Molecular Characterization of an Epidemic Clone of Panantibiotic-Resistant Pseudomonas aeruginosa

Abstract

We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired β-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-β-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.

Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe invasive diseases in critically ill and immunocompromised patients. Because of its ubiquitous nature, ability to survive in moist environments, and innate resistance to many antibiotics and antiseptics, P. aeruginosa is a common pathogen in hospitals and particularly in intensive care units (ICUs). A European survey on the prevalence of nosocomial infection in ICU patients showed that P. aeruginosa was one of the most frequent pathogens, isolated from 29% of ICU-acquired infections (27, 28). A multicentric study conducted in Belgium placed P. aeruginosa as the first gram-negative pathogen recovered in the ICU (6). Furthermore, the high frequency of multiple resistance among P. aeruginosa strains makes its eradication difficult (6, 27, 28), and mortality associated with P. aeruginosa infection is high compared to other bacteria.

P. aeruginosa exhibits intrinsic resistance to several β-lactams and may acquire additional resistance mechanisms, including decreased outer membrane permeability, penicillin binding protein modifications, production of extended-spectrum β-lactamases, acquisition of metallo-β-lactamases or other enzymes, increased expression of efflux pump systems, and decreased porin expression (5, 8, 16-19). These multiple-antibiotic-resistant P. aeruginosa strains caused sporadic nosocomial outbreaks (1, 8, 24), but several drug-resistant P. aeruginosa clones are now spreading among vulnerable patients, such as those with cystic fibrosis, as recently described (20).

Several typing methods have been used effectively for epidemiological studies of P. aeruginosa infection, including macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). We describe here the study of the emergence and propagation in the ICU of our tertiary-care hospital of multiresistant P. aeruginosa strains. These strains showed resistance to multiple antibiotics, including penicillins, cephalosporins, carbapenems, and aminoglycosides. Molecular studies were performed to study the clonal transmission and determine the antibiotic resistance mechanisms that could explain this panresistance.

MATERIALS AND METHODS

Nosocomial multiresistant P. aeruginosa case definition.

Cases of nosocomial colonization and infection with multiresistant P. aeruginosa were defined as any non-cystic fibrosis patients with an isolate of P. aeruginosa that was resistant or intermediately susceptible to meropenem, gentamicin, and ciprofloxacin acquired by a patient ≥48 h after his hospital admission.

Setting.

Erasme Hospital is a 858-bed tertiary-care university hospital located in Brussels, Belgium. The ICUs include four separate units with a total of 31 beds and had 2,355 admissions in 2002 (range, 166 to 222 admissions/month). In the ICUs, all patients were hospitalized in separate rooms.

Culture surveys.

Multidrug-resistant bacteria screening by rectal swab was routinely performed on each patient admitted to the ICU. Rectal swabs were cultured on selective MacConkey agar medium (BBL, Becton Dickinson, Le Pont de Claire, France) containing 5 μg of ceftazidime per ml for 48 h. All gram-negative bacilli were identified, and antibiotic susceptibility testing was performed. From July to December 2002, all multidrug-resistant P. aeruginosa isolates collected from patients hospitalized at the ICU (n = 18) and clinical P. aeruginosa isolates from patients admitted to other wards during the same period (n = 15) were analyzed. Environmental specimens (n = 102) collected from surfaces, water, sink, tap, antiseptic solutions, soap, “sterile” solutions, and swabbing contact plates of medical instruments were sampled on five occasions from August to September 2002. These specimens were incubated in Letheen broth (Difco, Becton Dickinson) and blood agar before subculture on cetrimide agar selective medium (Bio-Rad, Marnes-La-Coquette, France). Health care worker hand specimens (n = 10) were also cultured in September 2002 with the broth-bag sampling technique (3).

Identification and antimicrobial susceptibility testing.

Identification was based on Gram staining, oxidase test, arginine dihydrolase test, and growth on Kliegler medium (Merck, Darnstadt, Germany) and in brain heart infusion broth incubated at 42°C. Antibiotic susceptibility was determined for the following antimicrobial agents: ticarcillin/clavulanic acid, piperacillin, piperacillin/tazobactam, aztreonam, ceftazidime, cefepime, meropenem, imipenem, gentamicin, tobramycin, amikacin, ciprofloxacin, and colistin with the disk diffusion method (Rosco, Taastrup, Denmark). Isolates were classified as susceptible, intermediate, or resistant according to the standard criteria (14). MICs of cefepime, imipenem, meropenem, amikacin, gentamicin, tobramycin, isepamicin, and ciprofloxacin were determined by the E-test method (AB Biodisk, Solna, Sweden).

Typing.

O-serotyping was determined by slide agglutination test with polyvalent antisera and 16 monovalent antisera numbered O1 to O16 (Bio-Rad). Macrorestriction analysis resolved by PFGE was performed as previously described except that DNA was digested with SpeI and PFGE separation conditions were 2 to 40 s for 24 h (22). AFLP analysis was done on the ALF-Express sequencer (Amersham Biosciences, Roosendael, The Netherlands) as previously described (21). Computer analysis was achieved with the BioNumerics software (Applied Maths, Kortrijk, Belgium). For PFGE, DNA fragments ranging from 36 to 674 kb were considered, and analysis was performed with the Dice coefficient and the unweighted pair group method with average linkages clustering method. For AFLP, DNA fragments from 50 to 500 bp were analyzed with the Pearson correlation coefficient and unweighted pair group method with average linkages. The classification criteria used for PFGE analysis were described previously (22) and include a type, designated by a capital letter (e.g., A) and patterns showing zero to six DNA fragments. Any variant was indicated by a numerical suffix (e.g., A1). For AFLP, we classified a type designated by a capital letter and patterns showing ≥90% Pearson similarity as previously proposed (1).

Characterization of β-lactam resistance mechanisms.

To detect the presence of metallo-β-lactamases, the MICs of imipenem in the presence or not of EDTA were determined by E-test (AB Biodisk) as previously described (30). Carbapenemase detection was also performed by measuring hydrolysis of imipenem with UV spectrophotometry and PCR for detection of the imp and vim genes as described by Poirel et al. (17, 18). Detection of extended-spectrum beta-lactamases was performed by the double-disk synergy test and isoelectric focusing analysis as previously described (16). To counteract the effect of high-level expression of the naturally produced AmpC β-lactamase, the double-disk synergy test was also analyzed on cloxacillin-containing plates (16).

PCR for detection of the TEM and SHV penicillinases as well as OXA-1, OXA-2, OXA-9, and OXA-10 oxacillinases was performed as previously described (5). Isolates P8 and P17, belonging to PFGE types A1 and K1, respectively, were randomly selected for further analyses. Production of the chromosomally encoded ampC cephalosporinase and oprD porin was quantified as previously described (8). The expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pump systems was determined by quantitative real-time reverse transcription-PCR as previously described (8, 10). DNA sequencing of the putative repressor gene mexZ adjacent to the mexXY operon was done as described by Hocquet et al. (8).

RESULTS

Outbreak description.

From July to August 2002, a cluster of multiresistant P. aeruginosa colonizations were detected in the ICU. The incidence of colonization was 16 of 381 (4.2%) compared to 3 of 1,175 (0.25%) in the previous 6-month period (P < 0.001). From 8 August to 30 September 2002, the infection control team asked ICU staff to implement precautions, including the wearing of gowns and gloves for the care of ICU patients colonized or infected with multiresistant P. aeruginosa. Standard procedures using alcoholic chlorhexidine handrub solution for hand disinfection were reinforced. During the outbreak, 18 non-cystic fibrosis ICU patients were colonized (n = 9) or infected (n = 9) with multiresistant P. aeruginosa (Table 1). Nine patients (50%) developed infections, including pneumonia (n = 4), urinary (n = 1) or wound infections (n = 2), bacteremia (n = 1), and peritonitis (n = 1). All patients received colistin intravenous treatment. Three of the nine infected patients died, and mortality was attributable to their P. aeruginosa infection.

TABLE 1.

Origin and characteristics of multiresistant P. aeruginosa strains isolated from patients admitted to Erasme Hospital ICU and the ICU environment, July to December 2002

Origina	Isolation date (mmddyy)	Source	Resistant or intermediately susceptibleb to:	Serotype	PFGE type	AFLP typec
P1	090702	Skin	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	A
P2	110702	Rectal swab	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	A
P4	180702	Sputum	PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	A
P5	220702	Rectal swab	TIM, PIP, TZP, ATM, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	A
P6	240702	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	A
P7	250702	Peritoneal fluid	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	A
P8	290702	Urine	PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	ND
P9	010802	Wound	PIP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	A
P10	050802	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND
P11	050802	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND
P12	120802	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	A
P13	130802	Sputum	TIM, PIP, TZP, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND
P14	180802	Rectal swab	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	01	A1	A
P15	190802	Rectal swab	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	01	A1	A
P16	210802	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	K1	A
P17	270802	Rectal swab	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	K1	A
P18	160902	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND
P19	101202	Sputum	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND
E1	070802	Sink drain ICU5	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	06	C1	C
E3	070802	Tap ICU5	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	06	C1	C
E4	070802	Sink drain ICU5	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, CIP	11	A1	A
H1	040902	Nurse's hands	TIM, PIP, TZP, ATM, CAZ, FEP, MEM, IPM, GEN, TOB, AMK, CIP	11	A1	ND

^aP, patient; E, environment; H, health care worker.

^bAntibiotic codes: TIM, ticarcillin/clavulanic acid; PIP, piperacillin; TZP, piperacillin/tazobactam; ATM, aztreonam; CAZ, ceftazidime; FEP, cefepime; MEM, meropenem; IPM, imipenem; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; COL, colistin.

^cND, not done.

Antimicrobial susceptibility.

Most of the multiresistant P. aeruginosa isolates were panresistant to all antimicrobials tested except colistin (Table 1). The proportion of isolates showing resistance was as follows: cefepime, meropenem, imipenem, gentamicin, tobramycin, and ciprofloxacin, 100%; piperacillin, piperacillin/tazobactam, ceftazidime, and aztreonam, 94%; and ticarcillin/clavulanic acid, 83%. MIC determination confirmed the high-level resistance to cefepime (MIC = 16 to 32 μg/ml), imipenem (MIC > 32 μg/ml), meropenem (MIC = 8 to 32 μg/ml), amikacin (MIC = 32 to 64 μg/ml), gentamicin (MIC > 256 μg/ml), tobramycin (MIC = 128 to 256 μg/ml), isepamicin (MIC = 32 to 64 μg/ml), and ciprofloxacin (MIC > 256 μg/ml).

Typing.

Multiresistant P. aeruginosa isolates clustered in the same epidemic profile: serotype O:11, PFGE type A1, and AFLP type A (Table 1 and Fig. 1). Two serotype O:11 isolates were classified in another PFGE type, K1, and two serotype O:1 isolates showed the epidemic PFGE type A1/AFLP type A, which is predominantly associated with serotype O:11 (Table 1). Typing of P. aeruginosa isolates recovered from cystic fibrosis patients (n = 5) and patients hospitalized in other units (n = 10) showed distinct PFGE (Fig. 1) and AFLP profiles (data not shown). The epidemic curve of ICU patients infected or colonized with multiresistant P. aeruginosa from July 2002 to December 2002 showed that a clonal outbreak had occurred due to PFGE type A1 which peaked with 14 cases from July to August 2002 (Fig. 2). After implementation of precautions in August, clonal transmission was partially controlled, with only two epidemic cases over the September to December period (incidence of 0.25%, P < 0.001) (Fig. 2). Surveillance of multiresistant P. aeruginosa acquired in the ICU in 2003 revealed five cases (incidence of 0.2%), and typing showed the disappearance of the epidemic clone A1 in 2003 (data not shown).

FIG. 1.

Dendrogram indicating the similarity of PFGE profiles of P. aeruginosa strains (n = 46) and correlation with origin and resistance profile. These strains were isolated from patients (P), cystic fibrosis patients (CF), the environment (E), a health care worker's hand (H), in different ICU units (ICU), and in other units (Other). Resistance profiles were classified as multiresistant (MR), multisusceptible (MS), or resistant to one to five antimicrobials (non-MR).

FIG. 2.

Occurrence of new cases of nosocomially acquired multiresistant P. aeruginosa by PFGE type in the ICU, indicating the period when isolation precautions were implemented, July to December 2002. HCW, health care worker.

Environmental and health care worker culture surveys.

P. aeruginosa was recovered from 11 (11%) environmental samples. Eight isolates from tap water (n = 2), taps (n = 2), sink trap (n = 2), and room surfaces (n = 2) were either fully susceptible or resistant to ≤2 antibiotics and showed PFGE types different from that of the epidemic clone (Fig. 1). Multiresistant P. aeruginosa was recovered from the sink trap (n = 2) and tap (n = 1). Of these, one sink trap isolate belonged to epidemic type O:11/A1/A (Table 1 and Fig. 1). This sink was located in the room of a patient colonized with multiresistant P. aeruginosa. Health care worker screening detected multiresistant P. aeruginosa epidemic type O:11/A1/A on the hands of a nurse who had taken care of infected patient P9 before bacteriological screening (Table 1 and Fig. 1). This finding was used by the infection control team to emphasize the importance of careful hand protection and disinfection to the ICU staff.

β-Lactamase production.

Analysis by MICs determined by E-test with and without EDTA, spectrophotometric analysis of imipenem hydrolysis, and PCR detection for the vim and imp genes showed the absence of metallo-β-lactamases in multiresistant P. aeruginosa isolates. No TEM or SHV extended-spectrum beta-lactamases or oxacillinases (OXA-1, OXA-2, OXA-9, and OXA-10) were detected by PCR. Nevertheless, phenotypic and quantitative reverse transcription-PCR analysis detected hyperproduction of AmpC cephalosporinase. The putative function of AmpC in the resistance was evaluated by analysis of PFGE types A1 and K1 that showed expression values >50-fold higher than that of the wild-type PAO1 strain (Table 2).

TABLE 2.

Variations of resistance mechanisms in multiresistant P. aeruginosa isolates

Strain	β-Lactamase activity (nmol/min/mg of protein)	Expressiona		Sequence of mexZb
		oprD	mexX
PAO1	72	1.00	1.00
Positive control			>22c
PFGE type A1	5,100	0.02	49.4	GAC→G_C (164)
PFGE type K1	3,500	0.2	3.6

^amRNA quantification by RT-PCR. Numerical value are expressed relative to PAO1, which is set at 1.00.

^bNucleotide sequence compared with the PAO1 genome (www.pseudomonas.com).

^cMut-Gr1 strain overproducing MexXY was used as a positive control (29).

OprD porin.

The transcription level of the OprD porin was analyzed by quantitative real-time RT-PCR and compared to that of the reference strain PAO1. OprD appeared to be expressed 5- to 50-fold less than the wild-type PAO1 OprD in multiresistant P. aeruginosa types A1 and K1 (Table 2).

Efflux pump systems.

The contribution of the MexAB, MexCD, MexEF, and MexXY active efflux pump systems to the resistance of multiresistant P. aeruginosa was examined by comparison of PFGE types A1 and K1 with the reference strain PAO1. Transcription levels of mexA, mexC, and mexE were similar to those in wild-type PAO1 (data not shown). In contrast, epidemic type A1 P. aeruginosa overproduced MexXY compared with the wild-type PAO1 (49-fold). This was not observed for the type K1 isolate (Table 2).

To identify the mutations responsible for this MexXY upregulation, sequencing of the mexZ regulator was performed, and the nucleotide sequence was compared to the PAO1 sequence (www.pseudomonas.com). Both type A1 and K1 multiresistant P. aeruginosa exhibited two silent mutations resulting from a single nucleotide substitution at the isoleucine at position 37 (ATC→ATT) and the glutamic acid at position 113 (GAA→ GAG) (data not shown). The epidemic type A1 multidrug-resistant P. aeruginosa had an additional base deletion at aspartic acid position 164 (GAC→ G-C), introducing a frameshift outside the DNA binding region of MexZ (Table 2).

DISCUSSION

This study describes a clonal outbreak of panresistant P. aeruginosa in an ICU during a 6-month period. Typing characterized this epidemic panresistant P. aeruginosa clone as O:11/A1/A. PFGE and AFLP analysis appeared to be discriminant typing methods as demonstrated by others (23, 32). Transmission of this epidemic clone in the ICU was followed by its complete disappearance in 2003 following implementation of contact isolation measures. During the outbreak, this epidemic clone was recovered from ICU patients, from the sink of a colonized patient who was probably the source of this environmental contamination, and from the hands of a nurse, suggesting that transmission between patients could have occurred via health care worker hands.

P. aeruginosa is known to colonize the hospital environment, particularly moist sites (2). Sources such as tap water, sink, antiseptic solutions, respiratory equipment, and bronchoscopes are the most commonly incriminated nosocomial reservoirs of P. aeruginosa (7, 22, 25). Recent studies also reported health care workers as a transient reservoir and possible vehicle of nosocomial outbreaks (4, 12, 13, 31, 32). P. aeruginosa is not typically considered a skin-colonizing organism. The mechanism or source of hand colonization is presumably through contact with colonized patients or water. This mechanism has been linked to skin lesions and use of artificial nails (4, 13). Compliance with hand protection by gloves, washing, and hand disinfection after glove removal was emphasized as part of the effort to control the epidemic.

This particular multiresistant P. aeruginosa clone appeared to be very stable, with 90% of isolates showing identical PFGE/AFLP/serotype/antibiotic susceptibility profiles. It was recovered only in the ICU department. In contrast, an outbreak reported in Greece as being caused by multiresistant P. aeruginosa serotype O:11 showed distinct PFGE subtypes, which were also recovered from different units of the hospital (24). A recent study from France described the wide dissemination of an epidemic clone of multiresistant P. aeruginosa serotype O:6 inside and outside the hospital (8). Nosocomial P. aeruginosa strains are often multidrug resistant and acquire resistance determinants during the course of antimicrobial therapy in heavily colonized patients. This property probably contributes to their maintenance in the clinical environment under high antibiotic pressure, as was the case in the present study of a nosocomial outbreak limited to ICUs.

In the present study, multiresistant P. aeruginosa strains showed coresistance to 11 of the 12 antimicrobials tested and remained susceptible only to colistin. This broad-spectrum coresistance drastically restricted the choice of antimicrobial therapy, leaving colistin as the drug of last resort. Panresistance can be explained by accumulation of multiple resistance mechanisms, including gene mutation, overexpression of efflux pumps, loss or modification of porins, and acquired extended spectrum β-lactamases. In this study, overexpression of the AmpC chromosomal cephalosporinase was documented in the epidemic multiresistant P. aeruginosa. This mechanism contributed to resistance to β-lactams, as no acquired extended-spectrum beta-lactamase or oxacillinase was detected in these multiresistant P. aeruginosa isolates. However, differences in resistance to β-lactams between isolates were observed (for example, strain P4 was susceptible to ticarcillin/clavulanic acid and strain P5 was susceptible to ceftazidime) and these were probably due to various levels of AmpC cephalosporinase expression (8).

The carbapenem resistance of this epidemic strain could be explained by its decreased OprD porin expression rather than by AmpC overexpression (8). In contrast with European studies that showed the spread of the VIM metallo β-lactamase as the determinant of carbapenem resistance in P. aeruginosa isolates (9, 11, 15, 17), no VIM or IMP carbapenemase was detected in the strains in this investigation. A low prevalence of metallo-β-lactamase production was also observed among carbapenem-resistant P. aeruginosa in a Greek hospital (26).

We demonstrated by analysis of efflux pump systems that stable overexpression of the inducible mexXY system was present in the epidemic type A1. This altered phenotype could explain resistance to β-lactams and account for low-level coresistance to cefepime, aminoglycosides, and fluoroquinolones but also contribute to high-level resistance to these antibiotics in combination with other mechanisms (D. Hocquet, P. Plésiat, and the GESPA Group, Abstr. Int, Meet. Pseudomonas, abstr. 107, 2003). Sequencing analysis suggested that this overexpression was due to mutations in the mexZ repressor, recently described as agrZ (for aminoglycoside resistance dependent on mexZ) mutants by Llanes et al. (10). Double overexpression of the AmpC cephalosporinase and the inducible MexXY system combined with decreased expression of OprD could explain the panresistance displayed by our epidemic P. aeruginosa. Other mechanisms such as the presence of aminoglycoside-modifying enzymes or mutations in the quinolone-determining resistance region of topoisomerases can also contribute to resistance to aminoglycosides and fluoroquinolones, respectively, but were not explored in this study.

In conclusion, we demonstrated the epidemic spread of a panresistant P. aeruginosa clone involving 16 patients over a 6-month period in the ICUs of a university hospital. Increased expression of chromosomal AmpC cephalosporinase and the MexXY efflux pump system associated with a deficiency of OprD porin was implicated in this panresistance phenotype. Hand carriage of epidemic multidrug-resistant P. aeruginosa by a health care worker during the outbreak underlines the importance of careful hand hygiene in this high-risk ICU setting. This study illustrates the ability of multigene antibiotic-resistant mutants of P. aeruginosa to cause extensive outbreaks associated with significant morbidity and mortality. Our findings underscore the need to differentiate polyclonal emergence of multiple-antibiotic-resistant P. aeruginosa from clonal outbreaks, which require targeted infection control measures.