Portal de Periódicos da CAPES

Pseudomonas aeruginosa: antibiotic susceptibility and genotypic characterization of strains isolated in the intensive care unit

2001; Elsevier BV; Volume: 7; Issue: 12 Linguagem: Inglês

10.1046/j.1469-0691.2001.00348.x

1469-0691

Xavier Bertrand, Michelle Thouverez, Cyrille Patry, P. Balvay, D. Talon,

Bacterial Identification and Susceptibility Testing

A prospective study was carried out to assess the incidence and the local antibiotic susceptibility of Pseudomonas aeruginosa in intensive care units (ICUs) and to characterize cross-transmission by using pulsed-field gel electrophoresis as an epidemiologic tool. For this purpose, we screened surveillance cultures and routine clinical cultures from patients admitted to two adult ICUs during a 2-year period. Antibiotic susceptibility was determined by a disk diffusion method. The overall incidence of P. aeruginosa was 19.1 cases per 100 patients. Our findings concerning the antibiotic resistance of clinical isolates were concordant with those of other studies. Genotyping revealed that approximately 53.5% of P. aeruginosa colonization was acquired via cross-transmission; the other cases probably originated from endogenous sources. Cross-colonization seems to make a large contribution to the spread of P. aeruginosa in ICUs. A prospective study was carried out to assess the incidence and the local antibiotic susceptibility of Pseudomonas aeruginosa in intensive care units (ICUs) and to characterize cross-transmission by using pulsed-field gel electrophoresis as an epidemiologic tool. For this purpose, we screened surveillance cultures and routine clinical cultures from patients admitted to two adult ICUs during a 2-year period. Antibiotic susceptibility was determined by a disk diffusion method. The overall incidence of P. aeruginosa was 19.1 cases per 100 patients. Our findings concerning the antibiotic resistance of clinical isolates were concordant with those of other studies. Genotyping revealed that approximately 53.5% of P. aeruginosa colonization was acquired via cross-transmission; the other cases probably originated from endogenous sources. Cross-colonization seems to make a large contribution to the spread of P. aeruginosa in ICUs. Pseudomonas aeruginosa, a common hospital-acquired pathogen, is very important because of the number of infections caused and their gravity [1Shadberg DR Culver DH Gaynes RP Major trends in the microbial etiology of nosocomial infection.Am J Med. 1991; 91: 72S-75SGoogle Scholar,2Jarvis WR Martone WJ Predominant pathogens in hospital infections.J Antimicrob Chemother. 1992; 29: 19-24Crossref PubMed Scopus (482) Google Scholar]. Intensive care units (ICUs) are settings of high endemicity for this pathogen, which commonly causes bronchopulmonary infections, and less frequently urinary tract infections, infections of surgical wounds, and bacteremia [3Widmer AF Wenzel RP Trilla A Bale MJ Jones RN Doebelling BN Outbreak of Pseudomonas aeruginosa infections in a surgical intensive care unit: probable transmission via hands of health care workers.Clin Infect Dis. 1993; 16: 372-376Crossref PubMed Scopus (103) Google Scholar, 4Richards MJ Edwards JR Culver DH Gaynes RP Nosocomial infections in medical care units in the United States. National Nosocomial Infections Surveillance System.Crit Care Med. 1999; 27: 887-892Crossref PubMed Scopus (1301) Google Scholar, 5Talon D Mulin B Rouget C Bailly P Thouverez M Viel JF Risks and routes for ventilator-associated pneumonia with Pseudomonas aeruginosa.Am J Respir Crit Care Med. 1998; 157: 978-984Crossref PubMed Scopus (73) Google Scholar, 6Cattoen C Levent T Grandbastien B et al.Observatoire régional Pseudomonas aeruginosa du Nord-Pas-de-Calais: données épidémiologiques et microbiologiques.Med Mal Infect. 1999; 29: 160-166Crossref Scopus (10) Google Scholar]. Nowadays, endemic nosocomial infections are thought to originate mainly from patients' endogenous flora [7Grüner E Kropec A Huebner J Altwegg M Daschner F Ribotyping of Pseudomonas aeruginosa strains isolated from surgical intensive care patients.J Infect Dis. 1994; 167: 1216-1220Crossref Scopus (40) Google Scholar,8Bonten MJ Bergmans DC Speijer H Stobberingh EE Characteristics of polyclonal endemicity of colonization in intensive care units. Implications for infection control.Am J Respir Crit Care Med. 1999; 160: 1212-1219Crossref PubMed Scopus (121) Google Scholar]. We carried out a prospective study to assess the incidence and the local antibiotic susceptibility of P. aeruginosa in ICUs and to characterize cross-transmission by using pulsed-field gel electrophoresis (PFGE) as an epidemiologic tool. We studied two separate adult units: the medical and surgical ICUs at the University Hospital, Besançon. Each unit has 15 beds. These two units admit a total of 800 patients per year, giving a mean of 9500 patient-days per year. All patients admitted to these two ICUs between 1 January 1998 and 31 December 1999 were included in a prospective study and were tested for P. aeruginosa. Routine clinical specimens and surveillance specimens (rectal swabs, nasal swabs and tracheal aspiration) were screened for P. aeruginosa. Surveillance samples were collected from each patient on the day of admission and then once a week for the duration of hospitalization in the ICU. The disk diffusion method was used to determine the antibiotic susceptibility of the isolates, which were classified as susceptible, intermediate or resistant according to the criteria of the Antibiogram Committee of the French Society for Microbiology [9Report du comité de l'antibiogramme de la Société Française de Microbiologie.Clin Microbiol Infect. 1996; 2: S46-S48PubMed Google Scholar]. P. aeruginosa colonization (identified by clinical or surveillance culture) was considered to be ICU acquired if P. aeruginosa was not detected in any specimen during the first 48 h following admission to the ICU. Endogenous colonization was defined as colonization occurring with a strain of P. aeruginosa that had not previously been isolated from another patient. Exogenous colonization or cross-colonization was defined as colonization by a strain of P. aeruginosa with a PFGE pattern identical or very similar to that of isolates from another patient present in one of the ICUs. The genetic similarity of strains was investigated by PFGE (CHEF DRIII, Bio-Rad, Ivry sur Seine, France) using DraI (Boehringer, Mannheim, Germany) as previously described [10Talon D Capellier G Boillot A Michel-Briand Y Use of pulsed-field gel electrophoresis as an epidemiological tool during an outbreak of Pseudomonas aeruginosa lung infections in an intensive care unit.Intensive Care Med. 1995; 21: 996-1002Crossref PubMed Scopus (31) Google Scholar]. Samples of SmaI-restricted DNA of Staphylococcus aureus NCTC 8325 were included in each run as an internal reference. The banding patterns were analyzed by scanning photographic negatives. GelCompar software (version 4.1) was used for cluster analysis (Applied Maths, Kortrijk, Belgium). Each strain was first compared with all other strains, and the Dice correlation coefficient was used to calculate similarity. The strains were then grouped and the upgma clustering algorithm was used to depict the groups as a dendrogram. Major restriction patterns were defined as those differing by more than three fragments, with a similarity index <85%, as described by Tenover et al. [11Tenover FC Arbeit RD Goering RV et al.Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing.J Clin Microbiol. 1995; 33: 2233-2239PubMed Google Scholar]. During the study period, 1646 patients were admitted to the ICUs for a total of 19 544 days. One thousand three hundred and twelve specimens corresponding to 692 non-replicate sites (clinical and surveillance) of positive isolation tests for P. aeruginosa were collected from 314 patients (166 of these patients had positive clinical culture tests and 148 had positive surveillance culture tests only). The overall incidence of P. aeruginosa was 19.1 cases per 100 patients (17.2 in the SICU and 21.0 in the MICU) and 16.1 cases per 1000 days of hospitalization. Of 166 patients with at least one positive clinical culture, 69 (41.5%) had positive bronchoalveolar lavage tests and 57 (34.3%) had positive urine tests. The other clinical sites were superficial wounds, catheters, drainage fluids, and blood (Table 1). Resistance rates are shown in Table 1.Table 1Frequency of decreased susceptibility (% + R) of P. aeruginosa isolates in ICUsSites of isolation (number)TICPIPCAZATMIPMAMKCIPClinical specimens (232)42.226.719.434.934.522.838.8Urines (57)47.433.328.138.633.317.545.6Bronchoalveolar lavage (69)33.318.814.521.730.421.726.1Blood (7)71.457.114.371.428.628.657.1Drainage fluid (21)57.147.633.352.447.628657.1Superficial wounds (39)43.625.615.438.523.117.935.9Intravascular catheters (32)37.515.615.634.456.240.640.6Others (7)28.614.3028.614.3042.9Surveillance specimens (460)50.029.622.640.623.022.838.0Total (692)47.428.621.538.726.922.838.3TIC, ticarcillin; PIP, piperacillin; CAZ, ceftazidime; ATM, aztreonam; IPM, imipenem; AMK, amikacin; CIP, ciprofloxacin. Open table in a new tab TIC, ticarcillin; PIP, piperacillin; CAZ, ceftazidime; ATM, aztreonam; IPM, imipenem; AMK, amikacin; CIP, ciprofloxacin. We typed the initial ICU-acquired isolate from 155 patients (79 patients from the MICU and 76 from the SICU), randomly selected. The corresponding 155 non-replicate isolates yielded 71 major DNA patterns: 50 unique patterns, eight patterns including two isolates, five patterns including three isolates, two patterns including four isolates, one pattern including five isolates, three patterns including six isolates, one pattern including 10 isolates, and one pattern including 32 isolates. The outbreak involving the last clone, designated a major epidemic clone, has been reported previously [12Bertrand X Bailly P Blasco G Balvay P Boillot A Talon D Large outbreak in a surgical intensive care unit of colonization or infection with Pseudomonas aeruginosa that overexpressed an active efflux pump.Clin Infect Dis. 2000; 31: E9-14Crossref PubMed Scopus (40) Google Scholar]. It disseminated mostly in the SICU (28 cases of the 32). The dates of stay, the location of hospitalization and transfers of patients colonized by any of the strains of P. aeruginosa with a PFGE pattern including two or more isolates (21 patterns) are consistent with there being cross-transmission. Indeed, cross-transmission would have been possible, in both space and time, for all of these patterns except two. Eighty-three of the 155 studied cases of colonization (53.5%) were acquired by cross-transmission, and the 72 other cases probably originated from endogenous sources. The cross-transmission rates were 48.1% in the MICU and 59.2% in the SICU. Table 2 shows the association of DNA pattern with antibiotic susceptibility.Table 2Association of DNA pattern with antibiotic resistance for P. aeruginosa strains isolated in ICUsβ-Lactam resistance (%)PFGE DNA patternWild typeTicarcillin I/RCeftazidime I/RImipenem I/RUnique patternaPFGE pattern including a single isolate. (n = 51)40 (78.4)6 (118)2 (3.9)3 (5.9)Epidemic patternbPFGE pattern including two or more non-replicate isolates. (n = 104)50 (48.1)42 (40.4)2 (1.9)10 (9.6)Total90 (581)48 (310)4 (2.6)13 (8.4)a PFGE pattern including a single isolate.b PFGE pattern including two or more non-replicate isolates. Open table in a new tab Our study contributes to the knowledge concerning P. aeruginosa, for which little information is available about incidence and cross-transmission rate (except in epidemic situations), despite there being extensive published data concerning its resistance to antibiotics. The relative importance of endogenous and exogenous sources of P. aeruginosa is unclear. The possibility of cross-colonization in non-epidemic situations has rarely been studied using PFGE, the genomic fingerprinting method now regarded as the most accurate method for the typing of P. aeruginosa [10Talon D Capellier G Boillot A Michel-Briand Y Use of pulsed-field gel electrophoresis as an epidemiological tool during an outbreak of Pseudomonas aeruginosa lung infections in an intensive care unit.Intensive Care Med. 1995; 21: 996-1002Crossref PubMed Scopus (31) Google Scholar,13Speijer H Savelkoul PHM Bonten MJ Stobberingh EE Tjhie JHT Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit.J Clin Microbiol. 1999; 37: 3654-3661PubMed Google Scholar, 14Talon D Cailleaux V Thouverez M Michel-Briand Y Discriminatory power and usefulness of pulsed-field gel electrophoresis in epidemiological studies of Pseudomonas aeruginosa.J Hosp Infect. 1996; 32: 135-145Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 15Pradella S Pletschette M Mantey-Stiers F Bautsch W Macro***restriction analysis of Pseudomonas aeruginosa in an intensive care unit.Eur J Clin Microbiol Infect Dis. 1994; 13: 122-128Crossref PubMed Scopus (13) Google Scholar]. We show that clinical specimens identify only half of the patients positive for P. aeruginosa. In the absence of a screening program including surveillance samples, 47.1% (148/314) of all carriers would have remained undetected. Thus, P. aeruginosa clinical cultures do not present the full epidemiologic picture. Comparison of incidence rates between ICUs is difficult, mainly because of differences in patient populations. However, the incidences of P. aeruginosa colonization upon admission and during hospitalization in our institution are consistent with those elsewhere in France [6Cattoen C Levent T Grandbastien B et al.Observatoire régional Pseudomonas aeruginosa du Nord-Pas-de-Calais: données épidémiologiques et microbiologiques.Med Mal Infect. 1999; 29: 160-166Crossref Scopus (10) Google Scholar] and Europe [16Blanc DS Petignat C Janin B Bille J Francioli P Frequency and molecular diversity of Pseudomonas aeruginosa upon admission and during hospitalization: a prospective epidemiologic study.Clin Microbiol Infect. 1998; 4: 242-247Crossref PubMed Scopus (70) Google Scholar,17Bergmans DC Bonten MJ van Tiel FH et al.Cross-colonization with Pseudomonas aeruginosa of patients in an intensive care unit.Thorax. 1998; 53: 1053-1058Crossref PubMed Scopus (91) Google Scholar]. The rate of colonization upon admission was below 5%, and 74.2% of colonizations or colonizations/infections were ICU acquired. Our findings concerning the antibiotic resistance of clinical isolates are concordant with the French multicenter study of the GERPB (Workgroup on the resistance of P. aeruginosa to β-lactam antibiotics) including all types of ward [18Cavallo JD Fabre R Leblanc F Nicolas-Chanoine MH Thabaut A GERPB Antibiotic susceptibility and mechanisms of β-lactam resistance in 1310 strains of Pseudomonas aeruginosa: a French multicentre study (1996).J Antimicrob Chemother. 2000; 46: 133-136Crossref PubMed Google Scholar], except for imipenem resistance, which is higher in the ICU (18.5% for the GERPB versus 34.5% in our study). Moreover, the clinical sites most frequently associated with infection (bronchoalveolar lavage, blood, urines, drainage fluids) were not associated with a higher frequency of resistance. These results suggest that antibiotic resistance is not associated with virulence. It was clear that the emergence of antibiotic resistance within the P. aeruginosa population results from acquisition of resistance by susceptible strains following selective pressure exerted by antibiotics. However, our results show that this spread of resistance is also a consequence of the diffusion of epidemic multiresistant strains (Table 2). Our findings suggest that cross-colonization may be a major cause of P. aeruginosa colonization and infection in ICUs. We identified an epidemic clone of P. aeruginosa that persisted for at least 30 months in the SICU [12Bertrand X Bailly P Blasco G Balvay P Boillot A Talon D Large outbreak in a surgical intensive care unit of colonization or infection with Pseudomonas aeruginosa that overexpressed an active efflux pump.Clin Infect Dis. 2000; 31: E9-14Crossref PubMed Scopus (40) Google Scholar]. However, this epidemic clone led to an overestimate of cross-colonization rate. Excluding this clone, the rate of cross-transmission is very much lower, but nevertheless above 40%. The epidemiologic pattern of P. aeruginosa in ICUs may be described as a silent epidemic, similar to those of other nosocomial pathogens, such as vancomycin-resistant enterococci. Thus, the situation appears to be a succession of outbreaks affecting from three to 10 patients, against a background of sporadic cases. Larger outbreaks may then add to this underlying epidemiologic pattern [19Foca M Jakob K Whittier S et al.Endemic Pseudomonas aeruginosa infection in a neonatal intensive care unit.N Engl J Med. 2000; 343: 695-700Crossref PubMed Scopus (195) Google Scholar]. This work was supported by ‘Programme Hospitalier de Recherche Clinique 1996'.