< 0.1 0.1-< 0.5 0.5-< 1 1-< 5 5-< 10 10-< 20 20-< 50 50-< 100 100-< 500
Number of Isolates
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.
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
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
, Gabriel Ong
, Kenneth Neo
, Thean Yen Tan
Department of Laboratory Medicine, Changi General Hospital
School of Life Sciences & Chemical Technology, Ngee Ann Polytechnic
Prevalence of hypermutable strains
5 (16%) of the P. aeruginosa isolates were hypermutable.
Rifampicin mutation frequency distributions are shown in
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
of all strains tested
Poster No. AH16
Presented at CGH ASM 2014
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
MIC > 32µg/ml
MIC = 0.5µg/ml
Etest performed on Mueller
Hinton agar. Plates were
incubated at 35
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.
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.
% of Strains susceptible
Antimicrobial Susceptibilities for Hypermutable Strains
% of Strains susceptible
Antimicrobial Susceptibilities for Non-Hypermutable
susceptibilities of both
hypermutable (n = 5)
(n = 26) strains at 18 hr
and 48 hr.
Mutation frequencies of all strains derived from calculating mutant counts per
* Control strain PAO1 (mutation frequency of 2.1 mutants/10