Large Dissemination of VIM-2-Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains Causing Health Care-Associated Community-Onset Infections

Patient data and definitions.The study was conducted during a 3-year period (May 2005 to April 2008) and included patients who were referred to the outpatient department of Serres General Hospital with community-onset infections due to carbapenem-resistant P. aeruginosa isolates. This hospital is a 400-bed acute care hospital serving a population of ∼200,000 habitants, with ∼41,000 hospital admissions per year and ∼220,000 visits per year in the outpatient department. Standard infection control surveillance definitions were used (7). Patients with community-onset infections due to carbapenem-resistant P. aeruginosa were defined as those who were referred from the community to the outpatient department of the hospital for assessment and had symptomatic infections, with clinically significant isolation of imipenem- and/or meropenem-resistant P. aeruginosa (MIC > 8 μg/ml) from a specimen taken in the outpatient department. Episodes of community-onset infections which were identified from specimens taken in hospital wards were not included in the study. The community-onset infection was defined as healthcare associated if the patient fulfilled any of the previously proposed criteria (7). As recurrent urinary tract infection was defined as a repeated infection of the kidneys or bladder, with at least 1 week of follow-up observation after antimicrobial treatment discontinuation.

Microbiological data were recovered from the clinical laboratory of the hospital. Clinical data were obtained from the community patients or their closest relatives, their private practitioners, and the medical staff. Patient data were analyzed with respect to demographic data, underlying diseases, permanent urinary catheter usage, whether the patient had been admitted to a hospital or had had surgery in preceding year, whether the patient had an infection during a previous hospitalization or had received antibiotics within 30 days of the community-onset infection, and antibiotic treatment and immunoderepressor history.

Identification of bacterial isolates and susceptibility testing.Identification and initial susceptibility testing were performed with the MicroScan system (Dade Behring Inc., West Sacramento, CA). Identification was confirmed with the API 20NE system (bioMérieux, Marcy l' Étoile, France). The preliminary antibiotic resistance profile was confirmed by the agar dilution method using CLSI interpretative criteria (4) and P. aeruginosa ATCC 27853 as a control. Imipenem and meropenem MICs as well MICs of several antipseudomonal drugs (amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, colistin, gentamicin, netilmicin, piperacillin-tazobactam, tobramycin) were determined. Among carbapenem-resistant isolates, phenotypic detection of MBL production was performed using the imipenem-EDTA double-disk synergy test (28) and the Etest MBL assay (AB Biodisk, Solna, Sweden).

PCR amplifications and sequence analysis.PCR detection of various MBL, extended-spectrum β-lactamase (ESBL) and plasmidic AmpC gene types, including bla_IMP, bla_VIM, bla_SPM, bla_GIM, bla_TEM, bla_SHV, bla_CTX-M, bla_GES, and bla_CMY, was performed on the carbapenem-resistant isolates using consensus primers and amplification conditions described previously (3, 17, 23, 25). Primers amplifying the whole bla_VIM region were used for sequencing purposes (27). For integron mapping, PCR assays combining primers specific for 5′ and 3′ conserved sequences (13) with primers specific for bla_VIM, aacA, dfrA, aadA, qacEΔ1, and sul genes were performed. PCR products were purified using ExoSAP-IT reagent (USB Corporation, Cleveland, OH) and used as templates for sequencing on both strands with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA).

Serotyping and molecular typing.O serotypes were determined by the slide agglutination test of the International Antigenic Typing Scheme as previously described (24). Pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA was performed with a contour-clamped homogeneous electric field-direct repeat II system (Bio-Rad, Hemel Hempstead, United Kingdom) (10). Banding patterns were compared visually using previously described criteria (22). In addition, the carbapenem-resistant isolates recovered from community-onset infections were genotypically compared to those isolated in wards of the hospital where the outpatients with community-onset infections had previously resided.

Conjugation experiments and Southern blot analysis.The potential for conjugational transfer of imipenem resistance was examined in diparental filter matings using recipient strains of Escherichia coli strain 20R764 (lac⁺ Rif^r) and P. aeruginosa strain PU21 (Rif^r). P. aeruginosa clinical strains carrying transferable plasmids were used as controls. Transconjugants were selected on Mueller-Hinton agar containing imipenem (4 μg/ml) and rifampin (400 μg/ml).

Plasmid extraction was performed with two different lysis methods using Escherichia coli strain 39R861 as a control. The chromosomal location of the bla_VIM-2 allele was detected by Southern blotting after electrophoresis of the plasmid extract and gene-specific hybridization using digoxigenin-labeled bla_VIM-1 probe (24).

Statistics.To detect significant differences between groups, we used the two-tailed t test for parametric variables and the chi-square test or Fisher's exact test for nonparametric variables. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. The results were analyzed using a commercially available statistical software package (SPSS version 12.0; SPSS Inc., Chicago, IL).

Clinical isolates and antimicrobial susceptibilities.During the 3-year period of the study, 403 isolates of P. aeruginosa were cultured from clinical specimens of patients who visited the outpatient department; 97 (24.1%) of them that belonged to 45 outpatients were confirmed as resistant to imipenem and/or meropenem. The vast majority of the latter isolates were recovered from urine specimens (94 isolates; 96.9%) of patients who were admitted with symptoms and signs of urinary tract infection. The remaining three isolates were recovered from blood cultures of community-onset bacteremic cases. MICs of imipenem ranged from 32 to >128 μg/ml, while MICs of meropenem were from 16 to >128 μg/ml. One isolate per patient was selected for detailed susceptibility testing. The susceptibilities of the 45 carbapenem-resistant isolates to various antipseudomonal drugs are listed in Table 1. All isolates were susceptible to colistin, while 33 were susceptible to aztreonam, 24 to piperacillin-tazobactam, and 8 to gentamicin. In addition, 17 isolates exhibited intermediate susceptibility to gentamicin and 9 to aztreonam. All isolates exhibited resistance to ceftazidime, cefepime, ciprofloxacin, amikacin, netilmicin, and tobramycin. Susceptibilities of isolates from subsequent recurrences of infections were also tested. MICs of antipseudomonal antibiotics were not changed except in three isolates that showed an increase in colistin MIC to resistant levels (MIC of 4 μg/ml).

Clinical and epidemiological characteristics.The average age of each patient (42 male/3 female) was 70.4 years (range, 25 to 83 years). The distribution of new patients with community-onset infections is presented in Fig. 1. Characteristics of the 45 patients with community-onset infections due to MBL-producing P. aeruginosa are presented in Table 2. As many as 40 (88.9%) of the outpatients had a history of previous hospitalization or exposure to healthcare facilities during the preceding year, and therefore their infections were defined as healthcare-associated community-onset infections. The remaining five (11.1%) patients had not been previously admitted to our healthcare facilities or elsewhere and had not been residing in a nursing home or long-term-care facility during the preceding year. Two of the latter patients suffered from neurological disease, two had benign prostatic hyperplasia, and one suffered from papillary transitional carcinoma, which was diagnosed after visiting the outpatient department.

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FIG. 1.

Distribution of new patients with community-onset infections during the study period (May 2005 to April 2008). Jan, January; Feb, February; Mar, March; Apr, April; Jun, June; Jul, July; Aug, August; Sep, September; Oct, October; Nov, November; Dec, December.

All 40 patients with healthcare-associated infections were previously admitted to the hospital for genitourinary pathology, and 29 of them had undergone a surgical urological intervention. Among the patients with a history of previous hospital admission, the mean time of the hospital exposure before presentation with the community-onset infection at the outpatient clinic was 37.7 days (range, 10 to 123 days). It is of note that 4 of these 40 patients were also infected with carbapenem-resistant P. aeruginosa isolates during their previous hospitalization. In 21 of the 40 patients with a history of previous hospital admission, the first community-onset infection was documented within 30 days after hospital discharge, while in the remaining 19 patients the first community-onset urinary tract infection was documented more than 30 days after hospital discharge.

Recurrent community-onset infections due to carbapenem-resistant P. aeruginosa were detected in 18 outpatients. In five of these patients the recurrent infections lasted within 3 months, in six patients recurrent infections occurred for more than 3 months but lasted for up to 12 months, and in seven patients they occurred for more than 12 months. Among the 18 outpatients, the number of recurrent community-onset infections ranged from two to seven during the 3-year study period. The characteristics of the patients with recurrent infections compared to those without recurrent community-onset infections due to carbapenem-resistant P. aeruginosa isolates are presented in Table 3. The crude analysis showed that prostatic hyperplasia (OR, 6.25; 95% CI, 1.09 to 36.68; P = 0.026), diabetes mellitus (OR, 10.0; 95% CI, 1.06 to 94.7; P = 0.019), and MBL infection during a previous hospitalization (OR, 9.4; 95% CI, 1.23 to 71.6; P = 0.01) were significantly associated with recurrent infections due to carbapenem-resistant P. aeruginosa (Table 3).

The 40 outpatients with previous hospital exposure had been treated during their hospitalization with fluoroquinolones alone or received intravenous treatment (amikacin plus ticarcillin-clavulanic acid or piperacillin-tazobactam for 3 to 5 days) and subsequently fluoroquinolones or cefuroxime. Aztreonam with colistin or gentamicin as well as gentamicin alone were administered as etiologic therapies to treat the urinary tract and bacteremic community-onset infections.

Typing of isolates.PFGE analysis of XbaI-digested genomic DNA clustered the 97 carbapenem-resistant isolates of the study into four distinct clonal types, with the most prevalent type (type I) containing two subtypes differing from each other by one band. The prevalent type contained as many as 84 isolates, while three less-frequently detected clonal types (types II, III, and IV) contained 8, 3, and 2 isolates, respectively (Fig. 2). Serotyping revealed that all isolates belonged to type O11, except the three isolates of clonal type III, which were nontypeable with monovalent antisera. In all cases with repetitive isolates from the same patient, the same PFGE clonal type was detected. The five patients with no previous hospital exposure were infected with eight isolates that belonged to three clonal types (Fig. 2). As many as three of these patients had single isolates that belonged to the predominant clonal type I, while the remaining two patients had three and two isolates each that composed two of the minor clonal types (types III and IV). A collection of 12 contemporary carbapenem-resistant P. aeruginosa isolates causing hospital-acquired infections in our institution was run for comparison. The PFGE profile of the predominant clonal type I was identified among all of these isolates (Fig. 2).

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FIG. 2.

PFGE profiles of 18 VIM-2-producing P. aeruginosa isolates, which are representative of the four different clonal types. Lanes 1 to 4 and 6 to 13, isolates recovered from healthcare-associated community-onset infections; lanes 14, 15, 18, and 19, isolates recovered from non-healthcare-associated community-onset infections; lanes 17 and 20, isolates recovered from hospital-acquired infections; lanes M (5 and 16), multimers of phage lambda DNA (48.5 kb) molecular mass markers.

MBL characterization.Production of MBL was detected in all 97 isolates causing community-onset infections with both phenotypic methods. Application of the phenotypic methods in a random sample of 25 imipenem-susceptible or imipenem-intermediate (MIC ≤ 8 μg/ml) community P. aeruginosa isolates, collected during the same period, did not reveal any additional MBL-producing isolates. PCR assays showed that all 97 carbapenem-resistant isolates carried a bla_VIM gene, and sequencing analyses identified a bla_VIM-2 allele in all patients' representative 45 bla_VIM-positive isolates. Furthermore, PCR mapping of the implicated integron was performed in four representative bla_VIM-2-positive isolates of the predominant clone type and single representative bla_VIM-2-positive isolates of the three minor clonal types. It was found that in all seven isolates, the bla_VIM-2 cassette was flanked by two aacA29 cassettes, with aacA29a upstream of bla_VIM-2 and aacA29b downstream of bla_VIM-2. The Pc and P₂ promoters were strong and inactive (no GGG insertion), respectively. PCR testing for other bla genes, including various MBL, ESBL, and plasmidic AmpC genes, was negative in all cases. Conjugation experiments failed to detect that the MBL determinant could be transferred, and plasmid DNA was not visualized by either of the extraction methods in agarose gel electrophoresis. These findings, along with the hybridization of the chromosomal band with the bla_VIM probe, indicated the chromosomal location of the bla_VIM-2 gene.