ABSTRACT
During a 3-year period (May 2005 to April 2008), a series of 45 outpatients presented with community-onset urinary tract infections due to carbapenem-resistant Pseudomonas aeruginosa isolates. Forty of them had a history of previous hospitalization or exposure to healthcare facilities, while the remaining five had not been previously admitted to our healthcare facilities or elsewhere within the preceding 12 months. In 18 outpatients, the carbapenem-resistant organisms caused recurrent community-onset urinary tract infections, while in three outpatients the organisms were also implicated in bacteremic episodes. All 45 single-patient P. aeruginosa isolates harbored the blaVIM-2 metallo-β-lactamase (MBL) gene in a common class 1 integron structure. They belonged to one predominant pulsed-field gel electrophoresis type and three sporadically detected types; two of the sporadic clonal types were identified among outpatients without previous exposure to healthcare facilities, while the predominant clonal type was also identified to cause infections in hospitalized patients. This is the first study documenting that MBL-producing P. aeruginosa isolates cause community-onset infections that are related or not with exposure to healthcare facilities. Community-onset infections in our patients most likely resulted from the nosocomial acquisition of MBL producers, followed by a prolonged digestive carriage. The high rate of recurrent infections in the community underlies the difficulty of constraining infections caused by such microorganisms in the extrahospital setting.
Pseudomonas aeruginosa is a clinically significant gram-negative rod-shaped bacterium that may be selected and propagated within the hospital environment. Antimicrobial resistance in this species is a problem of growing concern and limits our therapeutic alternatives. Carbapenems are commonly used as last-resort drugs for the treatment of infections caused by multidrug-resistant P. aeruginosa isolates. However, intensive use of carbapenems in the treatment of nosocomial P. aeruginosa infections has facilitated the emergence of mechanisms that confer resistance to carbapenems, such as diminished permeability, overexpression of the intrinsic efflux systems, and production of carbapenemases (21). Acquisition of class B metallo-β-lactamases (MBLs) constitutes a growing family of carbapenem-hydrolyzing β-lactamases among P. aeruginosa strains (26). These enzymes efficiently hydrolyze all β-lactam compounds except aztreonam, and in most cases their genes reside within class 1 integrons of various compositions of gene cassettes (18). They are frequently classified in the IMP and VIM types, while other types, such as AIM, GIM, and SPM, are found only sporadically in some geographic regions (26).
MBL-producing P. aeruginosa bacteria are slowly but steadily increasing within hospitals, causing outbreaks and/or hyperendemic situations in several centers, mostly in the Far East and south of Europe (5, 9, 11, 15, 18). Studies have identified the risk factors for MBL acquisition (8, 14, 29) as well as the outcome of P. aeruginosa infections caused by MBL producers (12, 14). The increasing prevalence of nosocomial infections produced by MBL-possessing P. aeruginosa strains severely compromises the selection of appropriate treatments and is therefore associated with significant morbidity and mortality.
In Greece, the proportion of MBL-producing P. aeruginosa strains has been rapidly increasing among hospital-acquired infections since the beginning of the last decade (24). Previous studies have shown that this epidemic occurrence is frequently due to the clonal spread of VIM-type MBLs but also due to horizontal transmission of these enzymes between unrelated clonal strains (17, 20). In several European countries, also the increase of MBL production in P. aeruginosa is due primarily to the spread of VIM-type MBLs, suggesting a large reservoir of the respective blaVIM gene cassettes (5, 11, 15, 18). However, no previously published studies in Europe or elsewhere have described infections caused by MBL-producing P. aeruginosa strains among community patients with or without information on their association with healthcare facilities.
In our clinical laboratory, we observed during May 2005 the emergence of imipenem- and meropenem-resistant P. aeruginosa isolates, which were recovered from urine samples of elderly patients in the community. This event prompted the present observational cohort survey in which we describe the emergence and spread of MBL-producing P. aeruginosa strains among patients attending our outpatient community department. The route of the MBL-producing P. aeruginosa acquisition in outpatients was also investigated.
MATERIALS AND METHODS
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 blaIMP, blaVIM, blaSPM, blaGIM, blaTEM, blaSHV, blaCTX-M, blaGES, and blaCMY, was performed on the carbapenem-resistant isolates using consensus primers and amplification conditions described previously (3, 17, 23, 25). Primers amplifying the whole blaVIM 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 blaVIM, 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+ Rifr) and P. aeruginosa strain PU21 (Rifr). 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 blaVIM-2 allele was detected by Southern blotting after electrophoresis of the plasmid extract and gene-specific hybridization using digoxigenin-labeled blaVIM-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).
RESULTS
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).
Antimicrobial activity of VIM-2-producing P. aeruginosa isolates (n = 45) implicated in episodes of community-onset infections
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.
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.
Features of 45 patients with community-onset infections due to MBL-producing P. aeruginosa
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).
Patients' characteristics associated with recurrent community-onset MBL-producing P. aeruginosa infectionsa
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).
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 blaVIM gene, and sequencing analyses identified a blaVIM-2 allele in all patients' representative 45 blaVIM-positive isolates. Furthermore, PCR mapping of the implicated integron was performed in four representative blaVIM-2-positive isolates of the predominant clone type and single representative blaVIM-2-positive isolates of the three minor clonal types. It was found that in all seven isolates, the blaVIM-2 cassette was flanked by two aacA29 cassettes, with aacA29a upstream of blaVIM-2 and aacA29b downstream of blaVIM-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 blaVIM probe, indicated the chromosomal location of the blaVIM-2 gene.
DISCUSSION
To date, our understanding of the epidemiology of MBL-producing P. aeruginosa isolates is limited to outbreaks and infections reported from tertiary care medical centers (8, 9, 11, 12, 14, 15, 24, 29). The occurrence of community-onset infections by MBL-producing P. aeruginosa has not been previously recognized, and the epidemiology of these infections outside hospitals has not been studied. A previous study has identified two VIM-2-carrying P. aeruginosa strains from aquatic habitats and made implications that the hospital setting was the possible origin of these strains (19). In the present study, we showed the emergence of community-onset infections due to VIM-2-producing P. aeruginosa in a region of northern Greece, where most of the outpatients had a history of previous exposure to the healthcare system. Our hospital settings experience a high incidence of infections due to MBL-producing P. aeruginosa isolates (17, 20, 24), and this may have facilitated the large dissemination of such strains in our extrahospital setting.
As many as 45 outpatients were identified with infections due to carbapenem-resistant MBL-producing P. aeruginosa. These patients were of advanced age, and comprehensive analyses of their medical histories revealed previous hospital exposure in most cases (89.9%). Therefore, infections affecting these patients were characterized as healthcare-associated community-onset infections. The vast majority of these infections were in male patients, since all of them were previously admitted to the hospital for genitourinary pathology. In fact, the mean time to first isolation of a VIM-producing P. aeruginosa isolate was almost 40 days from the time of hospital discharge, suggesting that transmission of clonally related VIM-producing organisms was occurring during patients' hospitalization. This was further supported by the fact that P. aeruginosa isolates of the major clonal type were also identified to cause hospital-acquired infections during the study period. It should also be mentioned that these isolates belonged to serotype O11, which is associated with hospital-acquired infections. Thus, community-onset infections in our patients most likely resulted from nosocomial acquisition of MBL-producing P. aeruginosa strains followed by a prolonged digestive carriage. In addition, person-to-person transmission in the community is possible since the same clonal type was identified among three outpatients without previous exposure to the healthcare system.
It is also noteworthy that two distinct PFGE clonal types were detected among outpatients without a history of previous hospitalization, suggesting that the common integron structure bearing the blaVIM-2 gene cassette has been incorporated into different P. aeruginosa clones in the community. The integron structures of our representative blaVIM-2-positive isolates were similar to that of In59, initially detected in P. aeruginosa strain RON-2 in France (16) and identical to that of In59.2, recently described among blaVIM-2-bearing nosocomial P. aeruginosa strains in northern Greece (20). This fact further supports the horizontal migration of P. aeruginosa from the hospital environment to the community setting. Consistent with our observations, previous studies have also demonstrated that community-onset infections due to ESBL-producing gram-negative bacteria often result from nosocomial acquisition of the strains or their genetic elements (1). Moreover, genitourinary pathology, a finding common in our patients, has been recognized as an independent risk factor for community-onset infections caused by ESBL-producing organisms (2).
The present study provided evidence that VIM-producing P. aeruginosa clonal strains could easily disseminate outside the hospital environment. The spread of VIM-producing organisms causing healthcare-associated community-onset infections appears to have been rapid, perhaps because VIM infection and colonization were previously unrecognized in the hospital, resulting in an underestimation of the true incidence of VIM-producing P. aeruginosa organisms and the duration of the VIM-producing P. aeruginosa epidemic in the hospital setting. It is notable that rigorous infection control measures were undertaken in our hospital wards where outpatients were previously hospitalized. These practices were able to restrain the number of new community-onset case patients only during the third year of the study (Fig. 1).
The study also showed that the spread of VIM-producing P. aeruginosa organisms in the community represents a serious infection control and therapeutic challenge. In this context, it should be mentioned that we have previously documented in our extrahospital setting the transmission of acquired MBL-producing Proteus mirabilis isolates causing healthcare-associated community-onset infections (23). However, unlike in the present study, recurrent community-onset P. mirabilis infections were not detected, and all patients had previous exposure to healthcare facilities. It is possible that the ability of P. aeruginosa to produce biofilms at the site of infection, as well as the fact that several of our patients suffered from severe underlying diseases, were responsible for the large number of community-onset infections as well as the frequent recurrent infections in the community setting. In our study, prostatic hyperplasia and diabetes mellitus as well as infection caused by MBL-producing P. aeruginosa during a previous hospitalization were significantly associated with recurrent MBL infection in the community setting. It should be also mentioned that three patients with malignancy developed VIM-producing P. aeruginosa community-onset bacteremia that required hospitalization.
All of our MBL-producing P. aeruginosa isolates exhibited cross-resistance to many antipseudomonal drugs. Although retention of susceptibility to piperacillin-tazobactam by several of our isolates may seem unexpected, since MBLs hydrolyze this compound, similar observations have been reported previously for blaIMP- and blaVIM-bearing isolates (6, 9). Treatment of community-onset infections due to VIM-producing organisms had profound therapeutic and economic implications. In our patients, intravenous drugs with potential toxicity, such as gentamicin and colistin, were usually the only available antimicrobial agents. Moreover, the clinical and microbiological outcome was not desirable, since in several cases persistence of VIM-producing strains and recurrent infections were demonstrated over at least a 1-year period. Increased awareness and intensified infection control practices in the hospital as well as the community setting are the keys to curtailing the spread of these alarming carbapenem-resistant pathogens.
ACKNOWLEDGMENTS
None of the authors has any potential conflict of interest.
FOOTNOTES
- Received 5 June 2009.
- Returned for modification 8 June 2009.
- Accepted 11 September 2009.
- Copyright © 2009 American Society for Microbiology