Efux Pump Genes in β-lactam Isolates of P. aeruginosa

Armendáriz-Castillo et al.

p-ISSN 2477-9113

e-ISSN 2477-9148

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Volumen 38. No. 1, Mayo 2017

Analysis of Efux Pump Genes in β-lactam Resistant

Clinical Isolates of Pseudomonas aeruginosa from a Tertiary Level

Hospital in Ecuador

Isaac Armendáriz-Castillo

, Marcelo Grijalva

*, María José Vallejo

; Patricia Jiménez

Departamento de Ciencias de la Vida, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador

Centro de Nanociencia y Nanotecnología, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador

* rmgrijalva@espe.edu.ec

doi: 10.26807/remcb.v38i1.20

Recibido 12-01-2017; Aceptado 23-03-2017

ABSTRACT.- Pseudomonas aeruginosa is a nosocomial microorganism that causes a wide spectrum of infections

and is known as one of the primary multi-resistant microorganisms against β-lactam antibiotics. One of the main

resistance mechanisms found in P. aeruginosa is the efux pumps. This study is aimed at characterizing this mecha-

nism by analyzing the expression of four genes (mexA, mexX, oprJ and oprM) involved in antibiotic efux pumps

in Pseudomonas aeruginosa. Forty clinical isolates (20 resistant, 20 susceptible) were collected from the Bacte-

riology Laboratory at the Carlos Andrade Marin Hospital, in Quito-Ecuador. Expression levels for the selected ge-

nes were assessed by RT-qPCR assays using RpsL as a housekeeping gene for ΔΔCt adjusted relative quantitation

analysis. The importance of efux pumps as a resistance mechanism was corroborated through analysis of efux

pumps genes that showed overexpression in all phenotypically resistant isolates. Furthermore, phenotype/genotype

analysis was performed comparing Antibiotic Susceptibility Testing (AST) results with expression proles. Results

for the mexA genotype showed correlation with the TPZ resistance phenotype and the mexX genotype with the IPM,

MEM and FEP resistance phenotypes. In conclusion, the expression pattern of the efux pump genes suggests re-

sistance mechanisms that are due to horizontal transmission or pathogens spreading into the hospital environment.

KEYWORDS: Bacterial Resistance, Efux pumps, β-lactams, Pseudomonas aeruginosa, RT-qPCR.

RESUMEN.- Pseudomonas aeruginosa es un microorganismo responsable de una amplia variedad de infecciones y

es uno de los principales patógenos multiresistentes ante antibióticos β-lactámicos. Uno de los principales mecanis-

mos de resistencia en P. aeruginosa constituyen las bombas de eujo. El objetivo del presente estudio fue caracterizar

el mecanismo de bombas de eujo mediante el estudio de expresión de 4 genes (mexA, mexX, oprJ y oprM) involucra-

dos en este mecanismo en Pseudomonas aeruginosa. Cuarenta aislados clínicos (20 resistentes y 20 sensibles) fueron

recolectados en el Laboratorio de Bacteriología del Hospital “Carlos Andrade Marin” en Quito-Ecuador. Los niveles

de expression de los genes seleccionados fueron evaluados por RT-qPCR usando RpsL como gen constitutivo para el

análisis de cuanticación relativa basado en el método ΔΔCt ajustado. La importancia de las bombas de eujo como

mecanismos de resistencia fue conrmada ya que el estudio de expresión de los genes relacionados con bombas de

eujo mostró sobreexpresión de ellos en todos los aislados fenotípicamente resistentes. Se realizó además un análisis

fenotipo/genotipo comparando los resultados del antibiograma (AST) con los perles de expresión. La sobreexpre-

sión de mexA (genotipo) mostró correlación con el fenotipo de resistencia a TPZ, mientras que el genotipo mexX se

correlacionó con los fenotipos de resistencia a IPM, MEM y FEP. En conclusion, los patrones de expression de los

genes relacionados con bombas de eujo sugieren la presencia de mecanismos de resistencia basados en transmisión

horizontal que posibilitan la diseminación de patógenos en el ambiente hospitalario.

PALABRAS CLAVES: β-lactamasa, bomba de ujo, Pseudomonas aeruginosa, Resistencia bacteriana, RT-qPCR.

Artículo científico

REMCB 38 (1): 51-60, 2017

INTRODUCTION

Pseudomonas aeruginosa is one of the main no-

socomial microorganisms. It is responsible for

10% to 15% of nosocomial infections—such as

pneumonia, urinary tract infections, wound infec-

tions and bloodstream infections—and it is mainly

present in intensive care units and surgical wards

(Lister et al. 2009). According to Guzmán-Blanco

and Istúriz (2010), Pseudomonas aeruginosa in-

fections have a prevalence of 16% in studies con-

ducted in South American hospitals. In Ecuador,

infectious episodes due to Pseudomonas aerugino-

sa rate at 23% of all β-lactam resistant episodes.

Pseudomonas aeruginosa shows a high level of

resistance to the primary anti-pseudomonal anti-

biotics, such as β-lactams. Its genome is one of the

largest of all microorganisms, allowing it to mutate

and adapt to different stress conditions, such as ex-

posure to antibiotics and through several complex

resistance mechanisms (Meletis and Begkeri 2013).

Resistance mechanisms are divided into three cate-

gories: acquired, adaptive and intrinsic. Adaptive re-

sistance is due to environmental or nutritional stress,

intrinsic resistance is caused by the low permeabi-

lity of the outer membrane, while acquired mecha-

nisms are represented by horizontal gene transfer

and mutational events. The production of enzymes

(β-lactamases), mutations in regulatory porines in

the outer membrane and, most notably, overexpres-

sion of efux pumps-encoding genes may result

in a resistant phenotype (Breidenstein et al. 2011).

Efux pumps are part of the Nodular Resistance

Division (RND), a tripartite system that includes

a membrane fusion protein (MFP), an outer mem-

brane factor (OMF) and a cytoplasmic membrane

carrier. RND controls the inow and expulsion of

molecules from cytoplasm to periplasmic space.

RND complex genes are arranged in operons in the

P. aeruginosa chromosome. Twelve efux pumps

have been identied and are expressed in P. aerugi-

nosa strains. MexAB-oprM, MexCD-oprJ, MexEF-

OprN and MexXY have been widely studied, and

all of them were identied in multiresistant isola-

tes (Morita et al. 2012). Efux pumps use antibio-

tics as substrate and expel them out of the cell by

proton motive force using ions through the elec-

trochemical gradient of the membrane, in a pro-

cess known as chemiosmosis (Lister et al. 2009).

RT-qPCR assays allow RNA quantitation analy-

sis. Their high sensitivity, reproducibility and ef-

ciency have led qPCR to be the current main te-

chnique for gene expression analysis (Khan-Malek

and Wang, 2011). Quantitative PCR (qPCR) uses

specic primers and uorescent probes. When the

target sequence is detected, a uorescent signal is

emitted that has a higher intensity than the baseli-

ne-Threshold Cycle (Ct) (Nolan et al. 2006). Re-

lative quantitation is the method used for measu-

ring gene expression in quantitative PCR analysis.

It relies on a housekeeping gene—whose expres-

sion is constant under different conditions—and

utilizes the ratio of target gene expression over

housekeeping gene expression for the quanti-

tation of gene expression (Bolha et al. 2012).

The ΔΔCt adjusted method has been used for rela-

tive quantitation of gene expression in RT-qPCR.

The method takes into consideration the assay´s

Amplication Efciency (AE) value. The Ct values

of target and housekeeping genes are also consi-

dered in the equation and it allows for the calcula-

tion of gene expression ratios (Yuan et al. 2008).

The aim of this study was to implement RT-qP-

CR systems for quantitation of the expression

of gene encoding efux pumps in clinical isola-

tes of Pseudomonas aeruginosa and to compa-

re expression ratios in susceptible and resistant

isolates in order to evaluate the role of efux

pumps in antibiotic resistance mechanisms.

MATERIALS AND METHODS

Collection and Storage of Pseudomonas

aeruginosa Isolates.- Forty P. aeruginosa

isolates were collected from the Microbiology La-

boratory at the Carlos Andrade Marin Hospital in

Quito-Ecuador. Demographics and collection site

information for patients and isolates for this study

are shown in Table S1 (Supplementary Informa-

tion). Antimicrobial Susceptibility Testing (AST)

was performed in the hospital´s Bacteriology La-

boratory by qualied and experienced microbiolo-

gy technicians in accordance with to current stan-

dards (CLSI). This study was conducted with 20

resistant and 20 susceptible isolates (AST proles

for all isolates are shown in Table S2 (Supplemen-

tary Information). Suspensions in 15% v/v glyce-

rol/water were cryopreserved in our laboratory at

the Universidad de las Fuerzas Armadas-ESPE.

RNA Extraction, Purication and Quantita-

tion.- Prior to RNA extraction, 100µl of the cellu-

lar suspension in 15% glycerol was cultured in 4ml

of Brain Heart Infusion and incubated at 37ºC for

18 hours. Afterwards, the recommended protocol

for the Purelink RNA Mini Kit (Ambion), DNa-

Efux Pump Genes in β-lactam Isolates of P. aeruginosa

Armendáriz-Castillo et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

se treatment was performed using TURBO DNa-

se (Ambion), and a phenol/chloroform RNA ex-

traction protocol was then performed (Jacobs and

White, 2013). Puried RNA was quantied using

a Qubit 1.0 uorometer (Invitrogen) with RNA BR

kit (Invitrogen). Finally, an aliquot of the extrac-

ted RNA was run in a 2% agarose electrophoresis

gel in order to visually conrm RNA purication.

Design of Primers and Probes.- Five pairs

of primers and four probes (Table S3 Supple-

mentary Information) were designed with aid

of the Primer Express v3.0 software (Applied

Biosystems). The additional bioinformatics tools

BLAST, Clustal Omega and Oligo Analyzer were

used for ne tuning of design parameters (mel-

ting temperature (Tm), primer size, GC con-

tent, homo dimers and hetero dimers formation).

RT-qPCR.- All 40 strains of P. aeruginosa

were analyzed for each of the ve genes inclu-

ded in the study. TaqMan RNA-to-Ct 1-Step Kit

(Applied Biosystems) was used for mexA, oprM,

oprJ and rpsL genes (housekeeping gene used as

normalizer), and the Power SYBR Green RNA-

to-Ct 1-Step kit (Applied Biosystems) was used

for mexX. The assays were carried out in a 7300

Real Time PCR System (Applied Biosystems).

For the mexA, oprM, oprJ and rpsL amplication

assays, a 10µl nal reaction volume was used.

The reaction mix contained 5µl of 2X Taqman

RT-PCR Mix, 0.9µl of 10µM forward and rever-

se primers, 1µl of 2000nM MGB probe, 0.25µl

of TaqMan RT enzyme, 1µl of 10ng/µl RNA, and

0.95µl of nuclease-free water. The thermal cycler

program included a retro transcription step at 48ºC

for 20 minutes, an initial denaturation step at 95ºC

for 10 minutes, followed by 35 cycles consisting

of a denaturation step at 95ºC for 15 seconds, an

annealing step at 51ºC (for mexA and oprJ), 59ºC

(for oprM) and 53ºC (for rpsL) for 30 seconds,

and a nal extension step at 60ºC for 30 seconds.

For the mexX gene, the 10µl nal reaction mixture

contained 5µl of 2X Power SYBR green mix, 0.2µl

of 10µM forward and reverse primers, 0.08µl of

RT enzyme mix, 1µl of 10ng/µl RNA and 3.52µl

of nuclease-free water. The PCR thermal cycler

program consisted of a reverse transcription step

at 48ºC for 20 minutes, an initial denaturation step

at 95ºC for 10 minutes, followed by 35 cycles of

a denaturation step at 95ºC for 15 seconds, and

an annealing step at 55ºC for 30 seconds. A nal

extension step at 60ºC for 30 seconds was inclu-

ded. For Tm analysis, the thermal cycler was pro-

grammed as follows: denaturation at 95ºC for 15

seconds, renaturation at 60ºC for 1 minute, denatu-

ration at 95ºC for 15 seconds and nal renaturation

at 60ºC for 15 seconds. A dissociation curve was

used to determine if the predicted Tm (87ºC) for

the mexX amplicon corresponded to the actual Tm

of the cDNA PCR fragment obtained in the assay.

Amplication Efciency and Statistical Analy-

ses.- For calculation of the assay´s Amplication

Efciency (AE), two clinical strains were used as

assay controls, corresponding to a resistant and a

susceptible isolate (Wong and Medrano 2005). Four

RNA dilutions were prepared from the original con-

centrated RNA (10ng/µl), by adding DEPC treated

water to RNA nal concentrations of 5ng/µl, 1ng/

µl, 0.5ng/µl and 0.1ng/µl. A RT-qPCR reaction

was run for all gene systems with the above RNA

concentrations and Ct values were obtained for

each PCR run. Ct values of the housekeeping gene

(rpsL) were subtracted from the Ct values of the

four target genes, and a ΔCt value for each system

was obtained. A two-sample T-test was applied for

comparison of Ct’s for phenotypically resistant and

susceptible strains in each targeted gene. A p-value

of 0.05 was considered for statistical signicance.

Relative Quantitation of Gene Expression.- Ct

values were obtained and tabulated from the RT-qP-

CR for the ve genes (n= 40 isolates). The ma-

thematical model proposed by Yuan et al. (2008)

was used for calculation of expression ratios.

ΔΔCT

adjusted

= µ1 x AE1 - µ2 x AE2 - µ3 x AE3 +

µ4 x AE4 (1)

Being:

µ1: Target gene Ct for sample X

µ2: Control gene Ct for sample X

µ3: Target gene Ct of control sample

µ4: Control gene Ct of control sample

AE1: Amplication Efciency of target gene for

sample.

AE2: Amplication Efciency of control gene for

sample.

AE3: Amplication Efciency of target gene for

control sample.

AE4: Amplication Efciency of control gene for

control sample.

Ratio = 2-

ΔΔCt

(2)

Phenotype/Genotype Correlation.- A compa-

rison between dominant (most common) phe-

notypes and genotypes was performed to esta-

REMCB 38 (1): 51-60, 2017

blish the correlation and associations among the

genotypes responsible for antibiotic resistance.

RESULTS

RT-qPCR.- The assay tested the puried RNA of

all forty P. aeruginosa strains for each of the ve

target genes. Ct values, amplication and dissocia-

tion curves were obtained through the 7300-sof-

tware system (Applied Biosystems) from all forty

P. aeruginosa isolates. Amplication curves for

mexA and mexX resistant isolates (Figures 1A and

1B) had a lower Ct than susceptible ones. A mel-

ting curve analysis for mexX (87 ºC) was used for

conrmation of specic amplication of the PCR

fragment (Figure 1C). Finally, the constant expres-

sion of the housekeeping gene (rpsL) is showed for

both susceptible and resistant isolates (Figure 1D).

Amplication Efciency and Statistical Analy-

ses.- AE values were obtained from the resistant

and susceptible isolates used as controls for each

of the ve gene expression systems. The Ct va-

lues obtained in all serial dilutions were analyzed

by a two-sample T-test (Table 1 and Table 2).

Gene Expression.- Based on Yuan et al. (2008),

Statistical methods for efciency adjusted real-time

PCR quantication, gene expression was quanti-

ed using Eq (1) and (2) (see Materials and Me-

thods). Gene expression ratios for all strains and

genes are showed in Table S4 (Supplementary in-

formation). A comparison of the expression ratios

of resistant and susceptible phenotypes (average)

was performed for each gene as shown in Figure 2.

As expected, the expression ratios were higher in

resistant isolates in comparison to susceptible ones.

Over expression of efux pump genes in clinical

strains of P. aeruginosa was thus conrmed, stres-

sing its importance as one of the main resistance

mechanisms against β-lactam antibiotics. Further-

more, expression ratios for oprM and oprJ genes

are lower than expression ratios for mexA and

mexX genes, in spite of over-expression resistant

isolates being greater than that in susceptible ones.

CYCLE NUMBER

DISSOCIATION CURVE

TEMPERATURE (C)

DERIVATIVE

DELTA Rn

DELTA Rn VS CYCLE

DELTA Rn VS CYCLE DELTA Rn VS CYCLE

Figure 1 a) Duplicate amplication curves for mexA gene, dierence between resistant (red/violet) and sensitive (dark purple/

green) isolates. b) Duplicate amplication curves for mexX gene, dierence between resistant (blue/green) and sensitive (violet/fu-

chsia) strains are showed. c) Duplicate dissociation curve for mexX gene (red/green), showing a melting temperature of 87 ºC. d) 10

amplication curves for rpsL gene with constant expression for resistant (purple spectrum) and sensitive isolates (green spectrum).

Efux Pump Genes in β-lactam Isolates of P. aeruginosa

Armendáriz-Castillo et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Phenotype/Genotype Correlation.- Correlation

between genotype and phenotype was performed

using gene expression results as well as AST pro-

les. The results presented in Figure 3 showed

mexA and mexX as dominant genotypes, data that

was conrmed by the higher expression ratios

shown in Table S4 Supplementary information. Out

of the 20 resistant isolates tested in this study, in

95% of the isolates the mexA gene is overexpres-

sed and in 100% the mexX gene is overexpressed,

showing the presence of active genes encoding

efux pumps in clinical isolates of P. aeruginosa.

Furthermore, AST analysis showed that the main

resistance phenotypes were to MEM (meropenem)

and to IPM (imipenem), with a 100% resistance

prole, and to TPZ (tazobactam) and to FEP (ce-

fepime), with a 95% resistance prole (Figure 4).

AST proles for susceptible isolates showed that

more than 50% of the isolates express resistance

to at least one β-lactam antibiotic. When a com-

parison performed of the antibiotic susceptibili-

ty proles of the resistant strain with expression

ratios for both susceptible and resistant isolates

(Figure 5), we concluded that the mexA overex-

pression genotype is closely related to TPZ phe-

notype, while the mexX over expression genoty-

pe correlates to IMP, FEP and MEM resistance.

DISCUSSION

In this study, we developed RT-qPCR assays for

relative quantication of the expression of P.

aeruginosa genes encoding efux pumps (mexA,

mexX, oprJ and oprM). We then analyzed changes

in gene expression in both resistant and suscepti-

ble clinical isolates. This technique allowed us to

conrm the important role of efux pumps as one

of the main resistant mechanisms expressed in

P. aeruginosa clinical isolates in the collection

Gene Phenotype Slope AE (%)

Resistant -3.3058 100

Susceptible -3.3109 100

Resistant -3.3239 99.92

Susceptible -2.045 100

Resistant -3.5131 92.6

Susceptible -3.1858 100

Resistant -3.4104 96.44

Susceptible -3.4333 95.55

Resistant -3.4891 93.47

Susceptible -3.4549 94.73

rpsL

Table 1. Amplification Efficiency for each gene system

mexA

mexX

oprM

oprJ

Gene Phenotype Mean

Resistant 2.35

Susceptible 6.2

Resistant 7.89

Susceptible 12.86

Resistant -2,02

Susceptible 1.61

Resistant 2.05

Sensitive 4.39

Table 2. Two-sample t-test results for resistant and sensitive isolates (p-value is < 0.05 in all cases)

mexA

mexX

oprM

oprJ

Table 2. Two-sample t-test results for resistant and

sensitive isolates (p-value is < 0.05 in all cases)

Gene

Averange of gene expression ratio

analyzed. This nding is supported by the expres-

sion analysis of four genes performed in this study.

Despite different expression ratios, over-expression

in resistant isolates is much higher than

in susceptible ones (Bolha et al. 2012).

The RT-qPCR technique developed in this

study is as a robust evidence for gene ex-

pression analysis in susceptible and resistant

P. aeruginosa The model proposed by Yuan et

al. (2008) considers amplication efciency to be

reliable method for relative quantication, since

100% amplication value is not possible to achie-

ve because due to the different substrate conditions

and the efciency of the nucleic acid extraction

kit used. Usually, amplication efciencies for

RT-qPCR assays are within 70–100%. We obtained

an average efciency of 90% in all PCR systems

tested.

Figure 2. oprJ, oprM, mexA and mexX average expres-

sion ratios from sensitive and resistant strains.

REMCB 38 (1): 51-60, 2017

Expression ratios were considerably hi-

gher in all resistant isolates. mexA and mexX

genes are the dominant genotypes, being

over-expressed in almost all resistant isolates.

Overexpression of efux pump genes might suggest

the presence of a nosocomial strain of P. aeruginosa

in the hospital environment. However, it is impor-

tant to note that horizontal transmission might be

another mechanism for the spreading of infection

among patients coming from another health facility.

The evident difference in the expression ratios of

mexA and mexX in comparison with oprM and oprJ

is related with their function in the RND complex.

The mexA and mexX genes are important Membrane

Fusion Proteins (MFP) in the pump structure. They

act as assemble adaptors that ensure proper chan-

nel conformation in order to connect the cytoplasm

and the membrane. Therefore, the overexpression

of MFP’s in all strains suggests a well-conformed

and functional efux pump. Low expression of the

oprJ gene in comparison to the other three sys-

Figure 3. Eux pumps encoding genes expression ratios dierences in resistant strains of P. aeruginosa. A) mexA and

oprM, B) mexA and oprJ, C) mexX and oprJ and D) mexX and oprM.

Expression ratios

Isolate ID

Figure 5. Correlation among dominant phenoty-

pe and genotype for both sensitive and resistant

isolates. Genes mexA and mexX related with phe-

notypes resistant to tazobactam (TPZ), imipenem

(IMP), cefepima (FEP) and meropenem (MEM).

Antibiotics

Resistance percentage

Figure 4. Resistance percentages for the β-lac-

tams antibiotics: ceazidime (CAZ), Azithromy-

cin (ATM), tazobactam (TPZ), imipenem

(IMP), cefepima (FEP) and meropenem (MEM).

Genotype

Phenotype

Efux Pump Genes in β-lactam Isolates of P. aeruginosa

Armendáriz-Castillo et al.

p-ISSN 2477-9113

e-ISSN 2477-9148

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Volumen 38. No. 1, Mayo 2017

tems analyzed is due to the fact that it is acquired

from plasmids, rather than chromosomally encoded

in the P. aeruginosa genome (Lister et al. 2009).

The importance of analyzing the phenoty-

pe and genotype correlation in multiresistant

P. aeruginosa was rst stressed by Pommerenke

and collaborators (2010). In the present study, we

compared AST proles for susceptible and resistant

clinical isolates to the isolate’s expression ratios.

Our ndings show that a number of susceptible

isolates have measurable gene expression for the

resistance-related genes mexA and mexX and show

resistance to one or more of the phenotypically do-

minant antibiotics (tazobactam (TPZ), imipenem

(IMP), cefepima (FEP) and meropenem (MEM)).

The most remarkable case was the resistant isola-

te R11, which showed mexX overexpression, pro-

viding resistance to ATM, MEM and IPM. Phe-

notypically susceptible isolates (5, 7, 8, 10, 13,

15, and 20), which presented resistance to TPZ,

showed high mexA expression. Therefore, we

were able to establish a correlation for the pre-

sence of the mexA genotype in the TPZ-resistant

phenotype and a correlation of the mexX genoty-

pe the ATM, MEM and IPM-resistant phenotypes.

CONCLUSIONS

Gene expression (mexA, oprM, mexX and

oprJ) pattern analysis in resistant isolates con-

rmed the predominate role of efux pumps

as one of the most important β-lactam resistance

mechanisms.

Efux pumps encoding genes are mainly acquired

by mutations and horizontal gene transfer among

strains present in the inner hospital environment.

P. aeruginosa resistant isolates play an important

role on this acquired resistance mechanism inside

the hospital. This study found the same genetic pa-

ttern in more than 95% percent of all clinical iso-

lates studied. However, larger studies are recom-

mended in order to achieve a better comprehension

of the mechanisms involved in β-lactam resistan-

ce in Pseudomonas aeruginosa clinical isolates.

ACKNOWLEDGMENTS

We thank the Carlos Andrade Mari Hos-

pital Bacteriology Laboratory for kind-

ly providing the isolates for this study.

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REMCB 38 (1): 45-54, 2017

Isolate Age Sex Collection site Ward

67 F Pharynx Surgery

44 M Trachea Neurosurgery

74 F Trachea Neurosurgery

24 M Pharynx Surgery

25 M Femoral Surgery

48 M Blood culture Gastroenterology

74 F Trachea Neurosurgery

74 M Trachea Neurosurgery

71 F Trachea UCI

S10

25 M Femoral Surgery

S11

28 M Catheter Burns unit

S12

23 F Catheter Surgery

S13

27 F Trachea Burns unit

S14

27 M Blood culture Ortophedics

S15

74 M Trachea Neurosurgery

S16

72 M Trachea Surgery

S17

48 M Peritoneal Surgery

S18

74 M Trachea Neurosurgery

S19

48 M Secretion Surgery

S20

74 M Trachea Neurosurgery

62 M Femoral Ortophedics

67 M Pancreatic tissue Surgery

43 M Fracture Ortophedics

19 M Femoral head Surgery

19 M Left forearm Surgery

28 M Secretion Surgery

69 M Bile Gastroenterology

55 M Wound Surgery

50 M Limb Surgery

R10

44 F Skin (breast) Surgery

R11

11 M Secretion Preventive medicine

R12

28 M Sacral ulcer Surgery

R13

22 F Wound Surgery

R14

8 M Peritoneal Pediatrics

R15

46 M Trachea Neurosurgery

R16

58 F Peritoneal Neurosurgery

R17

42 M Catheter Dialysis unit

R18

25 M Trachea Chest medicine

R19

43 F Trachea Neurosurgery

R20

54 F Catheter Surgery

Table S1. Isolates collection sites and patient demographics (susceptible isolates-S, resistant isolates-R)

Suplementary information

Table S1. Isolates collection sites and patient demographics (susceptible isolates-S, resistant isolates-R)

Table S2. Primers and probes used in this study

Probes 5’ - 3’

Gene

MGB (Minor Groove Binding) with

FAM dye

Forward Reverse

Product length

(bp)

mexA CTGCTGCCCGGCAT GTTCCCCAACCCGAACAAC TTGACGCCTTCCTGCAACT 69

mexX N/A (Syber Green) CCATGCGTGCCCTGTTC TCGCCTGCGGGTTCAC 93

oprJ CTGCGTGCCAGCCT GATCGGCAGCGTCAACGT AATGTCCGGCCTGTTCGA 67

oprM CTCCAGGAGCGCGAG GGTTCGGGTTCCTGGTTGTT CGTTGATGTCCTTCTGGATCTTC 106

rpsL ACGCGGGTGCATAC TTTTCGGCGTGGTGGTGTA CAAAACTGCCCGCAACGT 58

Primers 5’ - 3’

Table S2. Primers and probes used in this study

Efux Pump Genes in β-lactam Isolates of P. aeruginosa

Armendáriz-Castillo et al.

REVISTA ECUATORIANA DE MEDICINA Y CIENCIAS BIOLOGICAS

Isolate CAZ FEP ATM MEM IPM TPZ

S S S S S S

R S S R S S

S S S S S S

S S S S S R

S S S R S S

S S S S R R

S S S S S R

S R S S S S

S10

S S S S S R

S11

S S S S R S

S12

S S S S R S

S13

S S S S S R

S14

S S S S S S

S15

S S S S S R

S16

S S S S R S

S17

S R S S S S

S18

S S S S S S

S19

S S S S S S

S20

S S S S S R

R R R R R R

S R R R R R

R R R R R R

S R R R R R

- R S R R R

R R R R R R

R10

R R R R R R

R11

S S R R R S

R12

R R R R R R

R13

R R R R R R

R14

S R R R R R

R15

R R R R R R

R16

R R R R R R

R17

R R R R R R

R18

R R R R R R

R19

R R R R R R

R20

R R S R R R

Table S3. AST profiles for susceptible (S) and resistant (R) isolates. Antibiotics used for the

analysis were: MEM (meropenem), IPM (imipenem), TPZ (tazobactam), FEP (cefepime) and

CAZ ceftazidima.

REMCB 38 (1): 45-54, 2017

mexA mexX oprJ oprM mexA mexX oprJ oprM

S-1

1,726 1,773 0,229 0,002

R-1

14,530 224,300 0,503 1,030

S-2

1,457 0,042 0,175 0,004

R-2

62,400 41,690 0,387 4,717

S-3

0,212 0,682 0,018 0,003

R-3

31,380 19,790 0,329 3,159

S-4

3,213 0,027 0,015 0,003

R-4

21,400 60,580 1,080 2,327

S-5

5,059 0,822 0,090 0,027

R-5

49,510 1920,000 1,247 2,954

S-6

2,729 29,370 0,139 0,042

R-6

65,190 36,830 1,003 3,748

S-7

1,138 0,307 0,125 0,036

R-7

34,560 1046,000 0,394 1,158

S-8

6,012 0,816 0,311 0,080

R-8

80,150 110900,000 1,487 34,210

S-9

3,561 19,330 0,379 0,023

R-9

8,535 612,300 0,947 0,300

S-10

1,574 0,456 0,013 0,026

R-10

65,580 753,000 0,795 4,332

S-11

3,409 6,220 0,295 0,040

R-11

0,003 27,120 0,000 0,091

S-12

1,701 6,813 0,124 0,014

R-12

182,200 7490,000 0,292 8,514

S-13

4,390 0,591 0,096 0,051

R-13

18,940 9,946 0,305 1,591

S-14

3,037 1,089 0,094 0,021

R-14

25,760 18,510 0,697 2,097

S-15

4,040 1,299 0,048 0,049

R-15

107,400 136,600 0,437 5,720

S-16

1,677 0,133 0,038 0,008

R-16

84,420 211,400 3,469 8,700

S-17

0,823 42,500 0,086 0,009

R-17

46,050 89,970 1,139 1,151

S-18

1,093 2,881 0,052 0,006

R-18

51,170 442,400 3,945 1,427

S-19

1,971 1,350 0,074 0,008

R-19

46,510 423,400 2,080 2,023

S-20

2,247 0,766 0,070 0,005

R-20

100,700 997,700 3,656 3,030

Average

2,553 5,863 0,124 0,023 54,819 6273,077 1,210 4,614

Strain

Susceptible

Strain

Resistant

Table S4. Expression ratios obtained for all efflux pumps encoding genes in susceptible and resistant strains.

Table S4. Expression ratios obtained for all eux pumps encoding genes in susceptible and resistant strains.