J Infect Chemother (2002) 8:59–63
© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2002
ORIGINAL ARTICLE Kou Takeyama · Yasuharu Kunishima Masanori Matsukawa · Satoshi Takahashi Takaoki Hirose · Nobumichi Kobayashi Intetsu Kobayashi · Taiji Tsukamoto
Multidrug-resistant Pseudomonas aeruginosa isolated from the urine of patients with urinary tract infection
Received: April 26, 2001 / Accepted: September 1, 2001
Abstract We report the clinical courses of 3 patients with urinary obstruction who developed acute pyelonephritis caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Genome ﬁngerprinting was performed to clarify the route of cross-infection, and an imipenemresistance gene was detected by the polymerase chain reaction (PCR) method. The study included 17 patients at our institute who had urinary tract infections caused by P. aeruginosa between January and December 1997. MDR was deﬁned as that when all the minimum inhibitory concentrations (MICs) were determined to show resistance according to the breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for P. aeruginosa. Pulse-ﬁeld gel electrophoresis (PFGE) was carried out for genome ﬁngerprinting. PCR was used to detect the metallo-β-lactamase gene (blaIMP). Three strains were revealed for MDR. The strains were isolated from the 3 patients with urinary tract obstruction who developed acute pyelonephritis. The treatment consisted of urinary drainage for the obstructed urinary tract and parenterally administered antimicrobials. Although none of the strains was susceptible to any antimicrobials, all patients had favorable outcomes. PFGE revealed that two strains had an identical genotype, implying cross-infection between the patients. The blaIMP gene was not detected in any of the three strains. In febrile patients with urinary tract infection caused by MDR P. aeruginosa, treatment for urinary obstruction is strongly recommended. Initial empirical chemotherapy with antimicrobials to which the organism is not K. Takeyama · Y. Kunishima (*) · M. Matsukawa · S. Takahashi · T. Hirose · T. Tsukamoto Department of Urology, Sapporo Medical University School of Medicine, S-1, W-16, Chuou-ku, Sapporo 060-8543, Japan Tel. ⫹81-11-611-2111 (ext. 3480); Fax ⫹81-11-612-2709 e-mail: [email protected]
N. Kobayashi Department of Hygiene, Sapporo Medical University, School of Medicine, Sapporo, Japan I. Kobayashi Chemotherapy Division, Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan
susceptible is often inevitable. Because there was epidemiological evidence of cross-infection with MDR P. aeruginosa, countermeasures against nosocominal infection are warranted. Key words Pseudomonas aeruginosa · Multidrug resistance · Urinary tract infection
Introduction Pseudomonas aeruginosa is well recognized as a nosocomial pathogen, and has inherent drug resistance.1 Recently, P. aeruginosa resistant to multiple antimicrobial agents has become a problem,2,3 particularly the strains that produce methalo-β-lactamase encoded by the blaIMP gene.4 There are several reports of outbreaks of multidrug-resistant (MDR) P. aeruginosa,2,3,5,6 but only one report of MDR P. aeruginosa that was isolated from urine.6 Therefore, the clinical signiﬁcance of MDR P. aeruginosa isolated from urine and the appropriate therapeutic regimen to combat infection with the organism have not been well established. We report our experience of the successful treatment of three patients with urinary tract obstruction who developed acute pyelonephritis caused by MDR P. aeruginosa. Chromosomal DNA genotyping of MDR P. aeruginosa was done by using pulsed-ﬁeld gel electrophoresis (PFGE) to identify the origin and route of infection. The expression of the metallo-β-lactamase (blaIMP) gene was also studied to clarify the mechanism of drug resistance.
Patients and methods Patients From January to December 1997, 425 patients were admitted to our urological ward. Of the 425 patients, 17 had urinary tract infections caused by P. aeruginosa, with 104
colony-forming units (CFU)/ml or greater density in urine. Three strains of MDR P. aeruginosa were isolated from three consecutive patients. The clinical courses of the patients were reviewed from medical charts. Bacterial isolation and susceptibility test Bacterial isolates were identiﬁed and determined by the analytical proﬁle index procedure, using a MicroScan Walk/ Away system (Dade Behring, Dade MicroScan, West Sacramento, CA, USA) according to the guidelines of the manufacturer. The following antibacterial agents were used for the susceptibility testing of P. aeruginosa: ceftazidime, cefoperazone, cefepime, imipenem, piperacillin, aztreonam, amikacin, gentamicin, tobramycin, ciproﬂoxacin and sulfamethoxazole-trimethoprim. The minimum inhibitory concentrations (MICs) were measured by the agar dilution method, using the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) document M7A4.7 In this study, MDR was deﬁned as that when all the MICs were determined to show resistance according to the breakpoints recommended by the NCCLS8 for P. aeruginosa. PFGE study Genomic DNA ﬁngerprinting by PFGE was carried out by the modiﬁed method of Ichiyama et al.9 Chromosomal DNA was prepared as described by Smith and Cantor.10 After P. aeruginosa strains had grown overnight, 0.5 ml of the cell suspension was added to 9.5 ml of brain heart infusion medium; this was incubated for 4 h at 35°C and spun down. The cell pellet was washed with saline ethylene diamine tetraacetic acid (EDTA) solution (0.15 M NaCl, 10 mM EDTA [pH 8.0]), and resuspended in Pett IV solution (0.1 M NaCl, 10 mM EDTA [pH 8.0], optical density (OD), 600; nm, 1.7). Bacterial suspension (1 ml), at 40°C–50°C, was mixed with 1 ml of 2% low-melting-point agarose (Wako Pure Chemical, Osaka, Japan), and poured into a 100-µl mold. The block was incubated overnight at 35°C in a lysis solution (1 M NaCl, 0.1 M EDTA [pH 8.0], 10 mM Tris-HCl [pH 8.0], 0.5% [w/v] Brij 58, 0.2% [w/v] deoxycholate, and 0.5% [w/v] Sarkosyl [Wako Pure Chemical]) supplemented with lysozyme (4 mg/ml [Seikagaku, Tokyo, Japan]). The block was then incubated overnight again, at 50°C, in ES solution (0.25 M EDTA [pH 8.0], 1% [w/v] Sarkosyl [Wako Pure Chemical]) supplemented with proteinase K (1 mg/ml [Sigma Chemical, St. Louis, MO, USA]). It was then treated with 1 mM phenylmethylsulfonyl ﬂuoride (Sigma Chemical) in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) for 4 h and washed four times with TE buffer. Thinly sliced sections of the block (about 10-µl) were digested with 10 U/plug of SpeI and XbaI (Takara Shuzo, Kyoto, Japan) at 37°C for 18 h. They were then electrophoresed through 0.8% agarose gel (Sigma Chemical) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA [pH 8.0]) at 10°C.
PFGE was performed using a CHEF DR II system (BioRad Laboratories, Richmond, CA, USA) at 6 V/cm for 18 h, with pulse times ranging from 0.1 to 20 s for XbaI digestion and at 6 V/cm for 18 h, and with pulse times ranging from 1 to 80 s for SpeI digestion. The gels were then stained with ethidium bromide, and DNA-digested patterns were photographed. Saccharomyces cerevisiae genomes and lambda DNA concatemers (Bio-Rad Laboratories, Richmond, CA, USA) were used as size standards. Polymerase chain reaction (PCR) to detect blaIMP gene Genome DNA was extracted with Instagene Puriﬁcation Matrix (Bio-Rad Laboratories, Hercules, CA). On detection of the blaIMP gene, we selected the next primers, 1241 (5⬘-CTACCGCAGCAGAGTCTTTG-3⬘) and 1808 (5⬘-AACCAGTTTTGCCTTACCAT-3⬘). The reaction mixture contained 10 µg of MgCl, 10 mM deoxynucleoside triphosphate and the primer (1241: 50 pM; 1808: 50 pM), 1 µg template DNA, and 2.5 U of AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT, USA). Ampliﬁcation was performed in a DNA thermal cycler (Perkin-Elmer) programmed for 24 cycles of 1 min at 94°C, 1 min at 57°C, 2 min at 72°C, 1 cycle of 1 min at 94°C, 1 min at 57°C, and 5 min at 72°C. Ampliﬁcation products were resolved by electrophoresis in 1% agarose gel and were detected by staining with ethidium bromide.
Results Susceptibility test Table 1 lists the MICs for the 17 isolates and their breakpoints determined by the NCCLS. Three strains were determined to be MDR, because all the antimicrobials tested showed MICs equal to or greater than the breakpoints for the three strains. Clinical courses of patients with MDR P. aeruginosa in urine Case 1 A 59-year-old man with muscle-invasive bladder cancer underwent radical cystectomy and ileal neobladder reconstruction. Three weeks after the surgery, the patient’s temperature was more than 38°C for 6 days. The serum white blood cell (WBC) court was 11 000/mm3 and C-reactive protein (CRP) level was 22.3 mg/dl on the second day of the febrile symptoms. Ultrasound and X-ray imaging examinations showed left hydronephrosis, caused by ureterointestinal anastomotic stricture. The patient had left ﬂank pain. We diagnosed acute pyelonephritis associated with hydronephrosis. He was treated empirically with aspoxicillin and arbekacin, and percutaneous nephrostomy (PNS) was performed for optimal urinary drainage. MDR P. aeruginosa
61 Table 1. Distribution of MICs for 17 strains of Pseudomonas aeruginosa and the breakpoints determined by NCCLSa Case no.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. NCCLSa
⬎128 ⬎128 ⬎128 128 4 4 4 4 2 2 4 1 1 4 1 4 4 32
⬎128 ⬎128 ⬎128 ⬎128 16 16 8 16 16 8 16 8 8 16 4 16 8 64
⬎128 ⬎128 ⬎128 64 4 16 4 4 4 2 2 1 4 2 1 2 2 32
64 64 64 2 1 32 2 2 2 2 2 2 32 2 1 2 2 16
⬎128 ⬎128 ⬎128 ⬎128 8 16 8 8 8 8 16 4 4 16 4 8 8 128
⬎128 ⬎128 ⬎128 64 8 16 8 16 8 4 8 4 4 8 4 8 8 32
128 128 128 16 4 16 4 4 32 4 4 8 8 4 4 4 4 32
32 32 32 ⬎128 2 16 4 4 16 2 2 4 8 2 4 4 2 8
⬎128 ⬎128 ⬎128 ⬎128 0.5 2 1 1 4 0.5 0.5 1 2 0.5 1 0.5 0.5 8
⬎128 128 128 32 0.25 128 0.25 0.5 8 0.12 0.12 0.12 0.25 0.12 0.12 0.12 0.12 4
⬎128 ⬎128 ⬎128 ⬎128 128 32 32 32 16 32 8 8 16 8 8 8 4 8/152
MIC, Minimum inhibitory concentration; CAZ, ceftazidime; CPZ, cefoperazone; CFPM, cefepime; IPM, imipenem; PIPC, piperacillin; AZT, aztreonam; AMK, amikacin; GM, gentamicin; TOB, tobramycin; CPFX, ciproﬂoxacin; ST, sulfamethoxazole-trimethoprim a NCCLS, Breakpoints determined by the National Committee for Clinical Laboratory Standards document M7-A47
was isolated from the PNS urine. The isolate from the patients’ urine was not susceptible to the antimicrobials (aspoxicillin and arbekacin) used, but the treatment for acute pyelonephritis was successful. After the acute pyelonephritis had completely resolved, MDR P. aeruginosa was persistently isolated from PNS urine for 3 months. Case 2 A 49-year-old man with muscle-invasive bladder cancer underwent radical cystectomy and ileal neobladder reconstruction. Two days after the beginning of intermittent trials with a clamp for the neobladder catheter for voiding training, he developed acute pyelonephritis. His temperature was more than 38°C for 2 days. The serum WBC count was 9500/mm3 and CRP level was 14.5 mg/dl. The patient had bilateral back dullness and tenderness of the bilateral costovertebral angles. MDR P. aeruginosa was isolated from urine. Although he was treated empirically with piperacillin (to which the isolate was not susceptible) and with release of the catheter clamp, he soon became afebrile, without developing any serious complications. Case 3 An 82-year-old man with bilateral hydronephrosis caused by muscle-invasive bladder cancer was hospitalized because of the development of acute pyelonephritis. His temperature had been more than 38°C for 14 days before he was hospitalized. On admission, his serum WBC count was 27 200/mm3, CRP level was 22.7 mg/dl, and the urine sediment was loaded with leukocytes. He had uncontrollable severe diabetes mellitus and this did not allow any surgical management for bladder cancer. Urine culture before admission indicated MDR P. aeruginosa. Bilateral PNS was done on the day of admission, with antimicrobials
(ceftazidime and arbekacin) being administered at that time. His symptoms were alleviated soon after the treatment, although the strain was not susceptible to the antimicrobials used. Chromosomal DNA analysis by PFGE Two distinctive restriction patterns were identiﬁed in the three strains of MDR P. aeruginosa in both conditions using the restriction enzymes XbaI and SpeI (Fig. 1). The restriction patterns of the strains isolated from cases 2 and 3 were considered to be identical, suggesting crossinfection between these patients. The strain isolated from case 1 had a different restriction pattern from that of cases 2 and 3. Detection of blaIMP gene The blaIMP gene was not detected in any of the three strains (Fig. 2).
Discussion In 1982, Rella and Hass11 ﬁrst reported that a nalidixic-acid resistant P. aeruginosa showed resistance to β-lactam antimicrobials. Since then there have been several reports12–14 of MDR P. aeruginosa. However, there is no well-established deﬁnition of “multidrug resistance”. In previous reports, the tested drugs have differed, and the majority of the studies used susceptibility tests revealing resistance to 8 or more antimicrobials as the criteria for deﬁning multidrug resistance.3,13–15 In this study, we found three strains with multidrug resistance; they were resistant to all 11 antimicro-
Fig. 1. Chromosomal DNA analysis by pulsed-ﬁeld gel electrophoresis. The restriction patterns of strains isolated from cases 2 and 3 were considered to be identical, suggesting cross-infection between these patients. The strain isolated from case 1 had a different restriction pattern from that of the strains isolated from cases 2 and 3. Y, Yeast chromosomes, Saccharomyces cerevisiae; L, lambda ladders; lane 1, case 1; lane 2, case 2; lane 3, case 3
Fig. 2. Detection of blaIMP gene by polymerase chain reaction (PCR). The blaIMP gene was not detected in any of the three strains. P1, Positive control 1; P2, positive control 2; lane 1, case 1; lane 2, case 2; lane 3, case 3; M, pHY marker
bials tested according to the breakpoints-MICs deﬁned by the NCCLS. P. aeruginosa can cause serious infections in an immunocompromised host, but not in healthy persons.16 All three of our patients were immunocompromised hosts, but, fortunately, there was no severe morbidity after the episodes of acute pyelonephritis. Several strains of MDR P. aeruginosa were isolated from case 1 after the antimicrobial chemotherapy had been completed, but the patient was asymptomatic. The MDR strain disappeared spontaneously, and had been replaced by another bacterium 3 months after completion of the treatment. Generally, antimicrobial chemotherapy is not necessary for patients with chronic complicated urinary tract infection caused by P. aeruginosa when they are asymptomatic. Even if MDR P. aeruginosa is isolated from the urine of such asymptomatic patients, it seems that chemotherapy is not required unless
they develop symptoms and signs originating from the urinary tract infection. In our study, when the patients manifested symptoms such as high fever, they all had urinary tract obstruction. Surgical relief of the urinary tract obstruction or improvement of urinary drainage is strongly recommended for symptomatic patients with urinary tract infection caused by MDR P. aeruginosa when they have urinary tract obstruction as well. Combinations of antimicrobial chemotherapy and improvement of urine ﬂow were all successful in alleviating the symptoms in our three patients, although none of the strains isolated from these patients were susceptible to the antimicrobials used (aspoxicillin, piperacillin, arbekacin, ceftazidime). In the treatment of patients with urinary tract obstruction who develop acute pyelonephritis caused by MDR P. aeruginosa, it would be worthwhile to start antimicrobial chemotherapy with these penicillins and cephalosporins even if the susceptibility tests show resistance to all available antimicrobial agents. If the chemotherapy is ineffective, combination chemotherapy, using antimicrobial agents such as fosfomycin with sulbactam/cefoperazone, fosfomycin with ceftazidine,17 and amikacin with aztreonam15 may be indicated, because these agents are reported to be effective against MDR P. aeruginosa in vitro. Unfortunately, the clinical efﬁcacy of such regimens has not been established. Polymyxin B sulfate and colistin may be the last choice for chemotherapy for MDR P. aeruginosa.18 Cross-infection with MDR P. aeruginosa in an intensive care unit and in a pediatric hospital2,3 and outbreaks, including urinary tract infection in a pediatric oncology ward, found to be related to bath toys,6 have been reported. In our patients, there may have been cross-infection, because the three isolates had almost the same antibiogram. However, DNA ﬁngerprinting, using PFGE, showed that only the isolates from cases 2 and 3 had identical genotypes. Case 3 was hospitalized at our institute 1 month before the admission of case 2. These ﬁndings imply that the MDR strain isolated was transferred from case 3 to case 2. The strain isolated from case 1 had a distinct genotype. This difference could be explained in terms of the strain in case 1 having a different origin from that in cases 2 and 3. Another explanation could be that the strain in case 1 had the same origin, but that its susceptibility to antimicrobials was somehow modiﬁed after cross-infection. In any event, these results suggest that adequate countermeasures are required to prevent the outbreak of urinary tract infections caused by MDR P. aeruginosa. Cross-transmission via transient contamination of the hands of medical personnel is the major route of nosocomial infection by such species. Strict handwashing protocols, as well as the cohorting and isolation of colonized and infected patients, are crucial practices for preventing the transmission of MDR P. aeruginosa. Recently, carbapenem-resistant P. aeruginosa producing metallo-β-lactamase that can efﬁciently hydrolyze carbapenems has become a problem.4 Senda et al.19 analyzed 132 stains of carbapenem-resistant P. aeruginosa and revealed that 15 strains (11.3%) had the blaIMP gene. We did not detect the blaIMP gene in our study, indicating that our strains may have a different mechanism for resistance to
imipenem, such as a decrease in the permeability of the cell membrane and different efﬂux systems in the bacterial membrane,19 from those strains with the blaIMP gene. In summary, we reported the clinical aspects of three patients with urinary tract obstruction who developed acute pyelonephritis caused by MDR P. aeruginosa. Although none of the causative microorganisms were susceptible to the antimicrobial agents used, the clinical outcomes were all favorable. Appropriate procedures to improve urinary drainage in patients with urinary tract obstruction may contribute to a favorable outcome. PFGE analysis indicated that there had been cross-infection between two of the three patients. The MDR P. aeruginosa reported in this study did not express the blaIMP gene.
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