A large sustained endemic outbreak of multiresistant Pseudomonas aeruginosa: a new epidemiological scenario for nosocomial acquisition

Setting and Definitions

The study was performed at the Hospital Universitari de Bellvitge, a 900-bed public tertiary-care institution for adult patients. The MRPA surveillance programme was initiated in January 2006 and all patients colonized or infected with MRPA were prospectively identified.

MRPA was defined as strains resistant to ≥ 3 of the following classes of antibiotics: antipseudomonal penicillins, antipseudomonal oxyimino-β-lactams, fluoroquinolones, aminoglycosides, and carbapenems [19]. A clinical sample positive for MRPA was considered to have been acquired in a specific ward if it appeared either during a stay in this ward or within a week of discharge from it. The remaining patients with clinical samples positive for P.aeruginosa and without the MRPA phenotype isolate from the time of admission to the time of discharge from hospital were considered non-MRPA.

Clinical assessment was determined in accordance with the definitions of the Centers for Disease Control and Prevention for nosocomial infections [20]. Patients with positive clinical samples for MRPA but without related signs or symptoms of infection were considered to be colonized.

Microbiological studies

P. aeruginosa strains were identified and tested for antimicrobial susceptibility by a MicroScan automated microdilution system using CN1S and CO1S panels (Dade International, West Sacramento, CA, USA). CLSI (Clinical and Laboratory Standards Institute) criteria [21] were used to define susceptibility or resistance to these antimicrobial agents.

We selected 284 MRPA phenotype strains isolated throughout the whole period (2006 [132], 2007 [101], 2008 [51]) for pulsed-field gel electrophoresis (PFGE) analysis using a method described previously [13]. DNA was digested with SpeI and DNA fragments were separated with a CHEF DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was run at 6 v/cm, 14°C for 20 hours with pulses ranging from 0,5 to 25 seconds. DNA restriction patterns generated by PFGE were interpreted according to the guidelines [22]. Isolates with PFGE patterns differing in more than four fragments were ascribed to distinct genotypes; differences in four restriction fragments or fewer were considered to be subtypes of a single genotype [23].

The mechanisms responsible for the multidrug-resistant phenotype detected were studied in three representative isolates from the epidemic clone. The potential presence of horizontally acquired β-lactamases was explored by phenotypic tests, which included Etest MBL strips (AB Biodisk, Solna, Sweden) for the detection of class B carbapenemases and double disk synergy tests (DDST) for the detection of ESBLs, using amoxicillin-clavulanate and ceftazidime, cefepime, aztreonam, imipenem, or meropenem disks (distance from 5 to 30 mm). To examine the involvement of mutation-driven resistance mechanisms in the multidrug-resistant phenotype, the relative mRNA expression of the genes encoding the major P. aeruginosa efflux pumps (mexB, mexD, mexF and mexY) and the chromosomal cephalosporinase (ampC) was determined by real-time PCR as described elsewhere [24, 25]. Isolates were considered to be hyperproducers if the relative expression of the corresponding gene was at least three times (mexB) or ten times (mexD, mexF mexY, and ampC) greater than the figure documented for the reference PAO1 strain. In addition, in order to detect inactivating mutations causing carbapenem resistance, oprD genes from these isolates were amplified by PCR and fully sequenced, using previously described primers and conditions [26]. The sequences obtained were compared with those available in the GenBank.

Environmental survey

To determine the possible route of transmission or reservoir in the hospital's physical environment, a total of 78 samples, 30 from room equipment (bedside tables, bed rails, phones, plug holes, tap handles, door handles, call buttons), 36 from sink surfaces, and 12 from water samples were taken from the urology and ICU wards, the wards which initially had the highest incidence. Twenty samples, (13 from urology wards and seven from the ICU), were obtained in February 2006 using moistened swabs, another 16 samples were obtained in September 2006 from the urology wards, and the remaining 42 samples were collected from the ICU during 2007. In addition, to improve our capacity to detect contamination, in 2008 we modified the swab technique by introducing the use of moistened sterile gauze pads [27]. Another 82 samples were collected (63 from ten ICU rooms and 19 from four non-ICU rooms). All these samples were taken from room equipment. During the surveillance procedure, all the non-ICU rooms had admitted patients with known MRPA colonization, compared with only three ICU rooms. The genetic similarity of MRPA strains isolated in the environmental survey was investigated by PFGE.

Active surveillance program

To determine the strength of the human reservoir, we carried out longitudinal active surveillance of rectal swabs in an ICU unit over a one-month period (March 2006). Weekly rectal swab samples were obtained on admission in order to identify digestive tract carriage of MRPA between ICU admission and discharge or the time to MRPA clinical sample detection. Patients admitted to the unit for less than 48 hours and those with MRPA digestive tract colonization at ICU admission were excluded.

Infection control interventions

Infection control measures: Intervention 1: Disposable aprons and gloves were used while caring for MRPA colonized/infected patients, and cleaning procedures were strictly supervised by infection control nurses (rooms were routinely cleaned once a day using water and liquid soap with 500 parts per million [ppm] hypochlorite, and rooms with colonized patients were cleaned twice a day in the same way but with a hypochlorite concentration of 1,000 ppm, and using a specific checklist for high-touch surfaces). ICU patients were in single rooms and patients in non-ICU wards were spatially segregated inside a single room. These infection control measures were introduced in February 2006. Intervention 2: Environmental cleanliness was reinforced, particularly in the ICU (which was closed for extensive decontamination, consisting in rigorous cleaning of the medical equipment and painting of the wards, in July-August 2006 and July-August 2007) and the urology ward (which was decontaminated in August 2007).

Restriction of carbapenem use in ICUs: In January 2007 the use of carbapenem for ICU patients was restricted. Compliance with this policy was monitored. Consumption was expressed as defined daily doses (DDD) [28].

Statistical analysis

An interrupted time series analysis was performed using the segmented linear regression procedure [29], evaluating the impact of the intervention on the level (abrupt change immediately following the intervention) and the secular trend of the series (change in the slope of the incidence rate following the intervention). The level and trend of the pre-intervention segments served as the control for the post-intervention segment.

MRPA patients included those who presented at least one MRPA specimen obtained during admission; previously known cases who tested positive on readmission were excluded, and newly identified cases attending the outpatient clinic were included from the date of prior admission. Colonization pressure was calculated monthly in the ICUs and the urology ward as follows: number of MRPA patient-days × 100/total number of patient-days [30].

To evaluate the effect of the interventions performed in the urology ward, four time segments were defined (Figure 1). Segments 1 and 3 served as the pre-intervention (baseline) periods. Time points before May 2006 were excluded from the analysis, since there were fewer than three observations before and after an intervention performed in February 2006 [31]. The interventions studied were: Intervention 1 "reinforcement of control measures and cleaning procedures" (October 2006) and Intervention 2 "extensive decontamination" (August 2007). The analysis was adjusted for the colonization pressure of the immediately preceding month and for the level and trend of the pre-intervention periods. Statistical analysis was performed with SPSS software, version 14.0.

Ethical considerations

The study formed part of the medical care provided in the setting of the surveillance and control of nosocomial outbreaks, and the physician had good reason to believe that participation in the research study would not adversely affect the health of the patients. All the samples and exams performed on patients were part of standard care procedures for epidemiological control. The study was approved by the local ethic committee of the hospital Universitari de Bellvitge and no informed consent was requested.

Description of the outbreak

From January 2006 to May 2008, 390 consecutive patients were colonized or infected by a main phenotype of MRPA; 288 (74%) patients were males with a mean age of 62.4 (SD ± 19.7) years; 85 (22%) were ICU patients and 305 patients acquired the MRPA strain in a non-ICU setting. The urology service [54 patients/305 (18%)] was the non-ICU ward with the highest rates of MRPA acquisition; the remaining patients were recorded in 30 other wards.

Among the 390 patients with clinical samples for MRPA, 165 (42%) patients were infected, with urinary tract infection [45 episodes (28%)] being the most frequent MRPA infection. Forty-six episodes (28%) were intra-abdominal infections: 36 (22%) surgical site infections, and 10 (6%) biliary infections. All 37 (23%) episodes of lower respiratory tract infection were observed in ICU patients. The remaining infections were: eleven (7%) primary bloodstream infections, 12 (7%) bone and joint infections, nine (6%) soft-tissue infections and five miscellaneous infections. Seventy-two (44%) out of 165 infected patients died. Of these deaths, 14 (19%) were attributed to MRPA infection.

Microbiological and genotypic analysis

All phenotypic tests for the detection of acquired β-lactamases yielded negative results. In contrast, the pan-β-lactam resistance phenotype was found to be driven by the interplay of the hyperproduction of the chromosomal cephalosporinase AmpC (238 ± 34-fold increased expression of ampC respect to wild-type reference strain PAO1) and the inactivation of OprD porin, caused by a C to T mutation in nucleotide 424 of oprD, which leads to a premature stop codon at amino acid 142. Furthermore, no significant modification of the expression of the efflux pump encoding genes mexB, mexD, mexF, or mexY was detected.

Environmental survey results

Three of 36 (8%) initial samples were positive for clonal MRPA strain. All three strains were isolated in wet areas in the urology ward. None of the remaining 42 samples (moistened surfaces, room equipment and water samples) from the ICU wards showed MRPA growth. Thirteen samples, five of them from water, showed growth for carbapenem-susceptible P.aeruginosa.

In addition, with the modified gauze technique [27], three of 19 (16%) samples from non-ICU rooms were positive for the MRPA clonal strain, while only four of 63 (6%) ICU samples were positive.

Active surveillance program

During the study period, 42 patients were admitted to the ICU; of these, six patients with an ICU stay of less than 48 hours and five carriers of digestive tract MRPA at ICU admission were excluded. Thirty-one patients were included, and six digestive tract carriers were colonized by MRPA (19%). The mean time to MRPA acquisition between ICU admission and digestive tract carriage was 22 days. The mean time between carriage of MRPA in the digestive tract and the detection of positive clinical samples was 11 days (range 1 to 21 days).

Response to infection control intervention programs

Before the interventions, there was a significant month to month increase in MRPA cases in the urology ward: segment 1: increase of 0.67 cases/1,000 patient-days (95%CI 0.21 to 1.12; p = 0.006) and segment 3: increase of 1.53 cases/1,000 patient-days (95%CI 0.84 to 2.23; p < 0.001). However, significant changes in the trends of incidence rates were noted after both interventions: intervention 1: -1.16 cases/1,000 patient-days (95%CI -1.86 to -0.46; p = 0.003) and intervention 2: -1.36 cases/1,000 patient-days (95%CI -1.88 to -0.84; p < 0.001). This represents a mean decrease of 5.4 MRPA cases/1,000 patient-days after intervention 1, and an even more marked reduction, 8.4 cases/1,000 patient-days, after intervention 2. Concomitantly, after intervention 2, an immediate effect was found in the first month: -1.58 cases/1,000 patient-days (95%CI -3.50 to 0.33; p = 0.09) (Figure 1).

In addition, restricted use of carbapenems was initiated in January 2007 (Figure 2). Carbapenem use decreased from 190-170 DDD/1,000 patient-days in the period immediately before the implementation of this restriction (October-December 2006) to 40-60 DDD/1,000 patient-days after its initiation (January-April 2007), with a reduction from 3.1 cases/1,000 patient-days in December 2006 to 2.0 cases/1,000 patient-days in May 2007. However, after this strong initial decrease, we observed a moderate increase in carbapenem consumption, which rose to around 100 DDD/1,000 patient-days from May to December 2007 and a progressive increase in incidence. Segmented linear regression analysis could not easily be applied in ICU intervention (carbapenem use restriction) since no evident linear patterns were found following the intervention.