a Panum Institute, Institute for Medical Microbiology and Immunology 24.1, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N; b Department of Gastrointestinal Infections, Statens Serum Institut; c Danish Cystic Fibrosis Centre; d Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
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Abstract |
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Introduction |
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Mucoid and non-mucoid phenotypes of P. aeruginosa, with apparent differences in their antimicrobial susceptibility pattern, are frequently isolated simultaneously from patients with CF and chronic lung infection.68 The reason for this difference in antibiotic susceptibility is not clear. The aim of the present study was to determine whether mucoid/non-mucoid paired isolates share the same genotype and to characterize their ß-lactamase activity (basal and induced levels), as this is as one of the most common mechanisms of resistance to ß-lactam antibiotics in CF P. aeruginosa strains.9 Outer membrane protein patterns for the mucoid/non-mucoid pairs were also analysed for possible changes associated with resistance to the antipseudomonal antibiotics.
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Materials and methods |
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Forty-two Danish CF patients (23 females, 19 males) with a mean age of 25 years (range 15.337.6 years) and an average duration of the chronic P. aeruginosa infection of 16.6 years (range 623 years) were included in this study. Thirty-two of these patients represent a cohort of patients infected with a multiresistant, non-mucoid strain, as described by Pedersen et al.10 All patients had been treated with regular courses of iv antipseudomonal therapy, as described previously.11,12
Sputum samples obtained by expectoration or endolaryngeal suction were Gram stained and examined under the microscope to confirm their origin from the lower airways. Material was cultured as reported previously.13 P. aeruginosa was identified by conventional biochemical tests. All these isolates were collected in 1997 and kept at 80°C in broth supplemented with 10% glycerol. One pair of mucoid/non-mucoid isolates was obtained from each patient.
Eight CF P. aeruginosa isolates from the strain collection of the Department of Clinical Microbiology, Rigshospitalet, isolated in 1986 (one isolate from each of two patients), 1988 (two isolates from each of two patients) and 1991 (one isolate from each of two patients) were included in the study for comparison.
All P. aeruginosa isolates were cultured overnight at 37°C in sterile beef broth.
MIC determination
The MIC of piperacillin (Lederle, Carolina, USA), ceftazidime (Glaxo-Welcome, Brøndby, Denmark), meropenem (Zeneca Limited, Macclesfield, UK), aztreonam (Bristol-Myers Squibb, Bromma, Italy), tobramycin (Tobramycinsulfate, Sygehusapotekerne, Denmark), colistin (Lundbeck, Valby, Denmark) and ciprofloxacin (Miles Inc., IL, USA) for each isolate was determined by the agar plate dilution method using IC50 agar (Statens Serum Institut, Copenhagen, Denmark), using an inoculum of c. 103104 cfu/spot.
The isolates were classified as susceptible or resistant according to the MICs of at least three antibiotics. The following breakpoints for resistance were used: piperacillin, MIC 128 mg/L; ceftazidime and aztreonam, MIC
32 mg/L; meropenem, MIC
16 mg/L; tobramycin, MIC
8 mg/L; and ciprofloxacin, MIC
4 mg/L.14
ß-Lactamase assay
ß-Lactamase production in basal conditions and after induction with benzyl-penicillin (500 mg/L) was measured spectrophotometrically using nitrocefin as substrate as described previously.9,15
Outer membrane proteins
Outer membrane proteins were prepared by the sarcosyl method,16 separated on 12.5% SDSPAGE gels and detected by Coomassie staining. The protein patterns were analysed visually.
Alginate assay
Alginate production was measured using a borate-carbazol method.17 d-Mannuronate lactone (Sigma, St Louis, MO, USA) was used to calibrate a standard curve.
Typing methods
All strains were genotyped by automated ribotyping in the RiboPrinter system, as recommended by the manufacturer (Qualicon, Wilmington, DE, USA). In brief, all strains were subcultured three times on 5% blood agar and a single colony from a 24 h culture was suspended in sample buffer and heated at 80°C for 15 min. Mucoid isolates were grown in BHI broth (SSI-Diagnostika, Hillerød, Denmark) for 6 h, and harvested from 1 mL of broth before suspension in sample buffer. After addition of lytic enzymes, samples were transferred to the RiboPrinter. Further analysis, which included EcoRI restriction of DNA, was carried out automatically. The RiboPrint profiles were aligned according to the position of the molecular size standard and compared with patterns obtained previously, then stored in a database consisting of 750 validated RiboPrints supplied from the manufacturer. The RiboPrint profiles were analysed in BioNumerics (Applied Maths, Kortrijk, Belgium).
Statistical analysis
The description and analysis of the data were carried out using StatView 4. 5 software for Apple Mac. Logarithmic transformation of the data was performed to stabilize the variance.
Paired t-test was used for comparison of the data of the paired mucoid/non-mucoid isolates. The data were presented as non-mucoid/mucoid ratios with 99% confidence intervals. The level of significance was 1%.
One-way analysis of variance (ANOVA) was used to determine the effect of the four most common ribogroups on the antibiotic susceptibility data. For the parameters that looked as though they could be statistically significant, Scheffés F-test was used as a comparison procedure. The level of significance was 5%.
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Results |
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The analysis of the genotypes determined by RiboPrinting showed that 13 different types existed among the 84 isolates. The same ribotype was observed in paired mucoid/ non-mucoid isolates in 18 of 42 patients.
The majority of the isolates were assigned to four types: 73-S2 (n = 39), 73-S1 (n = 16), 207-S3 (n = 8) and 227-S8 (n = 6). The isolates with the ribotype 73-S2 produced higher basal levels of ß-lactamase and presented a ß- lactamase hyperinducible phenotype compared with the isolates with the ribotype 73-S1 (P = 0.0075) (Table 1). This correlated with significantly higher MICs (P < 0.05) of piperacillin and meropenem compared with the other three ribotypes. The MICs of tobramycin were significantly lower for isolates with 73-S1 ribotype compared with 73-S2 ribotype (Table 1
). The median amount of alginate produced by P. aeruginosa isolates with ribotype 227-S8 was significantly higher (P = 0.03) than that produced by the isolates with ribotype 73-S2 [316 mg/L (range 0840 mg/L) versus 6.89 mg/L (range 0688 mg/L), respectively].
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Susceptibility to antibiotics, ß-lactamase production and phenotype
The mucoid isolates showed lower ß-lactamase activity and had lower MICs of all the antibiotics compared with non-mucoid isolates as indicated by the ratio values (Table 2).
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Outer membrane proteins
Outer membrane protein analysis showed the presence of a characteristic 54 kDa protein in all mucoid isolates, which was not present in the non-mucoid isolates (Figure). We have previously identified this protein as AlgE protein, which has a role in the alginate secretion.1,18
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Discussion |
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As 32 of the 42 patients included in our study belonged to a cohort of patients infected with a non-mucoid, multiresistant strain, our finding of a dominant ribogroup 73-S2 (39 isolates) was to be expected. This strain was resistant to all tested antibiotics, including tobramycin and ciprofloxacin. This ribogroup was also present in three resistant isolates collected before 1991. This is not surprising as it has been shown previously in longitudinal studies that once the chronic lung infection is established, the initially colonizing P. aeruginosa strain remains at all times.19 We suspect that intensive antibiotic treatment may have selected for this multiresistant strain. Although the induced levels of ß-lactamase of the isolates belonging to the ribogroup 73-S2 were significantly higher than those produced in the other isolates, this phenotype does not explain the additional resistance to tobramycin and ciprofloxacin. Therefore, other resistance mechanisms, such as enhanced efflux pumps, may be present in these isolates. Jalal et al.20 found changes in both gyrA and nfxB, and expression of OprN and OprJ in two ciprofloxacin-resistant isolates belonging to ribotype 73-S2.
RiboPrinting profiles of the two largest ribogroups, the resistant 73-S2 (39 isolates) and the susceptible 73-S1 (16 isolates) differed by only one band, indicating a degree of similarity between the two ribogroups. It is possible that the 73-S2 ribogroup might have evolved and spread in the CF population from the susceptible 73-S1 ribogroup under the selective antibiotic pressure. However, no association of these two ribotypes was observed among the 24 paired isolates with different ribotypes.
Isolates of ribotype 227-S8 appear to have maintained greater susceptibility to antibiotics. Interestingly, the isolates with 227-S8 ribotype produced significantly higher amounts of alginate compared with isolates belonging to the 73-S2 ribotype.
The ratios of the MIC of ß-lactam antibiotics for the non-mucoid/mucoid isolates showed that, generally, the isolates with mucoid phenotype were more susceptible than the non-mucoid paired isolates, and that this was associated with the levels of ß-lactamase production. This difference in susceptibility to anti-pseudomonal antibiotics between pairs of non-mucoid and mucoid P. aeruginosa, as well as the maintenance for a long period of time of the antibiotic susceptibility of isolates with ribotype 227-S8 overproducing alginate, indicates that the non-mucoid isolates are exposed to a relatively higher antibiotic selective pressure than the mucoid isolates. This might be due to the biofilm mode of growth.
As proposed previously, biofilm-embedded cells may have different degrees of susceptibility to antibiotics, depending on the site where each individual cell is located within the multiple layers of cells forming the biofilm.21 In the case of ß-lactam antibiotics, the ß-lactamase produced by the superficial layer in the biofilm will be able to inactivate the ß-lactam antibiotic before it reaches the deep layers.22,23 Bacterial microcolonies have recently been considered as organized communities with functional heterogeneity.24 In the current view of the biofilms as microbial societies with their own defence and communication systems,25 it is possible that the mucoid and non-mucoid phenotypes live in symbiosis within the biofilm. While the mucoid, alginate hyperproducing cells ensure the survival of the biofilm, the non-mucoid cells might play a protective role against antibiotics. Taking into account the fact that the biofilm mode of growth leads to a 1000-fold higher MIC of different antibiotics compared with the MIC that we have observed with planktonic cells, we can understand the therapeutic problems encountered in treating the P. aeruginosa chronic infection in CF patients.26
Interestingly, a significant difference in the MIC of colistin between the mucoid and non-mucoid isolates sharing the same ribotype was also found. In the Danish CF centre colistin is frequently administered for prophylaxis and for treatment of CF lung infection.27 Recently, the mechanism of colistin resistance in these strains has been shown to be related to structural modifications of lipid A.28
The presence of a 54 kDa outer membrane protein, AlgE, with a role in alginate secretion18 was found in isolates with high alginate production. The major porin OprF used by most ß-lactam antibiotics to penetrate the outer membrane was present in all the isolates, irrespective of their resistance pattern or alginate production, with the exception of one clinical P. aeruginosa isolate where OmpF was lacking. This OmpF isolate was mucoid and resistant to all ß-lactam antibiotics, as was its non-mucoid OmpF+ paired strain, but the OprF isolate had an MIC of ciprofloxacin four times higher than its paired OmpF+ strain. The lack of OmpF has previously been reported to be associated with resistance to quinolones in Gram-negative bacteria.29
In conclusion, our study has shown that mucoid isolates were generally more susceptible to antibiotics than their paired non-mucoid P. aeruginosa isolates, and this was associated with lower levels of ß-lactamase activity. We propose that the maintenance of the antibiotic susceptibility of alginate overproducing isolates might be explained by the co-existence in the biofilm of non-mucoid resistant isolates that might play a protective role.
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Acknowledgements |
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Notes |
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References |
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2 . Pedersen, S., Høiby, N., Espersen, F. & Koch, C. (1992). Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. Thorax 47, 613.[Abstract]
3 . Szaff, M., Høiby, N. & Flensborg, E. W. (1983). Frequent antibiotic therapy improves survival of cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. APMIS 72, 6517.
4 . Frederiksen, B., Lanng, S., Koch, C. & Høiby, N. (1996). Improved survival in the Danish center-treated cystic fibrosis patients: results of aggressive treatment. Pediatric Pulmonology 21, 1538.[ISI][Medline]
5 . Ciofu, O., Giwercman, B., Pedersen, S. S. & Høiby, N. (1994). Development of resistance as a side-effect of two decades of intensive antipseudomonal treatment in the Danish CF Centre. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 102, 67480.
6 . Thomassen, M. J., Demko, C. A., Boxerbaum, B., Stern, R. C. & Kuchenbrod, P. J. (1979). Multiple isolates of Pseudomonas aeruginosa with differing antimicrobial susceptibility patterns from patients with cystic fibrosis. Journal of Infectious Diseases 140, 87380.[ISI][Medline]
7 . Ballestro, S., Escobar, H., Villaverde, R., Elia, M., Ojeda-Vargas, M. & Baquero, F. (1993). Continuous monitoring of antimicrobial resistance in cystic fibrosis patients. In Proceedings of the Eighteenth European Cystic Fibrosis Conference. pp. 6372. (Escobar, H., Baquero, F. & Suarez, L., Eds). Elsevier Science Publishers, Madrid, Spain.
8
.
Shawar, R., MacLeod, D., Garber, R., Burns, J., Stapp, J. R., Clausen, C. R. et al. (1999). Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrobial Agents and Chemotherapy 43, 287780.
9 . Giwercman, B., Lambert, P., Rosdahl, V. T., Shand, G. H. & Høiby, N. (1990). Rapid emergence of resistance in Pseudomonas aeruginosa cystic fibrosis patients due to in-vivo selection of stable partially derepressed ß-lactamase producing strains. Journal of Antimicrobial Chemotherapy 26, 24759.[Abstract]
10 . Pedersen, S. S., Koch, C., Høiby, N. & Rosendahl, K. (1986). An epidemic spread of multiresistant Pseudomonas aeruginosa in a cystic fibrosis centre. Journal of Antimicrobial Chemotherapy 17, 50516.[Abstract]
11 . Pedersen, S. S., Jensen, T., Høiby, N., Koch, C. & Flensborg, E. W. (1987). Management of Pseudomonas aeruginosa lung infection in Danish cystic fibrosis patients. Acta Pathologica, Microbiologica, et Immunologica Scandinavica 76, 95561.
12 . Høiby, N. (1992). Prevention and treatment of the infections in cystic fibrosis. International Journal of Antimicrobial Agents 1, 22938.
13 . Høiby, N. (1982). Microbiology of lung infection in cystic fibrosis patients. Acta Paediatrica Scandinavica, Suppl. 301, 3354.
14 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility TestsSixth Edition. Approved Standard M2-A6. NCCLS, Villanova, PA.
15 . O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for detection of ß-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 2838.[ISI][Medline]
16 . Filip, C., Fletcher, G., Wulff, J. L. & Erhardt, C. F. (1973). Solubilization of the cytoplasmic membrane of Esherichia coli by the ionic detergent sodium-lauryl sarcosinate. Journal of Bacteriology 115, 71722.[ISI][Medline]
17 . Knutson, C. A. & Jeanes, A. A. (1968). A new modification of the carbazol analysis: application to heteropolysaccharides. Analytical Biochemistry 24, 47081.[ISI][Medline]
18 . Chu, L., May, T. B., Chakrabarty, A. M. & Misra, T. K. (1991). Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa. Gene 107, 110.[ISI][Medline]
19 . Römling, U., Fiedler, B., Boßhammer, J., Grothues, D., Greipel, J., von der Hardt, H. et al. (1994). Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis. Journal of Infectious Disease 170, 161621.[ISI][Medline]
20
.
Jalal, S., Ciofu, O., Høiby, N., Gotoh, N. & Wretlind, B. (2000). Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrobial Agents and Chemotherapy 44, 7102.
21 . Anwar, H., Strap, J. & Costerton, J. W. (1992). Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrobial Agents and Chemotherapy 36, 134751.[ISI][Medline]
22 . Dibdin, G. H., Assinder, S. J., Nichols, W. W. & Lambert, P. (1996). Mathematical model of ß-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released ß-lactamases. Journal of Antimicrobial Chemotherapy 38, 75769.[Abstract]
23
.
Ciofu, O., Beveridge, T., Kadurugamuwa, J., WaltherRasmussen, J. & Høiby, N. (2000). Chromosomal ß-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 45, 913.
24
.
Costerton, J. W., Steward, P. S. & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284, 131822.
25
.
Davis, D., Parsek, M., Pearson, J., Iglewski, B., Costerton, J. W., Greenberg, E. P. et al. (1998). The involvement of cell-to-cell signals in the development of bacterial biofilms. Science 280, 2958.
26
.
Ceri, H., Olson, M. E., Stremick, C., Read, R. R., Morck, D. & Buret, A. (1999). The Calgary Biofilm device: new technology for rapid determination of antibiotics susceptibilites of bacterial biofilms. Journal of Clinical Microbiology 37, 17716.
27 . Valerius, N. H., Koch, C. & Høiby, N. (1991). Prevention of chronic colonization with P. aeruginosa in patients with cystic fibrosis by early treatment with ciprofloxacin and inhalation with colistin. Lancet 338, 7256.[ISI][Medline]
28 . Moskowitz, S. M., Burns, J. L., Nguyen, C. D., Høiby, N., Ernst, R. K. & Miller, S. I. (2000). Polymyxin resistance and lipid A structure of Pseudomonas aeruginosa isolated from colistin-treated and colistin-naive cystic fibrosis patients. Pediatric Pulmonology, Suppl. 20, 272.
29
.
del Mar Tavio, M., Vila, J., Ruiz, J., Ruiz, J., Martin-Sanchez, A. & Jimenez de Anta, M. T. (1999). Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. Journal of Antimicrobial Chemotherapy 44, 73542.
Received 2 January 2001; returned 3 May 2001; revised 21 May 2001; accepted 28 June 2001