti microbial therapy. furU1 er add ing to the difficulty in interpretation .

Isolates of P aeruginosa from noncystic fibro is patients can be reproducibly serotyped in 92 to 99% of ca es (2.29). In contr ast. 68 to 80% or more of isolates from cystic fibrosis patients are nontypeable or polytypeabl by LPS-serotyping (2.30.31). Development of 0 -antigen specific monoclonal antibodies (MAbs) for serotyping may circumvent U1is problem and provide improved discrimination between strains (32-35). although reproducib ili ty for the MAbs was only 75% in a recent study (36). As noted earlier. changes in LPS serotype may occur in single strains over time (4.37). and some strains a re now known to synU1esize more Ulan one type of chemically and antigenically distinct LPS molecule (38). The reproducibility and discrimin atOJy power of several typing methods has been com pared. and use of more U1an one typing system has been advocated. at least for cystic fibrosis isolates (39. 40).

ll is unsual iliat P aeruginosa isolates from patient GICU-1 could not be serotyped while DNA probe studies demonstrate conclusively that this patient was co lonized wiU1 a single unique strain throughout the U1ree-week study period. Furthermore. serotyping iso lates of different colony morphologies in patient GICU -2 suggested the coexistence of two different strains. while DNA probe studies indicated U1at U1is reflected phenotypic valiability in a single strain as has been demonstrated previously (1) . Rapid adaptation with a lteration in an organism's phenotype in response to environmental stimuli Ulrough modulation of gene ex-

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