Characterization of clinical extensively drug-resistant Pseudomonas aeruginosa in the Hunan province of China
Li et al. Ann Clin Microbiol Antimicrob
Characterization of clinical extensively drug-resistant Pseudomonas aeruginosa in the Hunan province of China
Jun Li 0 2
Mingxiang Zou 0 2
Qingya Dou 1
Yongmei Hu 0 2
Haichen Wang 0 2
Qun Yan 0 2
Wen' en Liu 0 2
0 Department of Clinical Laboratory, Xiangya Hospital, Central South University , No. 87, Xiangya Road; Kaifu District, Changsha 410008, Hunan , China
1 Department of Infection Control Center, Xiangya Hospital, Central South University , Changsha 410008, Hunan , China
2 Department of Clinical Laboratory, Xiangya Hospital , Central South Univer- sity, No. 87, Xiangya Road; Kaifu District, Changsha 410008, Hunan , China
Background: Pseudomonas aeruginosa strains that are classed as extensively drug resistant (XDR-PA) are resistant to all antibiotics except for one or two classes and are frequently the cause of hard-to-treat infections worldwide. Our study aimed to characterize clinical XDR-PA isolates recovered during 2011-2012 at nine hospitals in the Hunan province of China. Methods: Thirty-seven non-repetitive XDR-PA strains from 37 patients were investigated for genes encoding antimicrobial resistance determinants, efflux pumps, outer membrane proteins, and movable genetic elements using polymerase chain reaction (PCR). The expression of genes encoding the efflux pump component MexA and the outer membrane protein OprD was measured using real-time PCR. In addition, clonal relatedness of these XDR-PA isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Results: Various genes encoding antimicrobial resistance determinants were found in all isolates. In particular, the blaTEM-1, blaCARB, armA, blaIMP-4, blaVIM-2, and rmtB, were found in 100, 37.8, 22, 22, 19 and 5 % of the isolates, respectively. Remarkably, two isolates coharbored blaIMP-4, blaVIM-2, and armA. In all 37 antibiotic-resistant strains, the relative expression of oprD was decreased while mexA was increased compared to the expression of these genes in antibioticsusceptible P. aeruginosa strains. All of the XDR-PA isolates harbored class I integrons as well as multiple other mobile genetic elements, such as tnpU, tnp513, tnpA (Tn21), and merA. A high genotypic diversity among the strains was detected by PFGE. Conclusions: Multiple antibiotic-resistance mechanisms contributed to the drug resistance of the XDR-PA isolates investigated in this study. Thus, the XDR-PA isolates in this area were not clonally related. Instead, multiple types of movable genetic elements were coharbored within each XDR-PA isolate, which may have aided the rapid development of these XDR-PA strains. This is the first report of XDR-PA strains that coharbor blaIMP-4, blaVIM-2, and armA.
Extensively drug-resistant Pseudomonas aeruginosa; Resistance mechanism; Movable genetic elements; Molecular epidemiology; PFGE
Pseudomonas aeruginosa, a rod-shaped, non-fermenting
gram-negative bacterium, causes nosocomial infections
that can lead to sepsis, pneumonia, endocarditis, and
urinary tract infections. The emergence of extensively
drug-resistant P. aeruginosa (XDR-PA) strains showing
resistant to all antimicrobial agents except for one or two
classes is becoming a major public health concern [
That was found by previous studies the mechanisms
of antibiotic resistance associated with clinical XDR-PA
isolates are complex [
]. The prevailing hypothesis is
that XDR-PA isolates acquire numerous drug-resistance
determinants through horizontal gene transfer that is
mediated by mobile genetic elements [
addition, genes encoding the antibiotic-inactivating enzymes
β-lactamases, aminoglycoside-modifying enzymes
(AMEs), and 16S rRNA methylases (16S-RMTases)
are frequently associated with antibiotic resistance in
XDR-PA strains. In recent studies, an over-expression of
drug-efflux pumps and diminished expression of outer
membrane proteins are suggested to play a role in drug
Although the prevalence of drug-resistance
determinants in XDR-PA strains isolated in other countries has
been determined [
], few studies have investigated
the resistance mechanisms and the epidemiological
profiles of clinical XDR-PA isolates found in China. Before
2011, XDR-PA strains were rarely found in China;
thereafter, a gradual emergence has occurred in some
hospitals. Therefore, the purpose of this study was to analyze
the resistance mechanisms and molecular epidemiology
of clinical XDR-PA strains isolated earlier in our region
recovered from 2011 to 2012.
Thirty-seven out of 482 (7.7 %) P. aeruginosa isolates
that were screened were XDR-PA strains displaying
resistance to all antimicrobial agents except colistin.
The P. aeruginosa strains were isolated from September
1, 2011 to June 30, 2012 from nine of fifteen different
hospitals in the Hunan province of China. Drug
susceptibility of XDR-PA isolates was determined by the
Kirby-Bauer (K-B) disk diffusion method and two
quality control strains (Escherichia coli ATCC25922 and P.
aeruginosa ATCC27853) were included in the analyses.
The results were analyzed and interpreted according
to the guidelines of Clinical and Laboratory Standards
Institute (CLSI) [
]. To prevent analysis of redundant
strains, only the first strain was collected when
duplicate strains were from the same patient.
Phenotypic tests for carbapenemase production
The modified Hodge test (MHT) was used to detect the
production of carbapenemase using an imipenem disc
(10 μg) as described by CLSI [
]. A combined-disc test
was carried out to detect the production of
metalloβ-lactamase (MBL). Two discs [One disc contained
imipenem (10 μg) and 5 μL of 0.5 M EDTA (Sigma
Chemicals), and the other disc contained only
imipenem (10 μg)] were placed 20 mm apart on a Mueller–
Hinton agar plate inoculated with each test strain. A
strain was considered positive for metallo-β-lactamase
production when the zone diameter around the
imipenem-EDTA disc was more than 4 mm of the
imipenemonly disc [
Detection of genes encoding antimicrobial resistance determinants and genes associated with movable genetic elements
The DNA templates used in polymerase chain
reactions (PCR) to amplify genes encoding antimicrobial
resistance determinants were obtained as follows:
bacterial suspensions were incubated for 10 min at 95 °C
followed by centrifugation at 10,000×g for 10 min to
remove cellular debris. Genes coding for carbapenem
β-lactamases (blaKPC, blaSME, blaGES, blaIMI/blaNMC,
blaNDM-1, blaVIM-2, blaIMP-4, blaSIM-1, blaGIM, blaSPM,
blaOXA-23, and blaOXA-51), extended-spectrum
β-lactamases (ESBLs) (blaTEM-1, blaCTX-M, blaSHV,
blaOXA-1, blaOXA-2, blaOXA-10, blaVEB, and blaPER), and
AmpC β-lactamases (blaMOX, blaFOX, blaDHA, blaCIT,
and blaEBC) were performed by PCR with previously
described primers [
]. In addition, the isolates
were screened by PCR for AME genes (aac(3)-IIa and
], 16S-RMTases (armA, npmA, rmtA,
rmtB, rmtC, rmtD, and rmtE) [
], a drug-efflux pump
component (mexA), and an outer membrane protein
(oprD). The PCR primers used to screen for these
genes are listed in Table 1. To detect genes associated
with the movable genetic elements, intI, traA, traF,
trbC, tnp513, ISpa7, ISEcp1, tnpU, tnpA (Tn21), tnsA,
and merA, PCR was performed with primers shown in
Table 1. All amplified DNA fragments were sequenced
and then analyzed using the BLAST program (http://
Quantifying expression of mexA and oprD
The expression of mexA and oprD in the XDR-PA
isolates was determined by real-time PCR (RT-PCR).
The experimental group consisted of the 37 XDR-PA
isolates and the control group was made up of 31 P.
aeruginosa isolates that were collected at the same
time as the XDR-PA strains but were shown to be
sensitive to all antimicrobial agents tested. The
primers used for quantifying mexA and oprD expression
are listed in Table 1. The RT-PCR reactions were
carried out by a QuantiFast SYBR Green RT-PCR Kit
from Qiagen and a Real-Time PCR System of
LightCycler 2.0 from Roche, Burgess Hill, UK, according
to the manufacturer protocols. All reactions were
repeated three times using 10 ng of RNA template
that was prepared using the RNeasy Mini Kit from
Qiagen, Crawley, UK and treated with Dnase. Gene
expression was normalized relative to that of the
16S rRNA gene using the 2−△△CT method [
expression of 16S rRNA gene was determined by the
primers shown in Table 1.
Pulsed‑field gel electrophoresis (PFGE)
Clonal relatedness of the XDR-PA isolates was analyzed
by PFGE. Preparation of genomic DNA was done in
agarose blocks. The DNA was then digested by the
restriction enzyme XbaI from Promega, USA, followed by
embedding into 1 % PFGE agarose gel. PFGE was
performed for 24 h using the GenePath System from
BioRad with the follow conditions: 5.5 V/cm, 12 °C, 120°,
and a switch time from 4 to 40 s. The molecular size
marker, Salmonella enterica strain H9812, was obtained
from the respiratory laboratory of infectious diseases,
CDC, China. The gel was stained for 30 min with
ethidium bromide and the gel image was documented
using the gel documentation system Gel Doc 2000 from
Bio-Rad. Finally, analyses of the results were performed
by the BioNumerics software platform (Applied Math,
Sint-Maten-Latem, Belgium) and visual inspection with
the criteria of relatedness proposed by Tenover et al.
WHONET software (version 5.4, WHO) was used to
analyze the patient demographic information and the
antibiotic resistance data using the 2014 CLSI criteria for
breakpoints for P. aeruginosa. Comparisons of different
groups were analyzed by a two-sided Chi square (χ2) test
with the SPSS13.0 software (SPSS Inc., USA). A P < 0.05
was regarded as statistically significant.
Phenotypic screening and resistance determinants
Of the 37 XDR-PA isolates, 13 (35.1 %) isolates were
positive for MTH and MBL, while all other isolates were
negative (Fig. 1).
Diverse types of genes encoding antibiotic-inactivating
enzymes were detected. Specifically, the genes blaTEM-1,
blaCARB, armA, blaIMP-4, blaVIM-2, and rmtB were found
in 100, 37.8, 22, 22, 19 and 5 % of the XDR-PA isolates,
respectively. The ESBL gene blaTEM-1 was found in all
isolates, while the other ESBL genes that were tested were
not found in any of the isolates. Among the 37 XDR-PA
isolates, 13 (35.1 %) isolates were positive for MBLs genes,
8 contained blaIMP-4, and 7 contained blaVIM-2. Two types
of 16S-RMTases genes were detected. Eight isolates
contained armA, while two isolates contained rmtB. Eight
isolates coharbored blaIMP-4 and armA and two isolates
coharbored blaIMP-4, blaVIM-2, and armA. No isolates
harbored genes encoding AmpC β-lactamases or AMEs.
The efflux-pump gene mexA and the outer membrane
protein gene oprD were detected in all 37 XDR-PA
isolates. However, no mutations were detected in any of the
oprD genes. Figure 1 lists the antibiotic resistance genes
detected in each isolate.
Expression of mexA and oprD
The expression of mexA was significantly higher in the 37
XDR-PA isolates than in the control group consisting of 31
antibiotic-sensitive isolates (1.95 ± 0.48 and 0.70 ± 0.13,
P = 0.018, respectively), while expression of oprD in the
XDRPA strains was significantly lower than the control group
(3.18 ± 0.60 and 0.94 ± 0.08; P = 0.002, respectively; Fig. 2).
Distribution of genes associated with movable genetic elements
Of the movable genetic element genes tested, intI, merA,
tnp513, tnpA (Tn21), and tnpU were detected in 100, 100,
81, 49, and 16 % of the isolates, respectively. Moreover,
most of the 37 XDR-PA isolates coharbored three or
more genes associated with movable genetic elements.
For instance, isolates xy36 coharbored five types of
movable genetic element genes (Fig. 1).
Clonal relatedness of the XDR‑PA isolates
Using PFGE, the 37 XDR-PA isolates were divided into
21 PFGE types (Fig. 1). The main PFGE type found was
A (five strains), including three strains from Xiangya
Hospital and two strains from the Third Xiangya Hospital. In
addition, an A1 subtype was found in both hospitals. The
B- and C-type isolates were found in the Xiangya
Hospital, while the D-type isolates were found in the Third
Hospital of Changsha as well as Chinese Medicine Hospital of
Liuyang. The E-type strains were isolated from the
Second People’s Hospital of Hunan province and the Third
Xiangya Hospital, and an E1 subtype was found in both
hospitals. The F-type strains were isolated from the
People’s Hospital of Liuyang; the G-type strains were isolated
from the Third Xiangya Hospital; and the H-type isolates
were found at Xiangya Hospital and Xiangtan Central
Hospital. The remaining 13 types, from I to U, were
collected from the Xiangya Hospital (eight strains), the
People’s Hospital of Chenzhou (one strain), the First Hospital
of Changsha (one strain), the Chinese Medicine Hospital
of Liuyang (one strain), the Third Xiangya Hospital (one
strain), and the Third Hospital of Changsha (one strain).
Pseudomonas aeruginosa is considered one of the
primary causes of hospital-acquired infections. An increase
in the prevalence of clinical XDR-PA isolates
correlates with a rise in mortality and morbidity rates.
Consequently, XDR-PA strains pose a considerable threat
to public health worldwide. In our study, 37 out of 482
(7.7 %) P. aeruginosa strains were found to be XDR-PA
strains. The XDR-PA strains were isolated from nine of
fifteen teaching hospitals detected in our region,
suggesting that the occurrence of XDR-PA isolates in our region
is low. To aid the prevention of the spread of XDR-PA
strains, we have analyzed the resistance mechanisms and
the molecular epidemiology of the XDR-PA strains.
Carbapenems are one of the most effective drugs
against severe infections caused by gram-negative bacilli.
Unfortunately, pathogens displaying resistance to
carbapenems are increasing due to the following three main
causes: production of carbapenemases, the
over-expression of efflux pumps, and the diminished expression of
the outer membrane porin OprD [
]. In P. aeruginosa,
production of carbapenemases, especially MBLs, is an
important antibiotic resistance mechanisms [
]. In this
study, 13 (35.1 %) strains produced the metallo-enzymes
IMP-4 and VIM-2, indicating that production of
carbapenemases played role in carbapenem-resistant in the
XDR-PA isolates. Notably, the prevalence of
carbapenemases in the 37 XDR-PA isolates in our study were higher
than that of other countries in the world [
from other regions in China . One isolate that
coharbored two MBL genes was reported in previous
]. This observation led to the emergence of a new
drug-resistant model for P. aeruginosa. In our study, two
isolates (xy35 and xy36) coharbored blaIMP-4 and blaVIM-2.
To our knowledge, this is the first report of the
co-existence of blaIMP-4 and blaVIM-2 in a P. aeruginosa strain.
Drug-efflux pumps have been correlated with
bacterial resistance since the 1980s [
]. The first efflux pump
found in P. aeruginosa is the MexAB-OprM pump, which
has a broad range of substrates, including carbapenems,
quinolones, aminoglycosides, tetracyclines, and
macrolides. Therefore, the presence of this efflux pump in
bacteria may lead to multi-drug resistance. In our study,
the mexA gene, encoding a component of MexAB-OprM
efflux pump, was found in all 37 XDR-PA isolates.
Furthermore, a significant over-expression of mexA in the
XDR-PA isolates was seen compared with the mexA
levels in antibiotic-susceptible strains. Thus,
over-expression of an efflux pump, especially MexAB-OprM, may
play a key role in the antibiotic resistance of these
Decreased expression of OprD can significantly reduce
the susceptibility of P. aeruginosa to carbapenems.
Previous studies showed that decreased expression of oprD is
primarily due to mutations in the OprD-encoding genes
]. In our study, the expression of oprD in the 37
XDRPA isolates was significantly lower than that in
susceptible strains confirming that a decreased level of this porin
plays a key role in carbapenem-resistance. However, no
mutations or deletions were detected for the oprD gene
in the 37 XDR-PA isolates. This finding was not
consistent with previous studies [
], suggesting that
decreased expression of oprD in the 37 XDR-PA isolates
might be due to regulation by small bioactive molecules,
amino acids, or efflux pump expression . Therefore,
in-depth studies on the role of outer membrane proteins
in bacterial drug resistance are needed in the future.
Production of AMEs and 16S-RMTases is the main
cause of bacterial resistance to aminoglycoside agents
]. In contrast to these reports, none of the 37
XDRPA isolates, which were resistant to aminoglycoside
antibiotics, harbored genes encoding AMEs and only 27.0 %
(n = 10) harbored either armA or rmtB 16S-RMTases
genes. These data suggest that 16S-RMTases play an
important role in aminoglycoside resistance in the
XDRPA isolates but also that some of these isolates may
utilize an alternative, unknown resistance mechanism, such
as new drug efflux pumps, new 16S-RMTases, or
mutational activation of the AmgRS two-component system
Movable genetic elements, including integrons,
plasmids, transposons, and insertion sequences, play a key
role in the horizontal transfer of resistance genes [
this study, most of the XDR-PA isolates coharbored three
or more genes associated with movable genetic elements.
The most frequently detected genes were intI, merA
tnp513, tnpA (Tn21), and tnpU, and their distribution
percentage among the XDR-PA isolates was 100, 100,
81, 49, and 16 %, respectively. Furthermore, two isolates,
cs15 and xy36, coharbored 5 types of genes associated
with movable genetic elements.
The XDR-PA strains previously isolated in other
countries were shown to be clonally related [
]. In contrast, the
XDR-PA strains isolated from hospitals within the Hunan
province of China were shown to be genetically diverse.
The 37 XDR-PA isolates were divided into 21 different
PFGE types and no one type predominated amongst the
isolates suggesting that none of the XDR-PA isolates are
considered to be an epidemic clone. Interestingly, the
A-type, D-type, E-type, and H-type were detected in two
different hospitals and the B-type, C-type, and F-type
were found in two different wards of the same hospital
during the same time period. Although prevalence of the
XDR-PA isolates in this area was sporadic, dissemination
of the same isolate was detected inter- and intra-hospital.
The high heterogeneity of the PFGE types suggests that
the mobile genetic elements may play a role in the
emergence of clinical XDR-PA strains.
In conclusion, this study shows that the extreme
antibiotic resistance of the clinical XDR-PA isolates is due to
strains coharboring multiple antibiotic resistance genes,
over-expressing drug-efflux pumps, and decreasing
expression of oprD, which encodes an outer membrane
porin. Moreover, this study has identified for the first
time a P. aeruginosa isolate that coharbors the blaIMP-4,
blaVIM-2, and armA antibiotic resistance genes. Although
the XDR-PA isolates were not clonally related, the strains
were shown to carry multiple genes encoding different
types of movable genetic elements, which may aid the
rapid development of XDR-PA isolates.
MZ contributed to the conception of the study. JL contributed significantly to
analysis and manuscript preparation; QD and YH performed the data analyses
and wrote the manuscript; HW, QY and WL helped perform the analysis with
constructive discussions. All authors read and approved the final manuscript.
We are thankful to the staff in the Laboratory of Xiangya Hospital of Central
South University who helped us in the collection and identification of these
XDR-PA isolates. In particular, we thank Xin Xia, a teacher at the Center for
Disease Control (CDC) in the Hunan Province of China, for helping us with the
The authors declare that they have no competing interests.
All experiments involving animals were performed according to the ethical
standards of the institute. The experimental protocols were signed and
approved by the ethics committee for each of the nine teaching hospitals in
This work was supported by the Hunan Development and Reform Investment
 No. 1493 from the Development and Reform Commission of the Hunan
Province, a Grant (14JJ7003) from the Natural Science Foundation of the
Hunan Province, and the Hunan Development and Reform Investment 
No. 658 from Development and Reform Commission of the Hunan Province.
A signed, informed consent was given by all participants included in the study.
1. Ciofi Degli Atti M , Bernaschi P , Carletti M , Luzzi I , Garcia-Fernandez A , Bertaina A , Sisto A , Locatelli F , Raponi M. An outbreak of extremely drugresistant Pseudomonas aeruginosa in a tertiary care pediatric hospital in Italy . BMC Infect Dis . 2014 ; 14 : 494 . doi: 10 .1186/ 1471 -2334-14-494.
2. Fonseca EL , Freitas Fdos S , Vicente AC . The colistin-only-sensitive Brazilian Pseudomonas aeruginosa clone SP (sequence type 277) is spread worldwide . Antimicrob Agents Chemother . 2010 ; 54 ( 6 ): 2743 . doi: 10 .1128/ aac.00012- 10 .
3. Jin JS , Kwon KT , Moon DC , Lee JC . Emergence of 16S rRNA methylase rmtA in colistin-only-sensitive Pseudomonas aeruginosa in South Korea . Int J Antimicrob Agents . 2009 ; 33 ( 5 ): 490 - 1 . doi: 10 .1016/j. ijantimicag. 2008 . 10 .024.
4. Lee YC , Ahn BJ , Jin JS , Kim JU , Lee SH , do Song Y , Lee WK , Lee JC . Molecular characterization of Pseudomonas aeruginosa isolates resistant to all antimicrobial agents, but susceptible to colistin , in Daegu, Korea. J Microbiology (Seoul, Korea). 2007 ; 45 ( 4 ): 358 - 63 .
5. Viedma E , Juan C , Acosta J , Zamorano L , Otero JR , Sanz F , Chaves F , Oliver A . Nosocomial spread of colistin-only-sensitive sequence type 235 Pseudomonas aeruginosa isolates producing the extended-spectrum betalactamases GES-1 and GES-5 in Spain . Antimicrob Agents Chemother . 2009 ; 53 ( 11 ): 4930 - 3 . doi: 10 .1128/aac.00900- 09 .
6. Livermore DM . Multiple mechanisms of antimicrobial resistance in, Pseudomonas aeruginosa: our worst nightmare? Clin Infect Dis . 2002 ; 34 ( 5 ): 634 - 40 . doi: 10 .1086/338782.
7. Poole K. Pseudomonas aeruginosa: resistance to the max . Front Microbiol . 2011 ; 2 : 65 . doi: 10 .3389/fmicb. 2011 . 00065 .
8. Beige F , Baseri Salehi M , Bahador N , Mobasherzadeh S. Plasmid mediated antibiotic resistance in isolated bacteria from burned patients . Jundishapur J Microbiol . 2015 ; 8 ( 1 ):e13567. doi: 10 .5812/jjm.13567.
9. Kiddee A , Henghiranyawong K , Yimsabai J , Tiloklurs M , Niumsup PR . Nosocomial spread of class 1 integron-carrying extensively drug-resistant Pseudomonas aeruginosa isolates in a Thai hospital . Int J Antimicrob Agents . 2013 ; 42 ( 4 ): 301 - 6 . doi: 10 .1016/j.ijantimicag. 2013 . 05 .009.
10. Stalder T , Barraud O , Casellas M , Dagot C , Ploy MC . Integron involvement in environmental spread of antibiotic resistance . Front Microbiol . 2012 ; 3 : 119 . doi: 10 .3389/fmicb. 2012 . 00119 .
11. Aghazadeh M , Hojabri Z , Mahdian R , Nahaei MR , Rahmati M , Hojabri T , Pirzadeh T , Pajand O . Role of efflux pumps: MexAB-OprM and MexXY(- OprA) , AmpC cephalosporinase and OprD porin in non-metallo-betalactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients . Infect Genet Evol . 2014 ; 24 : 187 - 92 . doi: 10 .1016/j. meegid. 2014 . 03 .018.
12. Lee JY , KO KS . OprD mutations and inactivation, expression of efflux pumps and AmpC, and metallo-beta-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from South Korea . Int J Antimicrob Agents . 2012 ; 40 ( 2 ): 168 - 72 . doi: 10 .1016/j.ijantimicag. 2012 . 04 .004.
13. Quale J , Bratu S , Gupta J , Landman D. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates . Antimicrob Agents Chemother . 2006 ; 50 ( 5 ): 1633 - 41 . doi: 10 .1128/AAC.50.5. 1633 - 1641 . 2006 .
14. Xavier DE , Picao RC , Girardello R , Fehlberg LC , Gales AC . Efflux pumps expression and its association with porin down-regulation and beta-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil . BMC Microbiol . 2010 ; 10 : 217 . doi: 10 .1186/ 1471 -2180-10-217.
15. Castanheira M , Deshpande LM , Costello A , Davies TA , Jones RN . Epidemiology and carbapenem resistance mechanisms of carbapenemnon-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries . J Antimicrob Chemother . 2014 ; 69 ( 7 ): 1804 - 14 . doi: 10 .1093/jac/dku048.
16. Polotto M , Casella T , de Lucca Oliveira MG , Rubio FG , Nogueira ML , de Almeida MT , Nogueira MC . Detection of P. aeruginosa harboring bla CTX-M-2, bla GES-1 and bla GES-5, bla IMP-1 and bla SPM-1 causing infections in Brazilian tertiary-care hospital . BMC Infect Dis . 2012 ; 12 : 176 . doi: 10 .1186/ 1471 -2334-12-176.
17. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing, 24th informational supplement . M100-S24 . Wayne: Clinical and Laboratory Standards Institute; 2014 .
18. Fang H , Ataker F , Hedin G , Dornbusch K. Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006 . J Clin Microbiol . 2008 ; 46 ( 2 ): 707 - 12 . doi: 10 .1128/ jcm.01943- 07 .
19. Perez-Perez FJ , Hanson ND . Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR . J Clin Microbiol . 2002 ; 40 ( 6 ): 2153 - 62 .
20. Queenan AM , Bush K. Carbapenemases: the versatile beta-lactamases . Clin Microbiol Rev . 2007 ; 20 ( 3 ): 440 - 58 . doi: 10 .1128/CMR.00001- 07 (table of contents).
21. Soleimani N , Aganj M , Ali L , Shokoohizadeh L , Sakinc T. Frequency distribution of genes encoding aminoglycoside modifying enzymes in uropathogenic E. coli isolated from Iranian hospital . BMC Res Notes . 2014 ; 7 : 842 . doi: 10 .1186/ 1756 -0500-7-842.
22. Doi Y , Arakawa Y. Ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides . Clin Infect Dis . 2007 ; 45 ( 1 ): 88 - 94 . doi: 10 .1086/518605.
23. Arabestani MR , Rajabpour M , Yousefi Mashouf R , Alikhani MY , Mousavi SM . Expression of efflux pump MexAB-OprM and OprD of Pseudomonas aeruginosa strains isolated from clinical samples using qRT-PCR . Arch Iranian Med . 2015 ; 18 ( 2 ): 102 - 8 . doi:015182/AIM.008.
24. Tenover FC , Arbeit RD , Goering RV , Mickelsen PA , Murray BE , Persing DH , Swaminathan B . Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing . J Clin Microbiol . 1995 ; 33 ( 9 ): 2233 - 9 .
25. Hong DJ , Bae IK , Jang IH , Jeong SH , Kang HK , Lee K. Epidemiology and Characteristics of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa . Infect Chemother . 2015 ; 47 ( 2 ): 81 - 97 . doi: 10 .3947/ic. 2015 . 47 .2.81.
26. Kalantar E , Torabi V , Salimizand H , Soheili F , Beiranvand S , Soltan Dallal MM. First survey of metallo-β-lactamase producers in clinical isolates of Pseudomonas aeruginosa from a referral burn center in Kurdistan Province . Jundishapur J Nat Pharm Prod . 2012 ; 7 ( 1 ): 23 - 6 . doi: 10 .17795/ jjnpp-3546.
27. Hong JS , Kim JO , Lee H , Bae IK , Jeong SH , Lee K. Characteristics of metallo-β-lactamase-producing Pseudomonas aeruginosa in Korea . Infect Chemother . 2015 ; 47 ( 1 ): 33 - 40 . doi: 10 .3947/ic. 2015 . 47 .1.33.
28. Zhang X , Gu B , Mei Y , Wen Y , Xia W. Increasing resistance rate to carbapenem among blood culture isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa in a university-affiliated hospital in China, 2004 - 2011 . J Antibiot (Tokyo). 2015 ; 68 ( 2 ): 115 - 20 . doi: 10 .1038/ja. 2014 . 119 .
29. Toval F , Guzmán-Marte A , Madriz V , Somogyi T , Rodríguez C , García F. Predominance of carbapenem-resistant Pseudomonas aeruginosa isolates carrying blaIMP and blaVIM metallo-β-lactamases in a major hospital in Costa Rica . J Med Microbiol . 2015 ; 64 (Pt 1): 37 - 43 . doi: 10 .1099/ jmm.0. 081802 - 0 .
30. George AM , Levy SB . Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a nonplasmid-determined efflux of tetracycline . J Bacteriol . 1983 ; 155 ( 2 ): 531 - 40 .
31. Shu JC , Chia JH , Siu LK , Kuo AJ , Huang SH , Su LH , Wu TL . Interplay between mutational and horizontally acquired resistance mechanisms and its association with carbapenem resistance amongst extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) . Int J Antimicrob Agents . 2012 ; 39 ( 3 ): 217 - 22 . doi: 10 .1016/j.ijantimicag. 2011 . 09 .023.
32. Shen J , Pan Y , Fang Y. Role of the outer membrane protein OprD2 in carbapenem-resistance mechanisms of Pseudomonas aeruginosa . PLoS One . 2015 ; 10 ( 10 ):e0139995. doi: 10 .1371/journal.pone. 0139995 .
33. Rojo-Bezares B , Estepa V , Cebollada R , de Toro M , Somalo S , Seral C , Castillo FJ , Torres C , Sáenz Y. Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons . Int J Med Microbiol . 2014 ; 304 ( 3-4 ): 405 - 14 . doi: 10 .1016/j.ijmm. 2014 . 01 .001.
34. Li H , Luo YF , Williams BJ , Blackwell TS , Xie CM . Structure and function of OprD protein in Pseudomonas aeruginosa: from antibiotic resistance to novel therapies . Int J Med Microbiol . 2012 ; 302 ( 2 ): 63 - 8 . doi: 10 .1016/j. ijmm. 2011 . 10 .001.
35. Ashenafi M , Ammosova T , Nekhai S , Byrnes WM . Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id, a streptomycin-inactivating enzyme . Mol Cell Biochem . 2014 ; 387 ( 1-2 ): 207 - 16 . doi: 10 .1007/s11010-013-1886-1.
36. Galani I , Souli M , Panagea T , Poulakou G , Kanellakopoulou K , Giamarellou H . Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a Greek university hospital . Clin Microbiol Infect . 2012 ; 18 ( 3 ): E52 - 4 . doi: 10 .1111/j.1469- 0691 . 2011 . 03738 .x.
37. Lau CH , Fraud S , Jones M , Peterson SN , Poole K. Mutational activation of the AmgRS two-component system in aminoglycoside-resistant Pseudomonas aeruginosa . Antimicrob Agents Chemother . 2013 ; 57 ( 5 ): 2243 - 51 . doi: 10 .1128/AAC.00170- 13 .
38. Ibrahim ME , Magzoub MA , Bilal NE , Hamid ME . Distribution of Class I integrons and their effect on the prevalence of multi-drug resistant Escherichia coli clinical isolates from Sudan . Saudi Med J. 2013 ; 34 ( 3 ): 240 - 7 .