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Detection and antimicrobial susceptibility testing of hypermutable strains of Pseudomonas aeruginosa in patients with clinical infections

Abstract

Aims Hypermutable strains of Pseudomonas aeruginosa have been associated with chronic lung diseases, and may be associated with increasing antimicrobial resistance. Our research evaluated the local prevalence of hypermutable strains of Pseudomonas aeruginosa in patients with clinical infections. We also analysed the difference in susceptibility between standard incubation time of 18 h and extended incubation time of 48 h on both hypermutable and non-hypermutable strains. Methods 31 P. aeruginosa strains were isolated from 24 patients (mean age = 70) with clinical infections such as bronchiectasis (54%) and tuberculosis (21%). Specimens collected included sputum (52%), endotracheal tube aspirates (13%) and bronchoalveolar lavages (10%). Detection of hypermutability was performed on 31 P. aeruginosa clinical isolates. Rifampicin mutation frequencies were calculated and compared against that of control strain PAO1; strains with frequencies 20-fold or higher were considered hypermutable. Antimicrobial susceptibility testing to aztreonam, ceftazidime, cefepime, ciprofloxacin, tazobactam-piperacillin, gentamicin, imipenem and meropenem was performed by Etest, and compared to microbroth dilution. Results were read at 18 h and 48 h. Results 5 out of 31 of the P. aeruginosa isolates were hypermutable. Etest had 98% agreement with the reference method for categorical susceptibility at 18h, with resistance present only to ciprofloxacin (6%), ceftazidime (3%) and cefepime (3%). Extended incubation of 48 h showed an increase in phenotypic resistance for all antimicrobials, ranging from 6.5% for aztreonam and meropenem to 48% for cefepime. Hypermutable strains were not significantly associated with antimicrobial resistance. Conclusions The local prevalence of hypermutable P. aeruginosa is low. There was an increase in phenotypic resistance following extended incubation for all antibiotics. However, the clinical significant of this phenotype remains uncertain.
0
2
4
6
8
10
12
< 0.1 0.1-< 0.5 0.5-< 1 1-< 5 5-< 10 10-< 20 20-< 50 50-< 100 100-< 500
Number of Isolates
Mutants/10^8 cells
Material and Methods
31 P. aeruginosa strains were isolated from 24 patients (mean
age = 70). The most common clinical infections were infective
exacerbation of bronchiectasis (54%) and tuberculosis (21%).
The study isolates were obtained from clinical respiratory
samples including sputum (52%), endotracheal tube aspirates
(13%), and brochoalveolar lavage (10%). Detection of
hypermutability was performed for all 31 isolates by calculating
rifampicin mutation frequencies against that of P. aeruginosa
control strain PAO1; strains with frequencies 20-fold or higher
were considered hypermutable.
1
Susceptibility testing to aztreonam, ceftazidime, cefepime,
ciprofloxacin, piperacillin-tazobactam, gentamicin, imipenem
and meropenem was performed by Etest (Figure 1) and
compared to microbroth dilution. Readings were taken at
intervals of 18 hours and 48 hours. The presence of resistant
mutant subpopulations in the 48 hours result were taken into
consideration when determining the minimum inhibitory
concentration (MIC).
Introduction
Hypermutable strains of Pseudomonas aeruginosa have been
associated with chronic lung diseases, and may be associated
with increasing antimicrobial resistance. This study aimed to
1) evaluate the local prevalence of hypermutable strains of
Pseudomonas aeruginosa in patients with clinical infections
2) analyze the difference in antimicrobial susceptibility test
results between standard incubation time of 18 hours and
extended incubation time of 48 hours for both
hypermutable (HM) and non-hypermutable strains (nHM).
Detection and antimicrobial susceptibility testing of hypermutable
strains of Pseudomonas aeruginosa in patients with clinical infections
Lily SY Ng
1
, Gabriel Ong
2
, Kenneth Neo
2
, Thean Yen Tan
1
1
Department of Laboratory Medicine, Changi General Hospital
2
School of Life Sciences & Chemical Technology, Ngee Ann Polytechnic
Results
Prevalence of hypermutable strains
5 (16%) of the P. aeruginosa isolates were hypermutable.
Rifampicin mutation frequency distributions are shown in
Figure 2.
Conclusion
The local prevalence of hypermutable P. aeruginosa is low. HM
strains were not significantly associated with antimicrobial
resistance. There was an increase in phenotypic resistance
following extended incubation for all antibiotics in both HM and
nHM strains. However, further work is required to evaluate the
clinical impact of resistance detected by extended-incubation
susceptibility testing in P. aeruginosa.
Categorical susceptibility for both Etest and microbroth dilution
was calculated using breakpoints from the Clinical Laboratory
Standards Institute.
2
Figure 2:
Mutation frequency
of all strains tested
using rifampicin
Poster No. AH16
Presented at CGH ASM 2014
Antimicrobial susceptibility
Etest achieved 98% agreement with the reference
microbroth dilution method for categorical susceptibility.
At 18hr, resistance was detected in ciproflxacin (20%) for
HM strains, and ciprofloxacin (6%), ceftazidime (3%), and
cefepime (3%) for nHM strains.
Extended incubation of 48hr showed an increase in
resistance for all tested antibiotics with the greatest
reduction in susceptibility to imipenem for HM strains, and
cefepime and piperacillin-tazobactam in nHM strains (Figure
3).
MIC > 32µg/ml
MIC = 0.5µg/ml
Figure 1:
Etest performed on Mueller
Hinton agar. Plates were
incubated at 35
o
C in ambient air,
with results read twice, at 18
and at 48 hours.
Etest for Meropenem (MP)
showing resistant sub-
colonies, on the left.
Etest for Imipenem (IP)
showing susceptible ellipse,
on the right.
References
1) Oliver, A. et al., 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung
infection. Science, 288, pp.1251-1254.
2) Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing;
Twenty-third Informational Supplement. CLSI document M100-S23. Wayne, PA: Clinical Laboratory
Standards Institute; 2013.
*PAO1 2.1
Non-hypermutable
isolates
Hypermutable
isolates
0%
20%
40%
60%
80%
100%
% of Strains susceptible
Antimicrobial Susceptibilities for Hypermutable Strains
18 Hour
48 Hour
0%
20%
40%
60%
80%
100%
% of Strains susceptible
Antimicrobial Susceptibilities for Non-Hypermutable
Strains
18 Hour
48 Hour
Figure 3:
Antimicrobial
susceptibilities of both
hypermutable (n = 5)
and non-hypermutable
(n = 26) strains at 18 hr
and 48 hr.
Mutation frequencies of all strains derived from calculating mutant counts per
10
8
viable CFU.
* Control strain PAO1 (mutation frequency of 2.1 mutants/10
8
cells)
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