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70. Comparison of PFGE and flagellin gene probe PCR to identify Pseudomonas aeruginosa strains
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84 2
Department of Chemical Pathology, St James’ s Hospital, Leeds, UK. Children affected with cystic fibrosis are treated aggressively with antibiotics, many of which are nephrotoxic. We investigated the urinary protein excretion in a cohort of patients before and after one course of antibiotic therapy. 18 children aged between 3 and 18 years were studied. They had all received regular intravenous antibiotics for a minimum period of the two previous years. Untimed urine samples were collected immediately before and then at the end of a two or three week intravenous course of antibiotics. Each course consisted of a combination of two antibiotics, one of which was always tobramycin or Colomycin. Samples were frozen soon after collection. They were analysed for total protein excretion and the results expressed as a ratio to creatinine (PCI). The mean PCI rose from 162 pre therapy to 280 post (p , 0.02). The patterns of proteins excreted were further evaluated by SDS polyacrylamide gel electrophoresis using the Pharmacia PHAST system. Tubular proteinuria was not observed in any of the pre therapy samples, but was present in 9 of the 18 post therapy samples. A significant band of a 1 microglobulin was present in all 9, retinal binding protein and b 2 microglobulin were observed in 6 and lysozyme in only three of the post therapy samples. These observations provide evidence of a transient disturbance in renal tubular function in children with cystic fibrosis treated with these antibiotics. There was no evidence of cumulative damage.
68. Comparison of genomic and antibiogram analyses of Pseudomonas aeruginosa isolates from cf sputum. S. McCallum, M.J. Gallagher, C. Cowperthwaite, C.A. Hart, M.J. Walshaw. Adult CF Unit, Cardiothoracic Centre, Liverpool, UK. Sputum cultures from CF patients chronically colonised with Pseudomonas aeruginosa (Pa) may contain isolates with different morphologies and antibiograms, and it is unclear whether these represent different strains. In order to explore this further, we collected Pa isolates from multiple sputum samples from 2 chronically colonised patients over a prolonged period of intravenous antibiotic therapy. The organisms were typed by pulse field gel electrophoresis (PFGE) using pulses of 5–33 at 6V/ cm for 20 hours at 148C following digestion with endonuclease Spe1. In one patient, 8 samples yielded 25 isolates with 4 distinct morphologies and 5 separate antibiograms. PFGE of 11 of these isolates showed their genomic fingerprints to be identical, confirming that all these samples were from the same organism. In the other patient however, 6 samples yielded 16 isolates with 6 distinct morphologies and 10 separate antibiograms. PFGE of 5 of these isolates demonstrated 3 distinct genomic fingerprints, confirming the presence of multiple organisms. Thus, whilst in some patients different antibiograms represent the same strain, in other patients they may represent multiple strains and this has implications for the correct choice of antibiotic therapy.
69. The diagnotic of Pseud. aerug. is laboratory dependent. R.M. Bertele-Harms, H.K. Harms, Children’ s Hospital of the University of Munich. In 1997, after 15 years cooperation with an experienced
S41
microbiologic laboratory in CF sputum an throat swab cultures, the complete laboratory staff changed. During the following year we noticed a surprising decline in pseudomonas (Ps.) positive results, which we could not explain by a sensational new therapy ore a changed technique in taking, preserving and mailing the probes. The table shows the Ps. related results of 220 CF patients during two 12 month periods in 1996 (old lab) and 1997 (new lab).
Throat
Swabs
year
1996
1997
number of cultures Ps. aerug. non mucoid Ps. aerug. mucoid
482
535
51%
28%
34%
24%
D
sputa D
1996
1997
284
285
2 23%
88%
64%
2 24%
2 10%
85%
74%
2 11%
D 5 differences in Ps. positivity between 1996 and 1997
As expected the percentage of Ps. positive cultures was higher in sputa than in throat swabs. However, in contrast to what is usually seen in CF, the frequency of Ps. positivity considerably declined from 1996 to 1997. 18 patients became Ps. free in 1997. The differences for the non mucoid Ps. were twice as much than for the mucoid strains. Similar declines in frequencies for throat swabs and sputa pointed to a systematic difference in the performance of the cultures. We propose to establish a European working group on the microbiology of CF with the intention to optimize the techniques and to elaborate a basis for more uniform and reliable results.
70. Comparison of PFGE and flagellin gene probe PCR to identify Pseudomonas aeruginosa strains. S. McCallum, M.J. Gallagher, C.M. Smyth, C.A. Hart, M.I. Walshaw. Adult CF Unit, Cardiothoracic Centre, Liverpool, UK. Whilst Pulse Field Gel Electrophoresis (PFGE) is the gold standard used to identify different strains of Pseudomonas aeruginosa (Pa), it is time consuming to perform. We therefore investigated the use of flagellin gene probe PCR analysis and compared it with PFGE in the identification of 22 Pa isolates taken from the sputum of 8 CF patients. PFGE was carried out using pulses of 5–33 at 6V/ cm for 20 hours at 148C, following digestion with the endonucleases Xba and Spe1. Polymerase chain reaction was carried out using a flagellin gene probe, and the amplicons digested using Taq, Mbo and Msp endonucleases. The fragments were analysed using metaphor gel electrophoresis, ethidium bromide staining and ultraviolet light transillumination. PFGE demonstrated that the isolates had 6 distinct patterns, and the same strain was identifiable in 4 patients. All 22 isolates were then subjected to flagellin gene probe PCR analysis. Analysis using Mbo or Msp restriction enzymes did not discriminate between isolates, but Taq produced results concordant with PFGE. These results show that amplification by PCR and RFLP analysis with Taq may be a useful method in the epidemiological study of Pa in cystic fibrosis patients.
Department of Chemical Pathology, St James’ s Hospital, Leeds, UK. Children affected with cystic fibrosis are treated aggressively with antibiotics, many of which are nephrotoxic. We investigated the urinary protein excretion in a cohort of patients before and after one course of antibiotic therapy. 18 children aged between 3 and 18 years were studied. They had all received regular intravenous antibiotics for a minimum period of the two previous years. Untimed urine samples were collected immediately before and then at the end of a two or three week intravenous course of antibiotics. Each course consisted of a combination of two antibiotics, one of which was always tobramycin or Colomycin. Samples were frozen soon after collection. They were analysed for total protein excretion and the results expressed as a ratio to creatinine (PCI). The mean PCI rose from 162 pre therapy to 280 post (p , 0.02). The patterns of proteins excreted were further evaluated by SDS polyacrylamide gel electrophoresis using the Pharmacia PHAST system. Tubular proteinuria was not observed in any of the pre therapy samples, but was present in 9 of the 18 post therapy samples. A significant band of a 1 microglobulin was present in all 9, retinal binding protein and b 2 microglobulin were observed in 6 and lysozyme in only three of the post therapy samples. These observations provide evidence of a transient disturbance in renal tubular function in children with cystic fibrosis treated with these antibiotics. There was no evidence of cumulative damage.
68. Comparison of genomic and antibiogram analyses of Pseudomonas aeruginosa isolates from cf sputum. S. McCallum, M.J. Gallagher, C. Cowperthwaite, C.A. Hart, M.J. Walshaw. Adult CF Unit, Cardiothoracic Centre, Liverpool, UK. Sputum cultures from CF patients chronically colonised with Pseudomonas aeruginosa (Pa) may contain isolates with different morphologies and antibiograms, and it is unclear whether these represent different strains. In order to explore this further, we collected Pa isolates from multiple sputum samples from 2 chronically colonised patients over a prolonged period of intravenous antibiotic therapy. The organisms were typed by pulse field gel electrophoresis (PFGE) using pulses of 5–33 at 6V/ cm for 20 hours at 148C following digestion with endonuclease Spe1. In one patient, 8 samples yielded 25 isolates with 4 distinct morphologies and 5 separate antibiograms. PFGE of 11 of these isolates showed their genomic fingerprints to be identical, confirming that all these samples were from the same organism. In the other patient however, 6 samples yielded 16 isolates with 6 distinct morphologies and 10 separate antibiograms. PFGE of 5 of these isolates demonstrated 3 distinct genomic fingerprints, confirming the presence of multiple organisms. Thus, whilst in some patients different antibiograms represent the same strain, in other patients they may represent multiple strains and this has implications for the correct choice of antibiotic therapy.
69. The diagnotic of Pseud. aerug. is laboratory dependent. R.M. Bertele-Harms, H.K. Harms, Children’ s Hospital of the University of Munich. In 1997, after 15 years cooperation with an experienced
S41
microbiologic laboratory in CF sputum an throat swab cultures, the complete laboratory staff changed. During the following year we noticed a surprising decline in pseudomonas (Ps.) positive results, which we could not explain by a sensational new therapy ore a changed technique in taking, preserving and mailing the probes. The table shows the Ps. related results of 220 CF patients during two 12 month periods in 1996 (old lab) and 1997 (new lab).
Throat
Swabs
year
1996
1997
number of cultures Ps. aerug. non mucoid Ps. aerug. mucoid
482
535
51%
28%
34%
24%
D
sputa D
1996
1997
284
285
2 23%
88%
64%
2 24%
2 10%
85%
74%
2 11%
D 5 differences in Ps. positivity between 1996 and 1997
As expected the percentage of Ps. positive cultures was higher in sputa than in throat swabs. However, in contrast to what is usually seen in CF, the frequency of Ps. positivity considerably declined from 1996 to 1997. 18 patients became Ps. free in 1997. The differences for the non mucoid Ps. were twice as much than for the mucoid strains. Similar declines in frequencies for throat swabs and sputa pointed to a systematic difference in the performance of the cultures. We propose to establish a European working group on the microbiology of CF with the intention to optimize the techniques and to elaborate a basis for more uniform and reliable results.
70. Comparison of PFGE and flagellin gene probe PCR to identify Pseudomonas aeruginosa strains. S. McCallum, M.J. Gallagher, C.M. Smyth, C.A. Hart, M.I. Walshaw. Adult CF Unit, Cardiothoracic Centre, Liverpool, UK. Whilst Pulse Field Gel Electrophoresis (PFGE) is the gold standard used to identify different strains of Pseudomonas aeruginosa (Pa), it is time consuming to perform. We therefore investigated the use of flagellin gene probe PCR analysis and compared it with PFGE in the identification of 22 Pa isolates taken from the sputum of 8 CF patients. PFGE was carried out using pulses of 5–33 at 6V/ cm for 20 hours at 148C, following digestion with the endonucleases Xba and Spe1. Polymerase chain reaction was carried out using a flagellin gene probe, and the amplicons digested using Taq, Mbo and Msp endonucleases. The fragments were analysed using metaphor gel electrophoresis, ethidium bromide staining and ultraviolet light transillumination. PFGE demonstrated that the isolates had 6 distinct patterns, and the same strain was identifiable in 4 patients. All 22 isolates were then subjected to flagellin gene probe PCR analysis. Analysis using Mbo or Msp restriction enzymes did not discriminate between isolates, but Taq produced results concordant with PFGE. These results show that amplification by PCR and RFLP analysis with Taq may be a useful method in the epidemiological study of Pa in cystic fibrosis patients.