Molecular Characterization of Extended-Spectrum Beta Lactamase- and Carbapenemase-Producing Pseudomonas aeruginosa Strains from a Malaysian Tertiary Hospital

Abstract

Pseudomonas aeruginosa infections account for high morbidity and mortality rates worldwide. Increasing resistance toward β-lactams, especially carbapenems, poses a serious therapeutic challenge. However, the multilocus sequence typing (MLST) of extended-spectrum beta lactamase (ESBL)- and carbapenemase-producing clinical P. aeruginosa has not been reported in Malaysia. This study aimed to determine the antibiotic susceptibility profiles, resistance genes, pulsotypes, and sequence types (STs) of clinical P. aeruginosa from a Malaysian tertiary hospital. These characteristics were analyzed by disk diffusion, minimum inhibitory concentration, polymerase chain reaction, pulsed-field gel electrophoresis (PFGE), and MLST for 199 nonreplicate clinical strains. The susceptibility of the strains toward the carbapenems and piperacillin–tazobactam was the lowest (≤90%), while ≥90% of the strains remained susceptible to all other classes of antimicrobial agents tested. The multidrug-resistant strains displayed high level resistance to cephalosporins (48 to ≥256 mg/L) and carbapenems (4–32 mg/L). Eleven strains harbored class 1 integrons containing bla_GES-13, bla_VIM-2, bla_VIM-6, bla_OXA-10, aacA(6′)-Ib, aacA(6′)-II, aadA6, and gcuD gene cassettes. Extra-integron genes, bla_GES-20, bla_IMP-4, bla_VIM-2, and bla_VIM-11, were also found. Overall, the maximum likelihood tree showed concordance in the clustering of strains having the same STs and PFGE clusters. ST708 was the predominant antibiotic-susceptible clone detected from the neonatal intensive care unit. The STs 235, 809, and 1076 clonal clusters consisted of multidrug resistant strains. ST235 is a recognized international high-risk clone. This is the first report of bla_GES-13 and bla_GES-20 ESBL-encoding gene variants and novel STs (STs 2329, 2335, 2337, 2338, 2340, and 2341) of P. aeruginosa in Malaysia.

Introduction

Pseudomonas aeruginosa, a ubiquitous aerobic gram-negative bacillus, is inherently resistant to many antimicrobial agents and tolerant to disinfectants.¹ It has been implicated in severe nosocomial infections such as bacteremia, pneumonia, postsurgical infections, and urinary tract infections among immunocompromised patients.^2,3 The extraordinary ability of the pathogen to acquire resistance to multiple classes of antimicrobial agents through mobile genetic elements or chromosomal mutations poses a serious therapeutic challenge in treating both community-acquired and nosocomial acquired infections.^4,5 Infections associated with the drug-resistant P. aeruginosa significantly increases surgical intervention events, duration of hospitalization, treatment costs, and mortality and morbidity rates, thus adversely affecting overall patient outcomes.²

In the United States, an estimated 51,000 P. aeruginosa infections were reported every year and 13% of these were caused by multidrug-resistant (MDRPA) strains.³ MDRPA are resistant to ≥1 antimicrobial agent from ≥3 antimicrobial classes according to Magiorakos et al.⁶ and classified as a serious threat by the U.S. CDC.³ In Malaysia, the antimicrobial resistance rates and emergence of MDRPA-associated clinical infections were previously reported.^7–10 Raja and Singh⁷ reported the resistance rates of P. aeruginosa to ceftazidime (CAZ), cefepime (FEP), imipenem (IPM), and meropenem (MEM) as 10.9%, 38.9%, 9.9%, and 36.8%, respectively, while Pathmanathan et al.⁸ reported 19.6%, 19.6%, 20.6%, and 22.7% resistance to the same antimicrobials. Incidences of MDRPA in Malaysian were varied at 5.7% in 2006, 69% in 2008, and 19.6% in 2009 based on studies by Raja and Singh, Pathmanathan et al., and Lim et al., respectively.^7–9 Since then, there has been no update on the MDRPA incidences in Malaysia.

Genotypic characterization of Malaysian P. aeruginosa strains has also been previously reported. Lim et al.⁹ showed that the clinical strains from six public hospitals in six different states in Malaysia were diverse and heterogeneous. Besides that, high levels of broad-spectrum antimicrobial resistance conferred by metallo-beta-lactamase (MBL)-encoding genes such as bla_IMP-7, bla_IMP-4, bla_VIM-2, and bla_VIM-11, as well as gene cassette-bearing class 1 and class 2 integrons were detected among Malaysian strains.^9–12 However, the multilocus sequence typing (MLST) of extended-spectrum beta lactamase (ESBL)- and carbapenemase-producing clinical P. aeruginosa has not been reported in Malaysia.

Therefore, this study aimed to determine the antimicrobial susceptibility profiles, the presence of (ESBL)- and carbapenemase-encoding genes, and the genetic relatedness of P. aeruginosa strains from a Malaysian tertiary hospital by pulsed-field gel electrophoresis (PFGE) and MLST. The data generated would be useful to elucidate the genotypes of Malaysian drug-resistant P. aeruginosa strains in relationship to previously reported international drug-resistant sequence types (STs).

Materials and Methods

Strains background and identification

We conducted a retrospective study of clinical P. aeruginosa collected in the 562-bedded tertiary hospital in Selangor, Malaysia. From April to November 2014, 199 consecutive and nonreplicate P. aeruginosa strains were isolated at the microbiology laboratory of the hospital. The strains were identified by basic biochemical tests and API20NE (bioMérieux, Marcy l'Etoile, France). We reconfirmed the identities of the strains by polymerase chain reaction (PCR) using PA SS-F and PA SS-R primers according to Spilker et al.¹³ P. aeruginosa ATCC 27853 was used as the positive control. In addition, the information of the strain background, such as specimen type, site of infection, locations or wards in the hospital, and the dates of culture and sensitivity testing, was recorded.

Antibiotic susceptibility testing

The antibiotic susceptibility of the strains was tested by disk diffusion and minimum inhibitory concentration (MIC), and the results were interpreted according to CLSI guidelines.¹⁴ The antibiotics used for the disk diffusion method were amikacin (AMK), CAZ, FEP, ciprofloxacin (CIP), gentamicin (GEN), IPM, MEM, polymyxin B (PMB), and piperacillin/tazobactam (TZP; Oxoid). Further MIC testing was done on strains that were intermediate or resistant to CAZ, FEP, IPM, and MEM using E-test strips (bioMérieux, Marcy l'Etoile, France). P. aeruginosa ATCC 27853 and Escherichia coli ATCC 35218 were used as the positive and negative controls, respectively. We used the International Expert Definitions for Acquired Drug Resistance to categorize the MDRPA strains.⁶

Detection of resistance genes, class 1 and class 2 integrons

The detection of selected ESBL genes (bla_TEM, bla_SHV, bla_{OXA-1 like}, and bla_CTXM for phylogenetic group 1, 2, and 9, bla_CTXM-8/-25, bla_VEB, bla_PER, and bla_GES) and carbapenemase genes (bla_IMP, bla_VIM, bla_SPM, bla_KPC, bla_NDM, bla_AIM, bla_GIM, and bla_SIM) was performed on all strains using multiplex PCR with primers and conditions as previously described.^15,16 The amplicons were purified and submitted to a commercial company for sequencing to validate the results. The resulting nucleotide sequences were aligned to reference sequences in the NCBI BLAST-n database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The positive control strains, Klebsiella pneumoniae strain KP C06 and K. pneumoniae ATCC BAA-1705, were used as reference for bla_TEM, bla_SHV, and bla_CTXM-1, and bla_KPC, respectively. PCR amplicons for bla_GES, bla_IMP, and bla_VIM were sequenced to validate the products, and the strains with these confirmed genes were used as positive controls in subsequent multiplex PCR tests. Ultrapure H₂O (ddH₂O) was used as the negative control for all multiplex PCR runs. Positive controls were unavailable for bla_{OXA-1 like}, bla_CTXM phylogenetic group 2 and 9, bla_CTXM-8/-25, bla_VEB, bla_PER, bla_SPM, bla_NDM, bla_AIM, bla_GIM, and bla_SIM. Therefore, negative results were treated with caution.

Strains that were resistant to any class of antimicrobial agents were subjected to PCR detection of class 1 and 2 integron-encoded integrases, intI1 and intI2, according to established protocols.¹⁷ The 5′CS/3′CS and attI2-orfX region primer pairs were used to amplify the integron variable region of the class 1 and class 2 integrons, respectively.¹⁷ Subsequent sequencing of the integron variable region to confirm the presence and content of gene cassette insertions was also performed.

Pulsed-field gel electrophoresis

PFGE was conducted according to established protocols⁹ with minor modifications using CHEF MAPPER (Bio-Rad Laboratories, Hercules, CA). Briefly, SpeI-restricted DNA plugs were electrophoresed for 23 hours with pulse times of 1 and 40 seconds, at 6 V/cm. Xba1-digested Salmonella serotype Braenderup H9812 was used as the DNA size marker.¹⁸ For strains that could not be typed because of Tris-dependent DNA degradation, the electrophoresis was repeated with 1× HEPES as the electrophoresis buffer.¹⁹ The gels were visualized in Gel Doc XR (Bio-Rad Laboratories) after staining in GelRed (Biotium). The similarity indices of the PFGE fingerprints were calculated with Dice coefficient at 1.0% optimization and 1.5% tolerance, while clustering was done using unweighted pair group method with arithmetic mean (UPGMA) algorithm in the BioNumerics 7 software (Applied Maths; bioMérieux, Schaerbeek, Belgium).

Multilocus sequence typing

Only 29 representative strains were selected for MLST. The selection was based on the PFGE analyses: (1) indistinguishable pulsotypes (n = 10), (2) pulsotypes with ≥85% genetic similarity (n = 6), (3) pulsotypes with ≤85% genetic similarity (n = 4), and (4) the MDRPA strains (n = 9). The primers and cycling conditions used were obtained from the MLST webpage (http://pubmlst.org/paeruginosa) and published procedures.²⁰

eBURST v3 (http://eburst.mlst.net) analysis using the most stringent definition, where the STs were identical or shared at least six or seven alleles, was used to detect clonal complexes or BURST groups (BGs) among the STs in this study (n = 29) and the P. aeruginosa PubMLST database (n = 5,265, http://pubmlst.org/paeruginosa).²¹ The STs were then classified as BG founders, single-locus variants (SLVs), double-locus variants, or singletons.²²

The nucleotide sequences of the STs were aligned to reference sequences from the P. aeruginosa PubMLST database using MUSCLE (MEGA7)²³ and concatenated with Sequence Matrix 1.8. To estimate the distances between the sequences, the maximum likelihood (ML) method based on the Tamura-Nei model was utilized.²⁴ The tree topology was determined by neighbor-joining method and the final phylogenetic tree was rooted using Acinetobacter spp. as the outgroup.²⁵

The BGs and ML tree were assessed using 2,000 bootstrap replicates and bootstrap percentages ≥70% were considered to be reliable.²⁶

Results

Strain distribution

All the 199 P. aeruginosa strains were confirmed as they harbored the 16S recombinant DNA (rDNA) variable regions 2 (V2) and 8 (V8) analyzed by PCR. These strains were isolated from various sources obtained from different locations in the hospital (Table 1). The top three sources were sputum (25.6%), wound swab (16.6%), and tracheal aspirate (15.6%); the majority of which were isolated from patients in the medical wards (30.2%), surgical wards (16.6%), and the intensive care unit (ICU; 13.6%). Sixty-one percent (n = 122) and 39% (n = 77) of the strains were from male and female patients, respectively.

Table 1.

Specimen Types and Sources of Clinical Pseudomonas aeruginosa Strains Studied

	Specimen types
Locations/wards	Abscess/Pus	Blood	Bone	BAL/NPA	Catheter tip	Eyes/cornea	Sputum	Throat swab	Tissue	Tracheal aspirate	Urine	Wound swab	Total
Intensive care units		3			1		4			18		1	27
Hematology	3	9		1			1		2	2	1	2	21
Emergency		1				1							2
Ophthalmology						1							1
Obstetrics and gynecology	1	2					1				1	3	8
Otorhinolaryngology	2												2
General medicine	4	3					37	1		3	8	4	60
Pediatrics	1	11		1		7			1	4		1	26
Orthopedics	2		1				1		3		2	10	19
General surgery	3					1	7		4	4	2	12	33
No. of specimens	16	29	1	2	1	10	51	1	10	31	14	33	199

BAL/NPA, bronchoalveolar lavage/nasopharyngeal aspirate.

Antibiotic susceptibility results

The antibiotic susceptibility results, based on the disk diffusion method, are summarized in Table 2. Twelve MDRPA strains (6.0%) were isolated from ICU (n = 3), surgical 1 (n = 1), gynecology (n = 2), medical 1 (n = 3), hematology 1 (n = 1), and hematology 3 (n = 2) wards.

Table 2.

Antibiotic Susceptibility Rates of Pseudomonas aeruginosa by Disk Diffusion Method

	No. of isolates, N (%)
Antimicrobial agents	Susceptible	Intermediate	Resistant
CAZ	180 (90.5)	5 (2.5)	14 (7.0)
FEP	186 (93.5)	0 (0)	13 (6.5)
TZP	176 (88.4)	7 (3.5)	16 (8.0)
IPM	175 (88.4)	0 (0)	23 (11.6)
MEM	173 (86.9)	3 (1.5)	23 (11.6)
GEN	175 (93.1)	0 (0)	13 (6.9)
AMK	190 (95.5)	1 (0.5)	8 (4.0)
NET	187 (94.0)	0 (0)	12 (6.0)
CIP	191 (96.0)	0 (0)	8 (4.0)
PMB	197 (100)	0 (0)	0 (0)

AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; FEP, cefepime; GEN, gentamicin; IPM, imipenem; MEM, meropenem; NET, netilmicin; PMB, polymyxin B; TZP, piperacillin/tazobactam.

The MIC values for CAZ (64 to ≥256 mg/L) and FEP (48 to ≥256 mg/L) confirmed that 12 of the strains were resistant. Twenty-three strains showed high level resistance toward IPM (16–32 mg/L) and 21 strains toward MEM (4–32 mg/L).

Prevalence and characterization of resistance genes and integrons

Table 3 summarizes the genetic contents of the gene cassettes, extra-integron ESBL- and carbapenemase-encoding genes, the resistant phenotypes, and MICs for cephalosporins and carbapenems. Of the 199 strains, only 2 strains possessed the extra-integron Ambler class A ESBL gene, bla_GES-20, while 7 possessed the Ambler class B MBL/carbapenemase genes, bla_IMP-4 (n = 3), bla_VIM-2 (n = 2), and bla_VIM-11 (n = 4). Two strains harbored both the bla_VIM-2 and bla_IMP-4. All these were MDRPA strains.

Table 3.

Gene Cassette Contents of Class 1 Integrons, Extra-Integron Resistance Genes, and Resistant Phenotypes of intI1-Bearing Pseudomonas aeruginosa from a Malaysian Tertiary Hospital

Integron locus/accession No.	Gene cassette contents	Amplicon size (bp)	Extra-integron resistance genes	Locus	Resistant phenotype	MIC (mg/L)	No. of strains (strain ID)
GU169702	bla _GES-13	1,200	bla _GES-20	CP022000	CAZ-FEP-TZP-IPM-MEM-GEN-NET-CIP	CAZ (>256), FEP (64, 128), IPM (>32), MEM (>32)	2 (PAC06, PAC08)
KR337993 (5′CS)	bla _VIM-6	1,200, 2,600	bla _VIM-11	NG_050338	CAZ-FEP-TZP-IPM-MEM-GEN-AMK-NET	CAZ (>256), FEP (64, 256), IPM (16, >32), MEM (6, 8)	2 (PAC138, PAC148)
KU839731 (3′CS)	bla_OXA-10-AAC(6′)-Ib	1,200, 2,600	bla _VIM-11	NG_050338	CAZ-FEP-TZP-IPM-MEM-GEN-AMK-NET	CAZ (>256), FEP (64, 256), IPM (16, >32), MEM (6, 8)	2 (PAC138, PAC148)
KR337993 (5′CS)	bla _VIM-6	2,600	bla _VIM-11	NG_050338	CAZ-FEP-TZP-IPM-MEM-GEN-AMK-NET-CIP	CAZ (>256), FEP (>256), IPM (>32), MEM (>32)	1 (PAC36)
KX241477 (3′CS)	bla_OXA-10-aac(6′)-II	2,600	bla _VIM-11	NG_050338	CAZ-FEP-TZP-IPM-MEM-GEN-AMK-NET-CIP	CAZ (>256), FEP (>256), IPM (>32), MEM (>32)	1 (PAC36)
MF168946	bla _VIM-2	1,200, 2,600	bla _VIM-11	NG_050338	CAZ-FEP-TZP-IPM-GEN-AMK-NET	CAZ (>256), FEP (48), IPM (>32), MEM (12)	1 (PAC200)
KY860572	aadA6-gcuD	1,400	bla _IMP-4	KX711879	CAZ-FEP-TZP-IPM- MEM-GEN-AMK-NET-CIP	CAZ (>256), FEP (128, >256), IPM (>32), MEM (>32)	2 (PAC45, PAC51)
KY860572	aadA6-gcuD	1,400	bla _VIM-2	FR695889/FR695890	CAZ-FEP-TZP-IPM- MEM-GEN-AMK-NET-CIP	CAZ (>256), FEP (128, >256), IPM (>32), MEM (>32)	2 (PAC45, PAC51)
KY860572	aadA6-gcuD	1,400	bla _IMP-4	KX711879	CAZ-FEP-IPM-MEM-GEN-NET-CIP	CAZ (>256), FEP (>256), IPM (>32), MEM (>32)	1 (PAC96)
KY860572	aadA6-gcuD	1,400	Not detected	Not applicable	GEN-AMK-NET-CIP	N/T^a	1 (PAC99)
KY860572	aadA6-gcuD	1,400	Not detected	Not applicable	CAZ-FEP-TZP-IPM-MEM-GEN-AMK-NET-CIP	CAZ (>256), FEP (128), IPM (>32), MEM (>32)	1 (PAC17)

Not test.

MIC, minimum inhibitory concentration.

Eleven (10 MDRPA and 1 non-MDRPA) strains harbored the intI1 integrase gene. However, none was positive for the intI2 integrase gene. None of the tested resistance genes and integrons was present in two MDRPA strains, PAC95 and PAC167.

Further analysis of the sequenced 5′CS and 3′CS variable region of the integron yielded eight different gene cassettes: bla_GES-13, bla_VIM-2, bla_VIM-6, and bla_OXA-10, which confer resistance toward beta-lactams; aacA(6′)-Ib, aacA(6′)-II, and aadA6, which confer resistance against aminoglycosides; and gcuD, which encodes for a hypothetical protein of unknown function. Strains with high MIC levels for CAZ, FEP, IPM, and MEM possessed the corresponding resistance genes, except for PAC17, which was resistant to beta-lactams and fluoroquinolone, but only harbored the aadA6-gcuD in the class 1 integron gene cassette.

Genetic diversity of P. aeruginosa

PFGE analyses

PFGE analysis of the 199 SpeI-digested chromosomal DNA yielded 163 reproducible pulsed-field profiles (F = 0.57–1.00) with 10–30 restriction fragments (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/mdr), indicating that the strains were genetically diverse. Cluster analysis generated 52 clusters (C1–C52) at ≥85% similarity or 3 band difference and 27 singletons (unique profile [UP] 1 to UP27). One untypeable strain (PAC 199) was excluded from the analysis. We observed three large clusters in the hospital. Two different clusters, C16 and C44, comprised strains that originated from the neonatal intensive care unit (NICU). Cluster C16, which was the largest cluster in the hospital, comprised nine indistinguishable strains, while cluster C44 contained six indistinguishable strains. Clonal strains in C35 (n = 5) were isolated from medical wards 1 and 3, and hematology 3.

MLST-eBURST analyses

Twenty-one STs were identified among the 29 P. aeruginosa, and eBURSTv3 analyses showed nine BGs or clonal complexes. The BGs comprised STs 235 (n = 3), 266 (n = 2), 381 (n = 2), 553 (n = 2), 708 (n = 4), 809 (n = 2), 1076 (n = 2), and1417 (n = 2), and the SLVs ST2338 and ST2341 (n = 2). The other STs were singletons (Fig. 1). In comparison with the global P. aeruginosa MLST database, the Malaysian P. aeruginosa strains, STs 111, 207, 235, 274, 381, 532, and 2033, were international predicted founders. STs 266, 708, 1076, 2329, 2338, and 2341 were SLVs, while STs 274, 553, 809, 1400, 1417, 2335, 2337, 2339, 2340, and 2341 were singletons. In this study, STs 2329, 2335, 2337, 2338, 2340, and 2341 were novel allelic profile strains.

FIG. 1.

Bootstrap cladogram of Pseudomonas aeruginosa MLST ST based on ML method (Tamura-Nei model) in comparison with PC. The tree with the highest log likelihood (−8281.5692) is shown. The bootstrap test (2,000 replicates) percentage of associated taxa clustered together is shown next to the branches. ^aST; ^bPFGE cluster; ^cresistance genes from class 1 integron GC and EI genes detected by PCR and confirmed by nucleotide sequencing: (ND) Not Detected, (I) GC: bla_GES-13, EI: bla_GES-20, (II) GC: aadA6-gcuD, EI: Not detected, (III) GC: bla_VIM-6- bla_OXA-10-aac(6′)-II, EI: bla_VIM-11, (IV) GC: aadA6-gcuD, EI: bla_IMP-4 and bla_VIM-2, (V) GC: aadA6-gcuD, EI: bla_IMP-4, (VI) GC: bla_VIM-6-bla_OXA-10-AAC(6′)-Ib, EI: bla_VIM-11, and (VII) GC: bla_VIM-2, EI: bla_VIM-11; ^dSource & location: tracheal aspirate (Tracheal asp.), bronchoalveolar lavage (BAL), intensive care unit (ICU), neonatal intensive care unit (NICU), emergency unit (Emer), surgical 1 (S1), surgical 3 (S3), gynecology (Gyn), medical 1 (M1), medical 2 (M2), medical 3 (M3), hematology 1 (H1), hematology 2 (H2), and hematology 3 (H3); ^eNot Tested (NT). EI, extra-integron; GC, gene cassettes; ML, maximum likelihood; MLST, multilocus sequence typing; PC, PFGE cluster; PCR, polymerase chain reaction; PFGE, pulsed-field gel electrophoresis; ST, sequence type.

MLST-ML analyses

Figure 1 illustrates the ML rooted tree integrated with STs, PFGE clusters, resistance genes, source, and location of the P. aeruginosa strains. The nucleotide changes per site are indicated by the 0–0.005 cladogram branch lengths (excluding the outgroup).²⁵ Strains on the clades with ≥70% bootstrap probabilities had identical STs, except ST381 (PAC47 and PAC165), which were located on distant branches. In contrast, SLVs, STs 2338 and 2341, which did not share identical STs, were positioned side by side with 98% clade probability.

Combined analyses based on MLST, PFGE, and resistance genes genotypes

Overall, based on the ML tree (Fig. 1), we observed concordance between the clustering of strains having the same STs or SLVs with the PFGE clusters, for example, ST553 with C6, ST1417 with C35, ST266 with C48, ST708 with C16, ST809 with C17, and ST2338/ST2341 with C44. However, ST235 and ST1076 were further subtyped by PFGE. Two antibiotic-susceptible clones detected from the NICU in the integrated analysis were ST708 and SLVs, STs 2338 and 2341.

The MDRPA genotypes were observed among the strains of STs 235, 809, and 1076 clonal complexes; however, these drug-resistant strains were of diverse backgrounds. In our study, the multidrug-resistant ST235 strains showed high-level MIC values for CAZ, FEP, IPM, and MEM (>256, >128, >32, and 32 mg/L, respectively) due to the presence of the bla_IMP-4 and bla_VIM-2 extra-integron resistance genes. Besides that, the multidrug-resistant characteristic for the strains of ST809 could be attributed to the presence of the class 1 integron bearing the bla_VIM-6-bla_OXA-10-AAC(6′)-Ib and the bla_VIM-2 gene cassette variants, as well as the extra-integron bla_VIM-11.

On the other hand, different types of class 1 integron gene cassettes and extra-integron resistance genes were detected for ST1076 MDRPA strains. Strain PAC08 harbored the bla_GES-13 gene cassette and bla_GES-20 extra-integron resistance gene, while strain PAC36 harbored the gene cassette containing the bla_VIM-6 and bla_OXA-10-aac(6′)-II genes, and the extra-integron bla_VIM-11. The high level of MICs in these two strains (>64 mg/L for CAZ, and FEP, and >32 for IPM, and MEM) could be due to the cumulative expression of their multiple resistance genes.

Discussion

The periodic investigation of antibiotic resistance profiles and the resistance genes among clinical P. aeruginosa strains in Malaysia is useful to gauge the level of activity among commonly prescribed antipseudomonal drugs. We found that the susceptibility of the strains toward the carbapenems and TZP was the lowest (≤90%), while ≥90% of the strains remained susceptible to all other classes of antimicrobial agents tested. These results concurred with the reported declining P. aeruginosa antibiotic resistance rates in the Malaysian National Antibiotic Guideline 2014.²⁷ Despite that, antibiotic usage may become limited due to selection pressure, which enhances the growth of antibiotic-resistant variants that possess genetic mutations²⁸ if judicious drug prescription is not followed. According to the hospital's 2014 Antibiotic Usage report (data not shown), MEM, IPM, FEP and CAZ were among the top 10 most frequently administered antibiotics in the wards. In a multicenter retrospective study by Micek et al.,²⁹ the initial inappropriate antibiotic prescription is significantly correlated (p < 0.001) to the incidence of MDRPA and P. aeruginosa pneumonia among hospitalized patients. Hence, the hospital's continuous judicious antibiotic therapy and review of the annual antibiotic susceptibility profiles are fundamental to detect and prevent emerging MDRPA.

In this study, the majority of P. aeruginosa strains were isolated from respiratory specimens from patients in the medical wards. Based on clinical reports (unpublished data), patients from these medical wards had underlying diseases such as diabetes mellitus (DM), chronic obstructive pulmonary disease (COPD), and cancers, which predisposed them to opportunistic P. aeruginosa infections. Results from an international study involving 12 hospitals from 5 countries showed that patients with DM or COPD comorbidities were significantly (p < 0.05) more likely to have MDRPA infections.²⁹ Our study concurred with these findings.

The high levels of resistance to beta-lactam antibiotics such as the cephalosporins and carbapenems were mediated by the bla_OXA, bla_VIM, and bla_IMP genes detected in our study. Our data concurred with findings by other Malaysian researchers^9,10,12 and previously published reports by Toval et al., Hansen et al., and Farshadzadeh et al.^30–32 The IMP-4, VIM-2, and VIM-11 subtypes of P. aeruginosa identified in this study were similar to Khosravi et al.'s study.¹⁰ However, to the best of our knowledge, this is the first report of the Ambler class A ESBL gene, bla_GES (bla_GES-13 and bla_GES-20 variants) detected in Malaysia. The bla_GES-bearing P. aeruginosa was first detected in Evgenidion General Hospital, Athens (2007–2008), and subsequently reported in studies from Turkey (2014) and Brazil (2012).^33–35 No resistance genes were detected from two MDRPA strains (PAC95 and PAC167). Meanwhile, the PAC17 strain, which only harbored the aadA6-gcuD, was resistant to beta-lactams and fluoroquinolone. Cephalosporin resistance may occur as a result of acquired beta-lactamases, total derepression of chromosomal AmpC, or upregulation in efflux systems, while carbapenem resistance is often due to loss of OprD porins, upregulation of efflux pump mechanisms, or acquisition of beta-lactamases.⁵ A cumulative combination of several other resistance mechanisms give rise to multidrug resistance. However, other mechanisms and genes of resistance were not exhaustively investigated in this study; hence, future work on the MDRPA strains is warranted.

Two different subtyping tools were used to analyze the genetic relatedness of the strains in this study. The PFGE method subtyped the strains from the NICU into two major clusters and this finding agreed with the MLST data. This implies that there was an undetected outbreak of antibiotic-susceptible P. aeruginosa clones at the time of sampling. PFGE subtyped the 199 strains into 52 clusters (≥85% similarity) and 27 singletons, which showed that the strains were genetically heterogeneous and there were multiple subtypes of P. aeruginosa in different locations or wards in the hospital.

PFGE genotyping is reliable, reproducible, and advantageous for local outbreak investigations. However, it is limited because clustering of the strains cannot be linked to international lineages. The investigation of the genetic linkage for drug-resistant international lineages is better mapped by MLST. P. aeruginosa ST111 and ST235 strains were previously reported to be multidrug-resistant or extensively drug-resistant high-risk international clones found in France, Germany, Japan, Spain, and Belgium.^31,36 The ST235 strains in our study were also multidrug resistant with high MIC values for CAZ, FEP, IPM, and MEM (>256, >128, >32, and 32 mg/L, respectively). However, the ST111 strain in this study was susceptible toward all antibiotics tested. ST235 strains harboring bla_VIM-2, bla_IMP-1, and bla_IMP-7 were reported from a Singaporean study.³⁷ Furthermore, bla_VIM-2-carrying-ST235 was widely distributed in a study involving six Asian countries, while ST111 was not detected.³⁸ Hence, the occurrence of the ST235 among Malaysian P. aeruginosa is indicative of a global dissemination of the drug-resistant clone. A 2013 Spanish study showed that the ST809 strains isolated from cystic fibrosis patients were non-MDRPA.³⁹ This report differs from our findings on MDRPA ST809 lineage, which harbored MBL-encoding genes. The ST1076 in this study is unique because one of the MDRPA strains possesses the ESBL-encoding bla_GES, while the other possesses bla_VIM-6 MBL and bla_OXA-10 ESBL genes concurrently. These characteristics also differed from the recently reported ST1076 from South Korea, which did not harbor any MBL-encoding genes.⁴⁰

Conclusion

The P. aeruginosa strains in this tertiary hospital were still susceptible to most of the antimicrobial agents tested. However, the presence of MDRPA strains is still a cause for concern because of the high-level cephalosporin and carbapenem resistance mediated by resistance genes, which can be easily disseminated. The clonal spread of the international high-risk clone ST235 in Malaysia requires future close monitoring. To the best of our knowledge, this is the first report of the bla_GES-13 and bla_GES-20 ESBL-encoding gene variants and novel STs (STs 2329, 2335, 2337, 2338, 2340, and 2341) of P. aeruginosa in Malaysia.

Footnotes

Acknowledgments

We thank the Director General of Health Malaysia for his permission to publish this article. This work was supported by the University of Malaya Postgraduate Research Fund PPP (PG073-2014B). H.Y.P.P. was supported by the postgraduate scholarship fund from the Ministry of Health, Malaysia. We also thank Dato’ Dr. Aishah Ahmad Makinuddin, Hospital Director, Dr. Melor @ Mohd Yusof Mohd Mansor, Head of Anesthesiology and ICU, Dato’ Dr. Chang Kian Meng, Head of Hematology Department, Dr. Muralitharan Ganesalingam, Head of O&G Department, Dr. Nor Aishah bt Mohd Arif, Head of General Surgery Department, Dr. Hjh Rosaida Hj Mohd Said, Head of General Medicine Department, Dr. Zuraidah Haji Abdul Latif, Head of Pediatric Department, and Matron Noor Hayati Kamaruddin of the Infection Control Unit, Selangor, Malaysia, for their assistance. The assistance of Dr. Elli Pinnock, curator of P. aeruginosa MLST database, in updating and assigning ST numbers to novel allelic sequences is also appreciated. We also thank Dr. Mahmoud Danaee for his guidance in statistical analysis.

Ethical Approval

We obtained ethical approval from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia.

Disclosure Statement

No competing financial interests exist.

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