35
AmpC, oprD Expression in β-lactam Resistant P. aeruginosa
Calvopiña et al.
p-ISSN 2477-9113
e-ISSN 2477-9148
REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS
Volumen 38. No. 1, Mayo 2017
AmpC, oprD Expression Analysis in β-lactam
Resistant Pseudomonas aeruginosa Clinical Isolates
from a Tertiary Level Hospital in Ecuador
Karina Calvopiña
1
, Marcelo Grijalva
2
*, María José Vallejo
2
, Rachid Seqqat
2
1
Laboratorio de Diagnóstico Molecular, Hospital Carlos Andrade Marín, Quito, Ecuador. Current afliation: School
of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.
2
Laboratorios de Nanomedicina y Nanobiología, Centro de Nanociencia y Nanotecnología y Departamento de Cien-
cias de la Vida, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador
* rmgrijalva@espe.edu.ec
doi: 10.26807/remcb.v38i1.19
Recibido 12-01-2017; Aceptado 01-03-2017
ABSTRACT.- Innate and acquired antibiotic resistance mechanisms in Pseudomonas present a challenge for clini-
cians looking for timely and effective chemotherapy. This is particularly important in critical care hospital settings.
This study is aimed at achieving a deeper understanding of two of the most important drug resistance mechanisms
in Pseudomonas aeruginosa at the molecular level. One hundred clinical isolates of Pseudomonas aeruginosa were
obtained from a tertiary level hospital in Quito, Ecuador. Expression of ampC and oprD was analysed through quan-
titative real-time PCR assays. A comparison between the ampC and oprD expression proles and the phenotypes
in antimicrobial susceptibility testing (AST) was conducted, with more than 50% of the isolates having concordant
proles for both ampC and oprD expression. Our results suggest that ampC and oprD expression might provide useful
information about molecular resistance mechanisms in strains which are circulating in Ecuador. However, larger scale
studies could clarify drug resistance mechanisms in order to guide targeted treatment.
KEYWORDS: ampC, oprD, β-lactams, Pseudomonas aeruginosa, RT-qPCR.
RESUMEN.- Los mecanismos innatos y adquiridos de resistencia a los antibióticos en
Pseudomonas representan un reto para los médicos que buscan una quimioterapia oportuna y ecaz. Esto es par-
ticularmente importante en las áreas de cuidados intesnsivos de los hospitales. Este estudio está dirigido a lograr
una comprensión a nivel molecular de dos de los más importantes mecanismos de resistencia a los fármacos en
Pseudomonas aeruginosa. Cien aislados clínicos de Pseudomonas aeruginosa se obtuvieron de un hospital de tercer
nivel en Quito, Ecuador. Se analizó la expresión de ampC y oprD mediante PCR cuantitativa en tiempo real. Se realizó
una comparación entre los perles de expresión ampC y oprD y los fenotipos obtenidos en la prueba de susceptibilidad
antimicrobiana (AST), con más del 50% de los aislados con perles concordantes para la expresión ampC y oprD.
Nuestros resultados sugieren que la expresión ampC y oprD podría proporcionar información útil sobre mecanismos
de resistencia molecular en cepas que están circulando en Ecuador. Sin embargo, los estudios a mayor escala pueden
aclarar los mecanismos de resistencia a los fármacos para establecer el tratamiento adecuado.
PALABRAS CLAVES: ampC, β-lactams, oprD, Pseudomonas aeruginosa, RT-qPCR.
Artículo científico
36
REMCB 38 (1): 35-44, 2017
INTRODUCTION
Pseudomonas aeruginosa is one of the most com-
monly isolated Gram negative bacteria in hospi-
tal-acquired infections. Multi-drug resistant strains
are a growing problem in health care settings, es-
pecially in extremely ill patients at intensive care
units (Obritsch et al. 2004). Eradication of the in-
fection is complex due to the intrinsic capacity of
P. aeruginosa to decrease its membrane per-
meability, to express efux pumps, to produ-
ce antimicrobial-degrading enzymes and to
acquire new resistance mechanisms throu-
gh mutations or plasmids (Luján Roca 2014).
The genome of the wild type P. aeruginosa strain
(PAO1) revealed the presence of an unusually high
number of genes involved in the regulation of bac-
terial pathogenicity (Kipnis et al. 2006 Stover et al.
2000 Woods 2004). For instance, the OprD chan-
nel allows access to carpabenems such as merope-
nem and imipenem (Hancock and Brinkman 2002,
Nikaido 1989). OprD-mediated resistance might
be related to different mechanisms that decrease
expression of a normally functioning porin, inclu-
ding mutations that cause translational disruptions
(Yoneyama and Nakae 1993). Furthermore, the pro-
duction of AmpC β-lactamase confers resistance to
most penicillin, cephalosporin and cefamicins and
shows variable results for aztreonam. However, the
expression at basal levels of ampC is not enough
alone to determine anti-pseudomonal β-lactams re-
sistance (Giwercman et al. 1990) Overexpression of
ampC and regulation of the factor AmpR are neces-
sary to acquire a resistant phenotype (Jacoby 2009).
A ve-year report (1997–2001) of the SENTRY
Antimicrobial Surveillance in the Latin American
region found a signicant rise of resistance rates to
commonly used anti-pseudomonal antibiotics over
a relatively short period of time (Andrade et al.
2003). In 2008 in Ecuador, the National Antimicro-
bial Resistance Network (REDNARBEC) reported
antibiotic resistance rates ranging from 25% for
amikacine to 45% for aztreonam, with even higher
rates in intensive care units. However, the majority
of the available local studies on antimicrobial resis-
tance are based on phenotypic proles (AST). For
this reason, we wanted to achieve a better unders-
tanding of the molecular mechanisms involved in
antimicrobial resistance in Pseudomonads in clini-
cal settings. The methodology used for this purpose
is described in the next section of this study.
The aims of this study were thus (i) to analyze
ampC and oprD expression levels, and (ii) to
compare gene expression proles for suscep-
tible and antibiotic-resistant Pseudomonas
aeruginosa isolates obtained from a referral hos-
pital in Ecuador in order to clarify their molecular
mechanisms of resistance.
MATERIALS AND METHODS
Clinical isolates and controls.- One hundred
P. aeruginosa clinical isolates were collected from
the Bacteriology Laboratory, Carlos Andrade Ma-
rin Hospital (HCAM) in Quito, one of Ecuador’s
largest hospitals. Due to their genotypic proles,
the clinical isolates P.a.119 is an antibiotic suscep-
tible and P.a.5 is an antibiotic resistant control. Ad-
ditionally, six of the isolates were unable to grow.
Consequently, this study was performed using 92
clinical isolates that were recovered either from
their corresponding culture plate from AST plates,
which included 5 anti-pseudomonal agents (ATM:
aztreonam; CAZ: ceftazidime; TPZ: piperacilin-ta-
zobactam; IMP: imipenem and MEM: meropenem)
or from the primary clinical isolate in MacConkey
agar, depending on isolate availability in the hos-
pital lab. AST was performed in the Bacteriology
Lab by qualied microbiology technicians. Sus-
ceptibility break points followed the accepted lab
standards at the time (CLSI). Results from the full
AST panels are not included in this study. Upon
collection, a single colony from each clinical iso-
late was transferred to BD Pseudomonas Isolation
Agar (DIFCO, Sparks). Bacterial suspensions (in
glycerol 15%) were then deep frozen at -80°C.
Primer and probe design.- Four sequence align-
ment runs were performed with the Clustal X 2.0
software (European Bioinformatics Institute, 2007)
for ampC, oprD and rpsL (used as normalizer in this
study) gene sequences of the P. aeruginosa reference
strains PAO1 (ID:878149, ID:881970, ID:881701),
PA7 (ID:5353514; ID:5358647; ID: 5356699,
UCBPP (ID:4382097;ID:4380780; ID:4381343 ),
and LESB58 (ID:7175766; ID: 7180952 ). Sequen-
ces were obtained from the GeneBank database
(National Centre for Biotechnology Information)
and the Pseudomonas Genome Database (Winsor
et al. 2011). A combination of primers and Taq-
man® probes were designed by using the Primer
Express® software package (Applied Biosystems,
USA), Olygo Analyzer (Integrated DNA Technolo-
gies) and BLASTn (National Library of Medicine).
(See Table S1 Supporting information.)
RNA isolation.- 100µL of the stored bacterial sus-
37
REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS
AmpC, oprD Expression in β-lactam Resistant P. aeruginosa
Calvopiña et al.
pensions was added to 5mL of Brain Heart Infusion
Broth Media (DIFCO, Sparks) followed by incu-
bation at 37°C with continuous shaking (200rpm)
until an optical density (OD600) of 0.8-1 was rea-
ched. RNA isolation from cultures was then carried
out with the PureLink Micro-to-Midi Total RNA
Purication System (Life Technologies, California)
according to manufacturer instructions, with a few
modications. Samples were treated with RNA Sta-
bilization Reagent (Qiagen) before RNA isolation
with a centrifugation step at 4°C. DNase treatment
was then performed (DNase I, Ambion, Austin,
USA) followed by a nal enzyme deactivation step
with EDTA 5mM and a purication step with 2M
LiCl. RNA quality and yield were assessed by spec-
trophotometry (NanoVue®, General Electric, Cam-
bridge, MA).
RT-qPCR.- RT-qPCR mixture (10µL) consisted of
100ng of RNA template. For rpsL nal concentra-
tions were 300nM for the forward primer and 50nM
for the reverse primer. Final probe concentration in
every system was 2000nM, (Applied Biosystems,
USA). Reaction mixture included 5µL of 2X Taq-
man mix from RNA-to-CT One Step TM One Step
Kit (Applied Biosystems, USA), 0.125µL of 40X
Taqman® RT Enzyme Mix (Applied Biosystems,
USA), and nuclease free water to complete the nal
volume. RT-qPCR was performed in a 7300 Real
Time PCR System (Applied Biosystems, USA),
with initial retro transcription at 48°C for 15 mi-
nutes, enzymatic activation at 95°C for 10 minutes,
followed by 40 cycles that consisted of a denatu-
ration step at 95°C for 15 seconds and an annea-
ling-extension step at 60°C for 60 seconds.
Expression analysis.- The adjusted ΔΔCt method
proposed by Yuan et al. (2006), was used in this
study. An effect on gene transcription was consi-
dered signicant when expression levels were ≥2.5
and ≤0.4-fold. A threshold level of 20-fold relative
to the ampC expression level in susceptible strain
P.a.119 allowed for discrimination of susceptible
and resistant strains. The expression level was cal-
culated relative to P.a.5 expression. Since the oprD
assay evaluated presence or absence of the porin,
for this assay, the threshold was set at 1-fold. ΔCt
values for susceptible (P.a.119) and resistant (P.a.5)
controls using four different concentrations (10; 8;
0.4 and 0.08ng/µL) were tested for the F varian-
ce test with a p value of >0,05. ΔCt median values
(see Table S2 Supporting Information) tested with
t and Wilcoxon tests showed a difference between
susceptible and resistant isolates used as controls
with p values of < 0.05. Expression levels were
then calculated by using the ΔΔCt adjusted method
and 2
-∆∆Ct
adjusted (Yuan et al. 2006). Qualitative
analysis and genotype assignment for susceptible
and resistant isolates were based on the above cut-
offs (20-fold for ampC and 1-fold for oprD). For
expression levels of the (0<X<1)-fold genotype as-
signment was based on the phenotypic AST prole
(see Table S3 Supporting Information).
Statistical analysis.- The means of ΔCt from sensi-
tive and resistant strains were compared using t and
Wilcoxon tests. Statistical analyses were performed
using the InfoStat package (Universidad Nacional
de Córdoba, Argentina), Microsoft Excel (Micro-
soft Corp.) and Origin 8.0 (OriginLab).
RESULTS
AST Proles.- Inhibition diameters were used for
denition of antibiotic susceptibility in compliance
with National Committee for Clinical Laboratory
Standards breakpoint recommendations. The re-
sults in Figure 1 show that more than 50% of the to-
tal isolates were susceptible to four of the drugs. In
contrast, the percentages for the aztreonam (ATM)
susceptible and resistant isolates were 43.5% and
44.6%, respectively.
Gene expression analysis.- Phenotype/genotype
correlation tests revealed discordance among an-
tibiotic susceptibility proles. Isolates with a sus-
ceptible/resistant genotypic prole matching their
appropriate AST prole were labelled as “concor-
dant”. Results on Figure 1 show average concor-
dance values (%) for each antibiotic. Qualitative
evaluation of susceptibility tests and ampC expres-
sion levels showed an average of 55.9% (Figure
1A) concordant strains, while for oprD this gure
was 50.1% (Figure 1B). For ampC, the average ex-
pression level was 5.6-fold for susceptible isolates
and 1234.42-fold for resistant isolates.
DISCUSSION
We measured oprD and ampC expression levels with
an qRT-PCR assay and compared the expression
proles obtained to the isolates’ AST proles. As ex-
pected, this study showed a discordant phenotype/
genotype prole in 53 % of the isolates. Similar re-
sults have been found in Ecuador (unpublished data)
where it was shown that 45% of clinical strains of
P. aeruginosa were discordant when comparing
AST and molecular testing.
Chromosomal expression of AmpC β-lactamase
38
REMCB 38 (1): 35-44, 2017
confers resistance to penicillin and cephalosporin
in P. aeruginosa. In this study, resistant strains for
CAZ (36.6%) and TPZ (48.7%) might be overex-
pressing AmpC. This resistance mechanism has
been previously described as a complete or partial
de-repression of this gene (LucDumas et al. 2006).
Despite the fact that aztreonam resistance in 53.7%
of the isolates may be mediated by a hyper-pro-
duction of AmpC, it is important to note that ATZ
resistance in P. aeruginosa involves other mecha-
nisms, such as the up regulation of efux pumps
(MexAB-OprM, MexCD-OprJ, MexEF-OprN and
MexXY-OprM) (Livermore 2002). An explanation
based solely on the study of the expression of one
gene will therefore not cover all the possibilities of
resistance mechanisms in P. aeruginosa.
Diffusion of carbapenems (MEM/IMP) inside cells
is mediated by the OprD porin. Resistant mutants
have reduced expression levels of this porin, or
have even led to a complete absence of the channel
(LucDumas et al. 2006). For instance, imipenem
resistance (38.2%) may have inactivating mutations
in the OprD channel that would reduce/block drug
intake. Similarly, in inmeropemem resistance for
ampC (45.5%) and oprD (37%), the presence of
two different mutations may increase the possibili-
ties of a multi-resistant strain.
Twelve clinical isolates were resistant to both
carbapenems, probably due to a synergistic
effect between the overexpression of AmpC
and an MexAB-OprM efux pump (Cabot
et al. 2011). This strong connection of the different
molecular mechanisms demonstrates the complex
nature of P. aeruginosa.
It is important to note that 47% of the isolates have
discordant phenotype-genotype proles. This dis-
cordance can be explained on the basis of trans-
criptional events such as disruptions of the oprD
promoter, premature termination of oprD transcrip-
tion or decreased transcriptional expression. Fur-
thermore, carbapenem resistance cannot be speci-
cally linked to AmpC overproduction, due to the
complex interplay between resistance mechanisms
and the high number of co-regulatory pathways, es-
pecially in clinical isolates with a wider genetic and
environmental background (Yoneyama and Nakae
1993)
Figure 1. Antibiotic Susceptibility Testing (AST) phenotype/genotype analysis for AmpC and OprD. Phe-
notype (AST): Red/Yellow-Resistant, White-Susceptible, Black-Not available. A) AST prole result (phenoty-
pe) and correlation with AmpC expression levels (genotype) for: Piperacilin-Tazobactam (TPZ), MEM (merope-
nem), Ceftazidime (CAZ) and Aztreonam (ATM) in 92 clinical isolates of P. aeruginosa. Concordance averages
for the above antibiotics are 57.3%, 58.6%, 53.5% and 54.3%, respectively. B) AST prole result (phenotype) and
correlation with OprD expression levels (genotype) for: Imipenem (IMP) and Meropenem (MEM) in 92 clini-
cal isolates of P. aeruginosa. Concordance averages for the above antibiotics are: 49.6% and 50.5%, respectively.
39
REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS
AmpC, oprD Expression in β-lactam Resistant P. aeruginosa
Calvopiña et al.
Those isolates which had a “susceptible” pheno-
type, but that were labelled resistant by molecular
analysis, could become resistant months after the
initial analysis. P. aeruginosa might develop resis-
tance during therapy, even with careful β-lactam
drug selection based on initial susceptibility data.
This inducibility feature in resistant P. aeruginosa
has been observed in patients treated with antipseu-
domonal penicillin, penicillin-inhibitor combina-
tions, AZT and extended-spectrum cephalosporin
(Lister et al. 2009). In those isolates that showed
genotypic resistance while being phenotypically
susceptible, the role of the MexAB-OprM efux
pump, or AmpC overproduction due to an inducer
such as IMP (Lister et al. 2009) could be of impor-
tance. High expression of mexA leavds to resistance
to AZT, TPZ and MEM (Lee and Ko 2012). Cef-
tadizime resistant isolates, however, might have a
high expression of mexB and mexY (Cabot
et al. 2011). P. aeruginosa strains with a high level
cephalosporinase phenotype show higher levels of
resistance to CAZ than AZT, which could be also
found in strains showing a metallo-β-lactamase
phenotype. Strains with the MexA–MexB–OprM
active efux phenotype had a higher level of AZT
resistance than CAZ resistance, which is similar
to the pattern observed in clinical isolates with the
high-level penicillinase phenotype (Vedel 2005).
P. aeruginosa possesses several innate and acqui-
red antibiotic resistance mechanisms (Shen et al.
2015). In this study we analyzed the expression of
ampC and oprD genes in phenotypically suscepti-
ble and resistant β-lactam clinical isolates. We then
assigned an S/R genotype based on gene expression
levels and compared the assigned genotypes with
the AST phenotypes. A high level of discordance
was found for phenotype/genotype. The presence of
other mechanisms such as efux pumps, plasmids
or gene up/down regulation would explain the dis-
cordance. Genetic information from this study mi-
ght be useful for understanding clinical outcomes
on the basis of the inducibility of resistance in ge-
netically “resistant” and phenotypically susceptible
isolates. Data from this study could form the basis
for the rational use of more complex and compre-
hensive platforms for the study of antibiotic resis-
tance mechanisms at the hospital level.
CONCLUSIONS
This study tested the value of using a limited
number of molecular resistance assays for the ra-
pid discrimination of susceptible and resistant
Pseudomonas aeruginosa clinical strains. The study
found that β-lactam resistance patterns, among tes-
ted clinical isolates, cannot be fully explained by
AmpC overexpression and the low expression of
oprD. In strains exhibiting phenotypic resistance
and genotypic susceptibility, other mechanisms or
interactions might be involved. Molecular biology
analyses revealed a genetic resistance background
in a number of AST susceptible strains, which may
play a role in the outcome of a number of infectious
episodes. AST phenotyping will need to be comple-
mented by rapid tests at the hospital level. Informa-
tion from studies like this one might be useful for
a comprehensive understanding of the mechanisms
involved in antibiotic resistance in Pseudomonas
aeruginosa, and for the guidance of infection con-
trol strategies and rational antibiotic usage.
REFERENCES
Andrade S, Jones R, Gales A, Sader H. 2003. In-
creasing prevalence of antimicrobial
resistance among Pseudomonas aerugi-
nosa isolates in Latin American medical
centres: 5 year report of the SENTRY An-
timicrobial Surveillance Program (1997-
2001). Journal of Antimicrobial Chemo-
therapy 52 (1): 140–141.
Cabot G, Ocampo-Sosa A, Tubau F, Macia M,
Rodríguez C, Moya B. 2011. Overex-
pression of AmpC and Efux Pumps in
Pseudomonas aeruginosa Isolates from
Bloodstream Infections: Prevalence and
Impact on Resistance in a Spanish Mul-
ticenter Study. Antimicrobial agents and
chemotherapy 55(5):1906–1911.
Giwercman B, Lambert P, Rosdahl V, Sha-
nd G, Hoiby N. 1990. Rapid emer-
gence of resistance in Pseudomonas
aeruginosa in cystic brosis patients due
to in-vivo selection of stable partially de-
repressed β-lactamase producing strains.
The Journal of antimicrobial chemothera-
py 26(2):247–259.
Hancock R, Brinkman F. 2002. Function of Pseu-
domonas aeruginosa: porins in uptake
and efux. Annual review of microbiolo-
gy 56(1):17¬–38.
Jacoby, G. 2009. AmpC β-lactamases. Clinical mi-
crobiology reviews 22(1):161–182.
Kipnis E, Sawa T, Wiener-Kronish J. 2006. Tar-
40
REMCB 38 (1): 35-44, 2017
geting mechanisms of Pseudomonas
aeruginosa pathogenesis. Médecine et
maladies infectieuses 36(2):78–91.
Lee J, and Ko K. 2012. OprD mutations and in-
activation, expression of efux pumps
and AmpC, and metallo-B-lactamases
in carbapenem-resistant Pseudomonas
aeruginosa isolates from South Korea.
International Journal of Antimicrobial
Agents 40(2):168–72.
Lister P, Wolter D, Hanson N. 2009. Anti-
bacterial-Resistant Pseudomonas
aeruginosa: Clinical Impact and Com-
plex Regulation of Chromosomally En-
coded Resistance Mechanisms. Clinical
microbiology reviews 22(4):582–610.
Livermore D. 2002. Multiple Mechanis-
ms of Antimicrobial Resistance in
Pseudomonas aeruginosa: Our Worst
Nightmare? Clinical infectious diseases,
34(5):634–40.
LucDumas J, VanDelden C, Perron K, Kohler T.
2006. Analysis of antibiotic resistance
gene expression in Pseudomonas aeru-
ginosa by quantitative real-time-PCR.
FEMS microbiology letters 254(2):217–
25.
Luján Roca D. 2014. Pseudomonas
aeruginosa: a dangerous adversary.
Acta bioquímica clínica latinoamericana
48(4):465–474.
Nikaido H. 1989. Outer membrane barrier as a
mechanism of antimicrobial resistance.
Antimicrobial agents and chemotherapy
33(11): 1831–1836.
Obritsch M, Fish D, MacLaren R, Jung R.
2004. National surveillance of anti-
microbial resistance in Pseudomonas
aeruginosa isolates obtained from inten-
sive care unit patients from 1993 to 2002.
Antimicrobial agents and chemotherapy
48(12): 4606–4610.
Shen J, Pan Y, Fang Y. 2015. Role of the Ou-
ter Membrane Protein OprD2 in
Carbapenem-Resistance Mechanisms
of Pseudomonas aeruginosa. PloS one
10(10):e0139995.
Stover C, Pham X, Erwin, A. 2000. Com-
plete genome sequence of
Pseudomonas aeruginosa PA01, an
opportunistic pathogen. Nature 406:
959–964.
Vedel G. 2005. Method to determine B-lac-
tam resistance phenotypes in
Pseudomonas aeruginosa using the disc
agar diffusion test. Journal of Antimicro-
bial Chemotherapy. 56(4):657–664.
Winsor G, Lam D, Fleming L, Lo R, Whiteside M,
Yu N. 2011. Pseudomonas Genome Da-
tabase: improved comparative analysis
and population genomics capability for
Pseudomonas genomes. Nucleic acids re-
search 39:D596-600.
Woods D. 2004. Comparative genomic analysis
of Pseudomonas aeruginosa virulence.
Trends in microbiology 12(10):437–439.
Yoneyama H, Nakae T. 1993. Mechanism of
efcient elimination of protein D2
in outer membrane of imipenem-re-
sistant Pseudomonas aeruginosa.
Antimicrobial agents and chemotherapy.
37(11): 2385–2390.
Yuan J, Reed, A, Feng C, Neal C. 2006. Statisti-
cal analysis of real-time PCR data. BMC
Bioinformatics, 7:85.
41
REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS
AmpC, oprD Expression in β-lactam Resistant P. aeruginosa
Calvopiña et al.
Table S1. Primers and probes used in this study
Name Start End Sequence 5’- 3’
rpsL F 109 129 GTA TAC ACC ACC ACG CCG AAA
Primers rpsL R 208 189 CAC CAC CGA TGT ACG AGG AA
ampC F 183 206 AGG AGA ACC GCA TTA CTT CAG CTA
ampC R 277 260 TGC TCA CCG AGC CGA TCT
oprD F 1062 1083 CCT GAC TTT CAT GGT CCG CTA T
oprD R 1161 1142 ATC CTC GCC GTA GCC GTA GT
Taqman Probes rpsL P 159 184 CCG TGT ACG TCT GAC CAA CGG TTT CG
5’FAM-
3’TAMRA
ampC P 215 235 CCT CGA AAG AGG ACG GCC GCC
oprD P 1098 1124 CAT CGA TGG CAC CAA GAT GTC TGA CAA
10 26093 10
191623 7047
2 29100 2
207571 8343
0.4 31450 0.4
23371 8079
0.08 33508 0.08
260866 7364
10 21161 10
197510 1409
2 23456 2
21629 1827
0.4 26035 0.4
243083 1727
0.08 282939 0.08
269142 1379
Table S2. Calculation of ΔCt, derived from subtracting Ct number of reference gene from that of the target gene using independent samples.
ngDNA/uL
ngDNA/uL
ΔCt
Isolate
Gene
Group
Isolate
Gene
Group

⬚

⬚
Supplementary information
Table S2. Calculation of ΔCt, derived from subtracting Ct number of reference gene from that of the target gene using independent samples.
Table S1. Primers and probes used in this study
42
REMCB 38 (1): 35-44, 2017
No. Isolate
Lab Sample
Code
ATM
CAZ
IMP
MEM
TPZ
ampC oprD
P.a.2 11021115 S:25 S:24 S:23 S:27 S:23
S:0.335 S:12.36
P.a.3 11021116 S:26 S:26 S:26 S:24 S:23
S:0.028 S:15.6
P.a.4 10273249 R:11 S:23 S:24 S:24 S:21
S:0.087
S:0.913
P.a.7 11131081 ND S:23 S:26 S:30 ND
R:110.445 S:38.08
P.a.8 11141338 R:16 S:23 S:24 S:26 S:22
S:5.692 R:0
P.a.9 11141248 R:6 R:18 S:27 S:24 R:6 S:1.608
R:0
P.a.10 11151194 S:23 S:26 S:28 S:24 S:22
S:0.016
S:1.974
P.a.11 11151193 ND ND S:23 S:23 S:21
S:16.90 R:0
P.a.12 11171255 S:23 S:23 S:25 S:26 S:28 S:1.507
R:0
P.a.14 11181146 S:22 S:26 S:28 S:32 S:24
S:2.696 R:0
P.a.15 11251191 R:16 R:14 S:23 S:28 S:22
S:2.637
S:2.334
P.a.16 11181252 ND R:14 S:23 S:26 R:16
S:2.596 R:0
P.a.18 12071319 R:14 S:23 S:24 S:23 R:10
S:0.262 R:0.880
P.a.19 12091227 R:6 S:18 S:18 R:6 R:12
R:150.270 R:0
P.a.20 12081157 R:21 S:23 S:23 S:29 R:16
S:5.430 R:0
P.a.21 12231243 S:24 S:26 S:24 S:22 S:22
R:3772.747 S:3780.6
P.a.22 12161191 ND R:14 R:10 R:10 R:14
R:6966.656 R:0
P.a.23 12201287 S:32 S:28 S:24 S:24 ND
S:13.474
S:1.75
P.a.24 12193049 R:14 ND S:30 S:25 R:15
R:108.961 R:0.02
P.a.25 12151218 R:12 S:23 S:25 S:26 S:23
R:30.116 S:6.56
P.a.26 12140634 ND S:26 S:23 S:25 S:21 S:2.132
R:0
P.a.27 12291217 R:10 S:22 R:10 R:6 R:15
R:59.506 R:0
P.a.28 1021188 R:6 R:10 S:23 S:24 ND
R:65.527 R:0
P.a.29 1031219 S:23 S:26 S:24 S:30 S:32
S:0.173 R:0
P.a.30 1031277 ND R:6 S:24 S:23 R:14
R:172.671 R:0
P.a.31 1031304 R:14 S:25 R:15 R:14 R:6
S:5.207
R:0.722
P.a.32 12301270 ND R:12 R:10 R:10 R:13
R:1766.666 R:0
P.a.34 1100659 R:6 R:6 R:10 R:6 R:6
R:76.033 R:0
P.a.35 1031363 R:10 S:22 S:24 R:6 R:15 S:2.022
R:0
P.a.36 1110461 S:30 S:30 S:30 S:30 S:32
S:0.268 R:0
P.a.49 1231373 S:24 S:24 S:29 S:29 S:21
R:33.255 R:0
P.a.51 1201208 ND S:28 S:26 S:30 S:28
S:0.302 R:0
P.a.52 1181297 S:23 S:27 S:25 S:30 S:25
S:11.877 R:0
P.a.53 1238029 R:6 R:6 R:12 R:10 R:11
R:215.432 S:2.55
P.a.54 1201226 R:13 S:18 R:12 R:6 S:20
R:718.823 S:740.44
P.a.55 1180460 R:6 S:22 S:30 S:20 S:18
R:359.920 R:0
P.a.58 1241228 R:12 R:16 R:14 R:11 R:11
S:12.328 S:3.10
P.a.59 1162013 S:24 S:26 S:30 R:14 ND R:49.583 S:0.52
P.a.60 1198025 ND S:24 R:14 R:11 R:11
S:3.070
R:2.382
P.a.61 1208034 R:12 S:21 R:14 R:12 R:10
R:20.207 R:0
P.a.72 1161270 R:6 R:6 R:10 R:15 R:10
R:160.548 S:29.89
P.a. 73 1161274 R:6 R:10 R:18 R:18 S:24
S:0,77 R: 0.65
P.a.74 1161262 R:6 R:6 R:10 S:16 R:10
S:0.011 S:35.96
P.a.75 1241286 R:6 R:6 R:10 R:12 S:28
S:0.364
S:1.728
P.a.77
1233168 R:8 S:23 S:24 S:18 S:18
S:4.516 R:0.014
P.a.78 1301303 S:24 S:25 ND ND S:24
S:11.029 R:0.12
P.a.79 1311208 R:6 S:22 R:12 R:12 S:22 S: 2.078
R:0
P.a.80 1201226 R:13 S:18 R:12 R:6 S:20
R:158.839 R: 0.55
P.a.82 1300431 R:10 S:22 R:10 R:10 S:23
R:38.935 R:0
P.a.83 2068047 S:24 R:17 R:15 R:6 R:10
R:288.281 R:0.17
P.a.84 2068036 S:24 R:16 S:17 R:6 R:16
R:6037.321 S:12.18
P.a.85 2081282 R:11 R:11 R:13 R:6 R:14
R:390.412
S: 2.336
P.a.86 2103266 S:28 S:23 S:26 S:28 S:24
S:9.525 R:0
P.a.87 2031228 S:22 R:15 R:14 R:12 S:21
S:0.00 R:0
P.a.88 2131285 R:6 R:6 R:6 R:6 R:11
R:186.993 S:22.58
P.a.89 2111135 R:6 R:6 R:8 R:6 R:11 S:0.716
R:0
P.a.90 2061320 S:24 R:16 S:25 S:20 R:17
S:0.398 S:13.47
P.a.91 2100658 S:25 S:25 S:25 S:30 S:25
R:86.398 S:14.43
P.a.92 2141215 R:16 R:18 S:28 S:28 S:24
S:0.343
S:1.636
P.a.93 2091239 R:16 S:23 S:28 S:28 S:30
R:193.169 R:0
P.a.94 3051324 R:6 R:6 R:12 R:10 ND
R:24.729 S:40.23
P.a.95 3051325 R:6 R:6 S:28 S:24 R:10
S:19.376 R:0
P.a.96 3051319 S:24 S:25 S:25 S:29 S:23
S:10.943 S:4.62
P.a.97 3063111 ND S:27 S:35 S:31 S:23
S:4.075
S:1.249
P.a.98 3051327 R:12 R:15 S:28 S:24 R:10
R:114.579 R:0
P.a.99 3060519 R:6 S:24 S:26 ND S:21
S:2.561 R:0
P.a.100 3091135 R:15 R:8 R:12 R:6 R:10
R:684.617 S:5.11
P.a.101 3050286 S:22 S:22 R:12 R:12 R:6
S:3.818 R:0.809
P.a.102 3138003 R:6 S:24 R:6 R:6 R:6
R:63.499 R:0.708
P.a.103 3181108 ND R:6 R:6 R:6 R:6
S:3.840 S:4.24
P.a.104 3161171 S:24 S:25 R:10 S:25 ND
R:24.198 R:0
P.a.105 3201336 R:12 S:18 R:12 R:6 R:15
S:9.046 R:0
P.a.106 3211222 R:18 S:24 R:15 R:10 R:6
R;552.38 S:18.33
P.a.107 3071352 S:26 S:26 R:12 R:13 R:16
S:0.149 R:0
P.a.111 4091355 S:27 ND S:25 S:28 S:23
S:12.431
S:1.267
P.a.112 4091345 S:34 S.35 R:12 S:16 S:24
R:392.668 S:8.46
P.a.113 4091339 S:32 S:30 R:14 S:16 S:24
S:3.120 R:0.024
P.a.114 4031360 S:28 S:30 R:15 S:20 S:23
S:14.808 R:0.35
P.a.115 4091347 S:34 S.35 R:12 S:16 S:24
R:341.736 S:9.27
P.a.116 4091418 S:32 ND S:30 S:30 S:24
S:9.248 R:0
P.a.117 4161402 S:26 ND S:30 S:28 S:28
R:250.292 R:0
P.a.118 4121276 S:25 S:30 S:25 S:28 S:24
R:23.588 R:0
P.a.120 4111697 S:30 S:25 S:28 S:30 S:24
R:900.241 S:221.31
P.a.121 3271265 S:25 S:28 S:24 S:30 S:22
R:226.113 R:0
P.a.122 4231360 R:20 R:6 S:23 S:25 R:12
R:18995.028 R:0
P.a.123 4171326 S:28 ND S:25 S:25 S:25
R:7062.392 S:224.86
P.a.124 4241507 S.28 S:27 S:28 S:28 S:28
R:170.923 R:0.33
P.a.125 4181242 S:25 S:25 S:28 S:30 S:25
S:8.658 S:3.63
P.a.126 4171314 S:22 S:23 S:25
S:28 S:23
R:116.421 S:3.28
P.a.127 4181238 S:25 S:25 S:38 S:30 S:25
R:205.875 S:33.20
P.a.128 4191192 S.28 ND S:25 S:25 S:25
S:0.00 R:0
P.a.129 4251282 S:24 S:28 S:28 S:28 S:22
R:80.724 R:0
PHENOTYPIC ANALYSIS
GENOTYPIC ANALYSIS
Table S3. Relative expression analysis for all isolates tested. AST analysis is based on CLSI standards. S represents sensitive and R resistant.
Genotypic analysis is based on cut off points for ampC =20-fold and for oprD=1-fold (values (0<X<1) required validation based on AST for
genotype assigment).
Table S3. Relative expression analysis for all isolates tested. AST analysis is based on CLSI standards.
S represents sensitive and R resistant. Genotypic analysis is based on cut o points for ampC =20-
fold and for oprD=1-fold (values (0<X<1) required validation based on AST for genotype assigment).
43
REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS
AmpC, oprD Expression in β-lactam Resistant P. aeruginosa
Calvopiña et al.
No. Isolate
Lab Sample
Code
ATM
CAZ
IMP
MEM
TPZ
ampC oprD
P.a.2 11021115 S:25 S:24 S:23 S:27 S:23
S:0.335 S:12.36
P.a.3 11021116 S:26 S:26 S:26 S:24 S:23
S:0.028 S:15.6
P.a.4 10273249 R:11 S:23 S:24 S:24 S:21
S:0.087
S:0.913
P.a.7 11131081 ND S:23 S:26 S:30 ND
R:110.445 S:38.08
P.a.8 11141338 R:16 S:23 S:24 S:26 S:22
S:5.692 R:0
P.a.9 11141248 R:6 R:18 S:27 S:24 R:6 S:1.608
R:0
P.a.10 11151194 S:23 S:26 S:28 S:24 S:22
S:0.016
S:1.974
P.a.11 11151193 ND ND S:23 S:23 S:21
S:16.90 R:0
P.a.12 11171255 S:23 S:23 S:25 S:26 S:28 S:1.507
R:0
P.a.14 11181146 S:22 S:26 S:28 S:32 S:24
S:2.696 R:0
P.a.15 11251191 R:16 R:14 S:23 S:28 S:22
S:2.637
S:2.334
P.a.16 11181252 ND R:14 S:23 S:26 R:16
S:2.596 R:0
P.a.18 12071319 R:14 S:23 S:24 S:23 R:10
S:0.262 R:0.880
P.a.19 12091227 R:6 S:18 S:18 R:6 R:12
R:150.270 R:0
P.a.20 12081157 R:21 S:23 S:23 S:29 R:16
S:5.430 R:0
P.a.21 12231243 S:24 S:26 S:24 S:22 S:22
R:3772.747 S:3780.6
P.a.22 12161191 ND R:14 R:10 R:10 R:14
R:6966.656 R:0
P.a.23 12201287 S:32 S:28 S:24 S:24 ND
S:13.474
S:1.75
P.a.24 12193049 R:14 ND S:30 S:25 R:15
R:108.961 R:0.02
P.a.25 12151218 R:12 S:23 S:25 S:26 S:23
R:30.116 S:6.56
P.a.26 12140634 ND S:26 S:23 S:25 S:21 S:2.132
R:0
P.a.27 12291217 R:10 S:22 R:10 R:6 R:15
R:59.506 R:0
P.a.28 1021188 R:6 R:10 S:23 S:24 ND
R:65.527 R:0
P.a.29 1031219 S:23 S:26 S:24 S:30 S:32
S:0.173 R:0
P.a.30 1031277 ND R:6 S:24 S:23 R:14
R:172.671 R:0
P.a.31 1031304 R:14 S:25 R:15 R:14 R:6
S:5.207
R:0.722
P.a.32 12301270 ND R:12 R:10 R:10 R:13
R:1766.666 R:0
P.a.34 1100659 R:6 R:6 R:10 R:6 R:6
R:76.033 R:0
P.a.35 1031363 R:10 S:22 S:24 R:6 R:15 S:2.022
R:0
P.a.36 1110461 S:30 S:30 S:30 S:30 S:32
S:0.268 R:0
P.a.49 1231373 S:24 S:24 S:29 S:29 S:21
R:33.255 R:0
P.a.51 1201208 ND S:28 S:26 S:30 S:28
S:0.302 R:0
P.a.52 1181297 S:23 S:27 S:25 S:30 S:25
S:11.877 R:0
P.a.53 1238029 R:6 R:6 R:12 R:10 R:11
R:215.432 S:2.55
P.a.54 1201226 R:13 S:18 R:12 R:6 S:20
R:718.823 S:740.44
P.a.55 1180460 R:6 S:22 S:30 S:20 S:18
R:359.920 R:0
P.a.58 1241228 R:12 R:16 R:14 R:11 R:11
S:12.328 S:3.10
P.a.59 1162013 S:24 S:26 S:30 R:14 ND R:49.583 S:0.52
P.a.60 1198025 ND S:24 R:14 R:11 R:11
S:3.070
R:2.382
P.a.61 1208034 R:12 S:21 R:14 R:12 R:10
R:20.207 R:0
P.a.72 1161270 R:6 R:6 R:10 R:15 R:10
R:160.548 S:29.89
P.a. 73 1161274 R:6 R:10 R:18 R:18 S:24
S:0,77 R: 0.65
P.a.74 1161262 R:6 R:6 R:10 S:16 R:10
S:0.011 S:35.96
P.a.75 1241286 R:6 R:6 R:10 R:12 S:28
S:0.364
S:1.728
P.a.77
1233168 R:8 S:23 S:24 S:18 S:18
S:4.516 R:0.014
P.a.78 1301303 S:24 S:25 ND ND S:24
S:11.029 R:0.12
P.a.79 1311208 R:6 S:22 R:12 R:12 S:22 S: 2.078
R:0
P.a.80 1201226 R:13 S:18 R:12 R:6 S:20
R:158.839 R: 0.55
P.a.82 1300431 R:10 S:22 R:10 R:10 S:23
R:38.935 R:0
P.a.83 2068047 S:24 R:17 R:15 R:6 R:10
R:288.281 R:0.17
P.a.84 2068036 S:24 R:16 S:17 R:6 R:16
R:6037.321 S:12.18
P.a.85 2081282 R:11 R:11 R:13 R:6 R:14
R:390.412
S: 2.336
P.a.86 2103266 S:28 S:23 S:26 S:28 S:24
S:9.525 R:0
P.a.87 2031228 S:22 R:15 R:14 R:12 S:21
S:0.00 R:0
P.a.88 2131285 R:6 R:6 R:6 R:6 R:11
R:186.993 S:22.58
P.a.89 2111135 R:6 R:6 R:8 R:6 R:11 S:0.716
R:0
P.a.90 2061320 S:24 R:16 S:25 S:20 R:17
S:0.398 S:13.47
P.a.91 2100658 S:25 S:25 S:25 S:30 S:25
R:86.398 S:14.43
P.a.92 2141215 R:16 R:18 S:28 S:28 S:24
S:0.343
S:1.636
P.a.93 2091239 R:16 S:23 S:28 S:28 S:30
R:193.169 R:0
P.a.94 3051324 R:6 R:6 R:12 R:10 ND
R:24.729 S:40.23
P.a.95 3051325 R:6 R:6 S:28 S:24 R:10
S:19.376 R:0
P.a.96 3051319 S:24 S:25 S:25 S:29 S:23
S:10.943 S:4.62
P.a.97 3063111 ND S:27 S:35 S:31 S:23
S:4.075
S:1.249
P.a.98 3051327 R:12 R:15 S:28 S:24 R:10
R:114.579 R:0
P.a.99 3060519 R:6 S:24 S:26 ND S:21
S:2.561 R:0
P.a.100 3091135 R:15 R:8 R:12 R:6 R:10
R:684.617 S:5.11
P.a.101 3050286 S:22 S:22 R:12 R:12 R:6
S:3.818 R:0.809
P.a.102 3138003 R:6 S:24 R:6 R:6 R:6
R:63.499 R:0.708
P.a.103 3181108 ND R:6 R:6 R:6 R:6
S:3.840 S:4.24
P.a.104 3161171 S:24 S:25 R:10 S:25 ND
R:24.198 R:0
P.a.105 3201336 R:12 S:18 R:12 R:6 R:15
S:9.046 R:0
P.a.106 3211222 R:18 S:24 R:15 R:10 R:6
R;552.38 S:18.33
P.a.107 3071352 S:26 S:26 R:12 R:13 R:16
S:0.149 R:0
P.a.111 4091355 S:27 ND S:25 S:28 S:23
S:12.431
S:1.267
P.a.112 4091345 S:34 S.35 R:12 S:16 S:24
R:392.668 S:8.46
P.a.113 4091339 S:32 S:30 R:14 S:16 S:24
S:3.120 R:0.024
P.a.114 4031360 S:28 S:30 R:15 S:20 S:23
S:14.808 R:0.35
P.a.115 4091347 S:34 S.35 R:12 S:16 S:24
R:341.736 S:9.27
P.a.116 4091418 S:32 ND S:30 S:30 S:24
S:9.248 R:0
P.a.117 4161402 S:26 ND S:30 S:28 S:28
R:250.292 R:0
P.a.118 4121276 S:25 S:30 S:25 S:28 S:24
R:23.588 R:0
P.a.120 4111697 S:30 S:25 S:28 S:30 S:24
R:900.241 S:221.31
P.a.121 3271265 S:25 S:28 S:24 S:30 S:22
R:226.113 R:0
P.a.122 4231360 R:20 R:6 S:23 S:25 R:12
R:18995.028 R:0
P.a.123 4171326 S:28 ND S:25 S:25 S:25
R:7062.392 S:224.86
P.a.124 4241507 S.28 S:27 S:28 S:28 S:28
R:170.923 R:0.33
P.a.125 4181242 S:25 S:25 S:28 S:30 S:25
S:8.658 S:3.63
P.a.126 4171314 S:22 S:23 S:25
S:28 S:23
R:116.421 S:3.28
P.a.127 4181238 S:25 S:25 S:38 S:30 S:25
R:205.875 S:33.20
P.a.128 4191192 S.28 ND S:25 S:25 S:25
S:0.00 R:0
P.a.129 4251282 S:24 S:28 S:28 S:28 S:22
R:80.724 R:0
PHENOTYPIC ANALYSIS
GENOTYPIC ANALYSIS
Table S3. Relative expression analysis for all isolates tested. AST analysis is based on CLSI standards. S represents sensitive and R resistant.
Genotypic analysis is based on cut off points for ampC =20-fold and for oprD=1-fold (values (0<X<1) required validation based on AST for
genotype assigment).
Table S3 continuation