# Antimicrobial Susceptibility and Genetic Characterisation ofBurkholderia pseudomallei Isolated from Malaysian Patients

Authors: Yalda Khosravi; Kumutha Malar Vellasamy; Vanitha Mariappan; Shet-Lee Ng; Jamuna Vadivelu
Journal: The Scientific World Journal
 (2014)

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## Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B).


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## Body

## 1. Introduction

Melioidosis, an infectious disease of major public health importance, is a multifaceted disease which is difficult to treat and results in high morbidity and mortality. It is mostly endemic in Southeast Asia, Northern Australia, the Indian subcontinent, and Central and South America [1, 2]. The causative agent of this fatal disease,Burkholderia pseudomallei, is an environmental saprophyte that can be isolated from soil and water and is known as a potential biological warfare agent [2]. Infection ofB. pseudomallei is acquired mainly through wound inoculation, inhalation, or ingestion [2, 3]. Melioidosis may arise many years after exposure, commonly in association with compromised immunity. Overall, the mortality rate of melioidosis is about 50% in Northeast Thailand (35% in children) [3] and 19% in Australia [4]. However, in Malaysia, it was associated with 65% mortality especially in the septicaemic form in the 1980s and reduced to 19–37% in the past 20 years [1, 5–7]. Recurrence of infection is the most important complication in survivors despite prolonged antimicrobial treatment and this has been reported in 10% of Thai patients who survived the primary infection episode [4].B. pseudomallei is intrinsically resistant to many antibiotics, including penicillin, first- and second-generation cephalosporins, macrolides, rifamycins, colistin, and aminoglycosides, but is usually susceptible to amoxicillin/clavulanic acid (AMC), chloramphenicol (CL), doxycycline (DOXY), trimethoprim/sulfamethoxazole (TS), ureidopenicillins, ceftazidime (CAZ), and carbapenems [8]. CAZ, AMC, or the carbapenem antibiotics are used for the initial parenteral phase of the therapy, followed by a prolonged course of oral antimicrobial therapy with either TS with or without DOXY or AMC [9]. However, CAZ-resistant (CAZR) and/or AMC-resistantB. pseudomallei have emerged, ultimately leading to treatment failure [9]. The carbapenems have been reported to have good bactericidal activities againstB. pseudomallei and have been used effectively to treat patients with septicaemic melioidosis [10, 11]. However, increased use of carbapenems may again give rise to resistance as observed with CAZ and AMC.Owing to the difficulty in eradication of the organism following infection, prolonged antibiotic therapy is needed and a high rate of relapse was observed if the therapy is incomplete [12]. Furthermore, recurrence of infection is common despite adequate antimicrobial therapy [13]. It has been shown thatB. pseudomallei isolated from relapse cases and persistent infections were resistant to antibiotics for treatment [3, 14].Thus, the aim of this study was to investigate the antimicrobial susceptibility profile ofB. pseudomallei against a panel of medically relevant antibiotics including CAZ, AMC, DOXY, and TS. Evaluation using other antimicrobials such as CL, meropenem (MERO), imipenem (IMP), tigecycline (TGC), and clarithromycin (CLA) were also included. The antimicrobial susceptibility was performed using two determination methods, that is, the agar disc diffusion and Etest. Additionally, polymerase chain reaction (PCR) technique utilising specific primers was also used for detection of antibiotics resistant genes as this proves to be reliable for detection of β-lactam, aminoglycosides, and CAZR genes in multidrug resistant (MDR) isolates. Further investigation using random amplification of polymorphic DNA (RAPD) analysis was performed to identify the genetic relationships among the isolates and the mode of dissemination of the antibiotic resistance genes.


## 2. Materials and Methods

### 2.1. Ethics Statement

Ethics approval is not required in this study. No consent was required since no human participant was involved. All 70 clinicalB. pseudomallei isolates used in this study were obtained from old archival bacterial collection and all the isolates used were anonymised. Since our institute is a teaching hospital, bacterial isolates obtained as a part of a diagnostic screening are archived and such cases are exempted from obtaining ethical clearances. The study however has an Institutional Biosafety Committee approval.


#### 2.1.1. Bacterial Strains

A total of 81B. pseudomallei isolates (70 clinical, 10 animal, and 1 soil) and oneBurkholderia thailandensis (ATCC 700388) isolates were investigated in this study. Of the 70 clinicalB. pseudomallei, 60 isolates were obtained from archival collections of strains isolated at the Medical Microbiology Diagnostic Laboratory, University of Malaya Medical Centre (UMMC, Kuala Lumpur), and 10 from Hospital Tengku Ampuan Afzan (HTAA, Kuantan, Pahang). The ten animal isolates were kindly provided by S Nathan (Universiti Kebangsaan Malaysia), Malaysia, and the soil isolate was obtained from the Bacteriology Unit, Institute of Medical Research (IMR), Malaysia. These isolates were confirmed using Gram-stain, morphological appearance on Ashdown agar, molecular identification using an in-house PCR method [18] and standard biochemical characterisation using API 20NE assay (Bio-Merieux, France).Escherichia coli (ATCC 25922) was used as control whileB. pseudomallei K96243 was used as reference strain. The isolates used in this study were obtained from different sources, that is, soil, animal and human (blood, pus, sputum, urine, spleen, and lungs).


### 2.2. Antibiotic Susceptibility Testing

#### 2.2.1. Disk Diffusion Test

In vitro antimicrobial susceptibility to nine antibiotics/antimicrobials, namely, CL, AMC, DOXY, MERO, IMP, CAZ, TGC, CLA, and TS, was determined using disc diffusion method according to the British Society of Antimicrobial Chemotherapy [19]. Nutrient agar plates were seeded with 100 μL of 106 colony forming unit per milliliter (CFU/mL) of the test isolates, which had been adjusted to 0.5 McFarland reading spectrophotometrically at 600 nm. The agar plates were then incubated at 37°C for 24 hrs.


#### 2.2.2. Minimum Inhibitory Concentrations (MICs)

Antimicrobial MICs were determined using the broth dilution method and Etest. The broth dilution method was performed using Luria-Bertani (LB) broth (Difco, Lennox) for 24 hrs at 37°C with 105 CFU/mL ofB. pseudomallei (previously washed using normal saline) as the inoculum. Interpretation of the broth dilution results was based on the NCCLS MIC breakpoints for non-Enterobacteriaceae and MIC forB. pseudomallei andB. thailandensis [19]. Inhibition of the bacterial growth was confirmed by spectrophotometric measurement at 600 nm and plating of the serial dilutions onto LB agar. Wells containing bacteria without antibiotics and fresh LB broth were included as positive and negative growth controls, respectively.E. coli ATCC 25922 was used as quality control organism in the antimicrobial MIC determinations. MIC determination using Etest strips (AB Biodisk, Sweden) was performed according to the manufacturer’s instruction.


#### 2.2.3. Polymerase Chain Reaction (PCR)

The DNA of all the isolates tested was extracted using Qiagen Mini Amp Kit (Qiagen, USA) according to the manufacturer’s instruction. Primers (PenA-F and PenA-R) were used to identify the presence ofpenA gene from class A β-lactamase which causes the CAZR (Table 1). Presence of the efflux pump, another main resistance mechanism, was also detected using the degenerate primers, MxBs, MxYs, and MxFs (Table 1). PCR amplification was performed in 25 μL mixture containing 1X PCR buffer (MBI Fermentas, USA), 2 mM MgCl2, 0.2 mM dNTP (MBI Fermentas, USA), 2.5 UTaq DNA polymerase (MBI Fermentas, USA), and 2 μL of DNA template. The PCR conditions used were as follows: initial denaturation at 95°C for 5 mins, 30 cycles of 95°C for 1 min, 55°C for 30 s and 72°C for 1 min, followed by a final extension for 10 mins at 72°C. The amplified PCR products were analysed using 1.5% agarose gel (Promega, USA) electrophoresis in 1X TBE buffer at 90 V for 1 hr and visualised using 3 μL SYBR safe (Life Technology, USA) staining under UV illumination. The limit of dilution was determined by subjecting the DNA of the targeted organisms to PCR after 10-fold serial dilution to produce DNA concentration ranging from 10 mg/mL to 10 fg/mL [17].Table 1
Primers used in this study.
Name
Target gene
Sequence
Reference
PenA-F
pen A
5′GTTCAGCAGATCTAACAGATCGCCGAGATGG3′
[15]
PenA-R
5′GCACCGCGATATCTCGCGCTCCGTGAACCTT3′
MxBs-F
bpeB
5′GATCTCCGCGCAGAACGT3′
[16]
MxBs-R
5′AGGCCGATCGCGAGCACG3′
MxYs-F
amrB
5′TTCATGCAGAACTTCCGC3′
[16]
MxYs-R
5′GAACGCCATCGGCACGAA3′
MxFs-F
BPSS1119
5′CGTCGTGATTTTCCTGTT3′
[16]
MxFs-R
5′ACGCGAACTCCTTGAACA3′
272
Variable
5′AGCGGGCCAA3′
[17]


#### 2.2.4. Genotypic Analysis Using Random Amplified Polymorphic DNA (RAPD)-PCR

The RAPD analysis was performed using primer 272 (Table1) in order to determine the fingerprinting patterns of theB. pseudomallei isolates [20]. The RAPD-PCR mixture (25 μL) consisted of 100 ng of genomic DNA, 0.5 μM primer, 1.25 U of Taq polymerase (MBI Fermentas), 0.2 mM of dNTP (Fermentas, USA), 1X Taq buffer with KCl (pH8), and MgCl2 (2 mM). The cycling condition employed includes (i) 4 cycles with each cycle consisting of 5 mins at 94°C, 5 mins at 36°C and 5 mins at 72°C, and (ii) 30 cycles with each cycle consisting of 1 min at 94°C, 1 min at 36°C, and 2 mins at 72°C, followed by a final extension step at 72°C for 10 mins. The RAPD products (one-ninth of each reaction mixture) were then separated on a 1.5% agarose gels at 90 V/cm for 2 hrs using 1 X TBE as the running buffer. Molecular size standards (VC1Kb ladder) (Fermentas, USA) were also included on the gels. The fingerprints were compared visually followed by analysis using the Gel Compare II V 4.0 software (Applied Maths, Kortrijk, Belgium).


## 2.1. Ethics Statement

Ethics approval is not required in this study. No consent was required since no human participant was involved. All 70 clinicalB. pseudomallei isolates used in this study were obtained from old archival bacterial collection and all the isolates used were anonymised. Since our institute is a teaching hospital, bacterial isolates obtained as a part of a diagnostic screening are archived and such cases are exempted from obtaining ethical clearances. The study however has an Institutional Biosafety Committee approval.


### 2.1.1. Bacterial Strains

A total of 81B. pseudomallei isolates (70 clinical, 10 animal, and 1 soil) and oneBurkholderia thailandensis (ATCC 700388) isolates were investigated in this study. Of the 70 clinicalB. pseudomallei, 60 isolates were obtained from archival collections of strains isolated at the Medical Microbiology Diagnostic Laboratory, University of Malaya Medical Centre (UMMC, Kuala Lumpur), and 10 from Hospital Tengku Ampuan Afzan (HTAA, Kuantan, Pahang). The ten animal isolates were kindly provided by S Nathan (Universiti Kebangsaan Malaysia), Malaysia, and the soil isolate was obtained from the Bacteriology Unit, Institute of Medical Research (IMR), Malaysia. These isolates were confirmed using Gram-stain, morphological appearance on Ashdown agar, molecular identification using an in-house PCR method [18] and standard biochemical characterisation using API 20NE assay (Bio-Merieux, France).Escherichia coli (ATCC 25922) was used as control whileB. pseudomallei K96243 was used as reference strain. The isolates used in this study were obtained from different sources, that is, soil, animal and human (blood, pus, sputum, urine, spleen, and lungs).


## 2.1.1. Bacterial Strains

A total of 81B. pseudomallei isolates (70 clinical, 10 animal, and 1 soil) and oneBurkholderia thailandensis (ATCC 700388) isolates were investigated in this study. Of the 70 clinicalB. pseudomallei, 60 isolates were obtained from archival collections of strains isolated at the Medical Microbiology Diagnostic Laboratory, University of Malaya Medical Centre (UMMC, Kuala Lumpur), and 10 from Hospital Tengku Ampuan Afzan (HTAA, Kuantan, Pahang). The ten animal isolates were kindly provided by S Nathan (Universiti Kebangsaan Malaysia), Malaysia, and the soil isolate was obtained from the Bacteriology Unit, Institute of Medical Research (IMR), Malaysia. These isolates were confirmed using Gram-stain, morphological appearance on Ashdown agar, molecular identification using an in-house PCR method [18] and standard biochemical characterisation using API 20NE assay (Bio-Merieux, France).Escherichia coli (ATCC 25922) was used as control whileB. pseudomallei K96243 was used as reference strain. The isolates used in this study were obtained from different sources, that is, soil, animal and human (blood, pus, sputum, urine, spleen, and lungs).


## 2.2. Antibiotic Susceptibility Testing

### 2.2.1. Disk Diffusion Test

In vitro antimicrobial susceptibility to nine antibiotics/antimicrobials, namely, CL, AMC, DOXY, MERO, IMP, CAZ, TGC, CLA, and TS, was determined using disc diffusion method according to the British Society of Antimicrobial Chemotherapy [19]. Nutrient agar plates were seeded with 100 μL of 106 colony forming unit per milliliter (CFU/mL) of the test isolates, which had been adjusted to 0.5 McFarland reading spectrophotometrically at 600 nm. The agar plates were then incubated at 37°C for 24 hrs.


### 2.2.2. Minimum Inhibitory Concentrations (MICs)

Antimicrobial MICs were determined using the broth dilution method and Etest. The broth dilution method was performed using Luria-Bertani (LB) broth (Difco, Lennox) for 24 hrs at 37°C with 105 CFU/mL ofB. pseudomallei (previously washed using normal saline) as the inoculum. Interpretation of the broth dilution results was based on the NCCLS MIC breakpoints for non-Enterobacteriaceae and MIC forB. pseudomallei andB. thailandensis [19]. Inhibition of the bacterial growth was confirmed by spectrophotometric measurement at 600 nm and plating of the serial dilutions onto LB agar. Wells containing bacteria without antibiotics and fresh LB broth were included as positive and negative growth controls, respectively.E. coli ATCC 25922 was used as quality control organism in the antimicrobial MIC determinations. MIC determination using Etest strips (AB Biodisk, Sweden) was performed according to the manufacturer’s instruction.


### 2.2.3. Polymerase Chain Reaction (PCR)

The DNA of all the isolates tested was extracted using Qiagen Mini Amp Kit (Qiagen, USA) according to the manufacturer’s instruction. Primers (PenA-F and PenA-R) were used to identify the presence ofpenA gene from class A β-lactamase which causes the CAZR (Table 1). Presence of the efflux pump, another main resistance mechanism, was also detected using the degenerate primers, MxBs, MxYs, and MxFs (Table 1). PCR amplification was performed in 25 μL mixture containing 1X PCR buffer (MBI Fermentas, USA), 2 mM MgCl2, 0.2 mM dNTP (MBI Fermentas, USA), 2.5 UTaq DNA polymerase (MBI Fermentas, USA), and 2 μL of DNA template. The PCR conditions used were as follows: initial denaturation at 95°C for 5 mins, 30 cycles of 95°C for 1 min, 55°C for 30 s and 72°C for 1 min, followed by a final extension for 10 mins at 72°C. The amplified PCR products were analysed using 1.5% agarose gel (Promega, USA) electrophoresis in 1X TBE buffer at 90 V for 1 hr and visualised using 3 μL SYBR safe (Life Technology, USA) staining under UV illumination. The limit of dilution was determined by subjecting the DNA of the targeted organisms to PCR after 10-fold serial dilution to produce DNA concentration ranging from 10 mg/mL to 10 fg/mL [17].Table 1
Primers used in this study.
Name
Target gene
Sequence
Reference
PenA-F
pen A
5′GTTCAGCAGATCTAACAGATCGCCGAGATGG3′
[15]
PenA-R
5′GCACCGCGATATCTCGCGCTCCGTGAACCTT3′
MxBs-F
bpeB
5′GATCTCCGCGCAGAACGT3′
[16]
MxBs-R
5′AGGCCGATCGCGAGCACG3′
MxYs-F
amrB
5′TTCATGCAGAACTTCCGC3′
[16]
MxYs-R
5′GAACGCCATCGGCACGAA3′
MxFs-F
BPSS1119
5′CGTCGTGATTTTCCTGTT3′
[16]
MxFs-R
5′ACGCGAACTCCTTGAACA3′
272
Variable
5′AGCGGGCCAA3′
[17]


### 2.2.4. Genotypic Analysis Using Random Amplified Polymorphic DNA (RAPD)-PCR

The RAPD analysis was performed using primer 272 (Table1) in order to determine the fingerprinting patterns of theB. pseudomallei isolates [20]. The RAPD-PCR mixture (25 μL) consisted of 100 ng of genomic DNA, 0.5 μM primer, 1.25 U of Taq polymerase (MBI Fermentas), 0.2 mM of dNTP (Fermentas, USA), 1X Taq buffer with KCl (pH8), and MgCl2 (2 mM). The cycling condition employed includes (i) 4 cycles with each cycle consisting of 5 mins at 94°C, 5 mins at 36°C and 5 mins at 72°C, and (ii) 30 cycles with each cycle consisting of 1 min at 94°C, 1 min at 36°C, and 2 mins at 72°C, followed by a final extension step at 72°C for 10 mins. The RAPD products (one-ninth of each reaction mixture) were then separated on a 1.5% agarose gels at 90 V/cm for 2 hrs using 1 X TBE as the running buffer. Molecular size standards (VC1Kb ladder) (Fermentas, USA) were also included on the gels. The fingerprints were compared visually followed by analysis using the Gel Compare II V 4.0 software (Applied Maths, Kortrijk, Belgium).


## 2.2.1. Disk Diffusion Test

In vitro antimicrobial susceptibility to nine antibiotics/antimicrobials, namely, CL, AMC, DOXY, MERO, IMP, CAZ, TGC, CLA, and TS, was determined using disc diffusion method according to the British Society of Antimicrobial Chemotherapy [19]. Nutrient agar plates were seeded with 100 μL of 106 colony forming unit per milliliter (CFU/mL) of the test isolates, which had been adjusted to 0.5 McFarland reading spectrophotometrically at 600 nm. The agar plates were then incubated at 37°C for 24 hrs.


## 2.2.2. Minimum Inhibitory Concentrations (MICs)

Antimicrobial MICs were determined using the broth dilution method and Etest. The broth dilution method was performed using Luria-Bertani (LB) broth (Difco, Lennox) for 24 hrs at 37°C with 105 CFU/mL ofB. pseudomallei (previously washed using normal saline) as the inoculum. Interpretation of the broth dilution results was based on the NCCLS MIC breakpoints for non-Enterobacteriaceae and MIC forB. pseudomallei andB. thailandensis [19]. Inhibition of the bacterial growth was confirmed by spectrophotometric measurement at 600 nm and plating of the serial dilutions onto LB agar. Wells containing bacteria without antibiotics and fresh LB broth were included as positive and negative growth controls, respectively.E. coli ATCC 25922 was used as quality control organism in the antimicrobial MIC determinations. MIC determination using Etest strips (AB Biodisk, Sweden) was performed according to the manufacturer’s instruction.


## 2.2.3. Polymerase Chain Reaction (PCR)

The DNA of all the isolates tested was extracted using Qiagen Mini Amp Kit (Qiagen, USA) according to the manufacturer’s instruction. Primers (PenA-F and PenA-R) were used to identify the presence ofpenA gene from class A β-lactamase which causes the CAZR (Table 1). Presence of the efflux pump, another main resistance mechanism, was also detected using the degenerate primers, MxBs, MxYs, and MxFs (Table 1). PCR amplification was performed in 25 μL mixture containing 1X PCR buffer (MBI Fermentas, USA), 2 mM MgCl2, 0.2 mM dNTP (MBI Fermentas, USA), 2.5 UTaq DNA polymerase (MBI Fermentas, USA), and 2 μL of DNA template. The PCR conditions used were as follows: initial denaturation at 95°C for 5 mins, 30 cycles of 95°C for 1 min, 55°C for 30 s and 72°C for 1 min, followed by a final extension for 10 mins at 72°C. The amplified PCR products were analysed using 1.5% agarose gel (Promega, USA) electrophoresis in 1X TBE buffer at 90 V for 1 hr and visualised using 3 μL SYBR safe (Life Technology, USA) staining under UV illumination. The limit of dilution was determined by subjecting the DNA of the targeted organisms to PCR after 10-fold serial dilution to produce DNA concentration ranging from 10 mg/mL to 10 fg/mL [17].Table 1
Primers used in this study.
Name
Target gene
Sequence
Reference
PenA-F
pen A
5′GTTCAGCAGATCTAACAGATCGCCGAGATGG3′
[15]
PenA-R
5′GCACCGCGATATCTCGCGCTCCGTGAACCTT3′
MxBs-F
bpeB
5′GATCTCCGCGCAGAACGT3′
[16]
MxBs-R
5′AGGCCGATCGCGAGCACG3′
MxYs-F
amrB
5′TTCATGCAGAACTTCCGC3′
[16]
MxYs-R
5′GAACGCCATCGGCACGAA3′
MxFs-F
BPSS1119
5′CGTCGTGATTTTCCTGTT3′
[16]
MxFs-R
5′ACGCGAACTCCTTGAACA3′
272
Variable
5′AGCGGGCCAA3′
[17]


## 2.2.4. Genotypic Analysis Using Random Amplified Polymorphic DNA (RAPD)-PCR

The RAPD analysis was performed using primer 272 (Table1) in order to determine the fingerprinting patterns of theB. pseudomallei isolates [20]. The RAPD-PCR mixture (25 μL) consisted of 100 ng of genomic DNA, 0.5 μM primer, 1.25 U of Taq polymerase (MBI Fermentas), 0.2 mM of dNTP (Fermentas, USA), 1X Taq buffer with KCl (pH8), and MgCl2 (2 mM). The cycling condition employed includes (i) 4 cycles with each cycle consisting of 5 mins at 94°C, 5 mins at 36°C and 5 mins at 72°C, and (ii) 30 cycles with each cycle consisting of 1 min at 94°C, 1 min at 36°C, and 2 mins at 72°C, followed by a final extension step at 72°C for 10 mins. The RAPD products (one-ninth of each reaction mixture) were then separated on a 1.5% agarose gels at 90 V/cm for 2 hrs using 1 X TBE as the running buffer. Molecular size standards (VC1Kb ladder) (Fermentas, USA) were also included on the gels. The fingerprints were compared visually followed by analysis using the Gel Compare II V 4.0 software (Applied Maths, Kortrijk, Belgium).


## 3. Results

### 3.1. Antibiotic Sensitivity Testing

#### 3.1.1. Disk Diffusion

The results indicated that, of the 81B. pseudomallei isolates tested, six were found to be MDR (resistant to MERO, IMP, and CAZ). However, the remaining 75 isolates were resistant to at least one of the antimicrobial agents (non-MDR). The MDR isolates had susceptibility results that were distinct from other isolates tested (Table 2). The MERO- and IMP-resistant isolates had smaller inhibition zone (15.7 mm) compared to the sensitive isolates (20 to 25 mm). These isolates were also persistently mucoid and the inhibition zone diameter was not distinct. Among the 81B. pseudomallei isolates, only 4.94% were CAZR and 2.47% showed intermediate susceptibility to CAZ, which is among the drug of choice for treatment of melioidosis. The CAZR isolates had no inhibition zone compared to the CAZ-sensitive isolates (24 to 29 mm).Table 2
Origin, resistogram (disk diffusion), and RAPD of 81B. pseudomallei isolates and a B. thailandensis isolate.
StrainID
Origin of isolationa
Antimicrobial susceptibility testing
PCR detection
CL
CLA
DOXY
IMP
MERO
TGC
TS
CAZ
AMC
PenA, bpeB, amrB & BPSS1119
RAPD
Bp1
Blood
I
S
S
S
S
R
S
S
S
Negative
B
Bp2
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp3
Blood
I
I
R
R
S
R
S
S
I
Negative
B
Bp4
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp5
Blood
R
R
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp6
Blood
S
S
S
S
S
R
S
S
I
Negative
B
Bp7
Blood
I
S
S
S
S
R
S
S
R
Negative
B
Bp8
Blood
S
S
S
S
S
I
S
S
S
Negative
B
Bp9
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp10
Blood
S
S
S
S
S
R
S
S
R
Negative
B
Bp11
Urine
S
S
S
S
S
R
S
S
R
Negative
B
Bp12
Blood
I
S
S
S
S
S
S
S
I
Negative
B
Bp13
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp14
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp15
Blood
S
I
S
S
S
S
S
S
I
Negative
B
Bp16
Sputum
I
I
S
S
S
R
S
S
I
Negative
B
Bp17
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp18
Pus
R
I
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp19
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp20
Blood
S
I
S
S
S
R
S
S
I
Negative
B
Bp21
Blood
I
S
S
S
S
R
R
S
R
Negative
B
Bp22
Blood
S
S
S
S
S
R
R
S
S
Negative
B
Bp23
Blood
I
I
S
S
S
R
I
S
I
Negative
B
Bp24
Lung
S
S
S
S
S
R
I
S
I
Ngative
B
Bp25
Blood
I
R
R
R
R
R
R
I
R
PenA, bpeB, amrB & BPSS1119
A
Bp26
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp27
Blood
S
S
S
S
S
S
R
S
I
Negative
B
Bp28
Blood
S
I
S
S
S
S
I
S
I
Negative
B
Bp29
Blood
I
S
S
S
S
S
R
S
I
Negative
B
Bp30
Spleen
I
S
S
S
S
S
S
S
I
Negative
B
Bp31
Blood
S
S
S
S
S
R
R
S
I
Negative
B
Bp32
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp33
Blood
I
I
S
S
S
S
R
S
I
Negative
B
Bp34
Blood
S
I
S
S
S
S
I
S
S
Negative
B
Bp35
Blood
R
R
R
R
R
S
R
I
S
PenA, bpeB, amrB&BPSS1119
A
Bp36
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp37
Blood
R
I
R
R
R
R
R
R
I
PenA, bpeB, amrB, & BPSS1119
A
Bp38
Blood
I
I
S
S
S
R
I
S
S
Negative
B
Bp39
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp40
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp41
Sputum
I
S
S
S
S
R
S
S
R
Negative
B
Bp42
Blood
I
I
S
S
S
S
I
S
I
Negative
B
Bp43
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp44
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp45
Blood
I
I
S
S
S
S
S
S
S
Negative
B
Bp46
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp47
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp48
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp49
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp50
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp51
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp52
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp53
Blood
I
R
S
S
S
R
I
S
I
Negative
B
Bp54
Blood
I
R
S
S
S
S
S
S
R
Negative
B
Bp55
Blood
R
S
S
S
S
S
I
S
I
Negative
B
Bp56
Blood
I
I
S
S
S
S
I
S
R
Negative
B
Bp57
Pus
I
R
S
S
S
S
I
S
R
Negative
B
Bp58
Sputum
S
S
S
S
S
S
R
S
R
Negative
B
Bp59
Sputum
S
I
S
S
S
R
I
S
I
Negative
B
Bp60
Sputum
S
S
S
R
R
R
R
S
R
Negative
B
Bp61
Animal
S
I
S
S
S
S
I
S
S
Negative
B
Bp62
Animal
I
S
S
S
S
I
I
S
R
Negative
B
Bp63
Animal
S
S
S
S
S
S
I
S
R
Negative
B
Bp64
Animal
S
S
S
S
S
S
S
S
S
Negative
B
Bp65
Animal
I
S
S
S
S
I
S
S
R
Negative
B
Bp66
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bp67
Animal
S
S
S
S
S
S
S
S
I
Negative
B
Bp68
Animal
I
S
S
S
S
R
S
S
R
Negative
B
Bp69
Animal
I
S
S
S
S
S
S
S
S
Negative
B
Bp70
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bt71
Soil
I
I
S
S
S
R
I
S
R
Negative
B
Bp72
Blood
I
I
S
S
S
R
S
S
R
Negative
B
Bp73
Blood
R
I
S
S
S
R
S
S
R
Negative
B
Bp74
Blood
I
R
S
S
S
R
I
S
R
Negative
B
Bp75
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp77
Blood
R
R
S
S
S
R
S
S
S
Negative
B
Bp78
Blood
R
I
S
S
S
I
S
S
S
Negative
B
Bp79
Blood
R
R
I
S
S
R
I
R
R
PenA, bpeB, amrB&BPSS1119
A
Bp80
Blood
R
R
I
S
S
R
I
S
I
Negative
B
Bp81
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp82
Soil
I
S
S
S
S
R
S
S
R
Negative
B
Foot note: R: resistant; I: intermediate; S: sensitive; CL: chloramphenicol; AMC: amoxiclin/clavulanic acid; DOXY: doxycycline; MERO: meropenem; IMP: imipenem; CAZ: ceftazidime; TGC: tigecycline; CLA: clarithromycin; TS: trimethoprim/sulfamethoxazole.
Intermediate is counted as resistant.
aBlood, pus, sputum, urine, spleen, and lungs are of human origin.
Bp:B. pseudomallei; Bt: B. thailandensis.


#### 3.1.2. MIC Etest

The highest reading for the Etest results (0.125–128 mg/L) was demonstrated for the MICs of CAZ, which also correlated with the most resistant isolate identified by disk diffusion (Tables2 and 3). The Etest results also demonstrated that 100% of the CAZR isolates were shown to be resistant to MERO. The isolate with the highest measurable CAZ Etest result was also found to confer highest resistance towards MERO.Table 3
Broth microdilution and Etest MICs of 81B. pseudomallei isolates.
Antimicrobial agent
MIC (mg/L)
Broth dilution
Etest
Range
50%
90%
Range
50%
90%
Chloramphenicol
4–16
8
16
0.25–24
3
8
Amoxiclin/clavulanic acid
4–8
8
8
0.125–2
0.25
0.25
Doxycycline
0.5–1
0.5
1
0.19–16
0.19
16
Meropenem
3-4
2
4a
1–4
1
1.5
Imipenem
0.5–1
0.5
1a
0.094–8
0.094
0.125
Ceftazidime
2–64
2
4a
0.125–128
0.250
0.5
Tigecycline
3-4
2
6
0.5–32
3
6
Clarithromycin
4–16
4
16
1.5–48
1
32
Trimethoprim/sulfamethoxazole
4–64
16
64
0.003–0.25
0.032
0.125
Foot note:amultidrug resistant isolates.


#### 3.1.3. MIC Broth Microdilution

Conventional broth microdilution MIC results for CAZ, AMC, MERO, IMP, and TS were found to correlate with the Etest MIC results (Table3). However, there was no significant correlation between MICs of MERO Etest and MICs determined by the disc diffusion and Etest.


#### 3.1.4. PCR Amplification

PCR results demonstrated that the efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) were present in all the six MDR isolates found in this study (Table 2, Figure 1). We also observed that isolates which showed the presence ofpenA also demonstrated the presence ofbpeB,amrB, andBPSS1119.Figure 1
PCR results demonstrating the agarose gel electrophoretic analysis of PCR products generated from amplification of efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) fromB. pseudomallei. The presence of a 594 bp, 303 bp, 356 bp, and 124 bp fragments in the agarose gel indicated a positive result forbpeB,amrB,BPSS1119, andpenA, respectively. Ladder: 100 bp ladder (MBI Fermentas, USA).


#### 3.1.5. Molecular Typing of Antibiotic-ResistantB. pseudomallei Strains Using RAPD

A total of six different PCR fingerprinting patterns were generated from the six MDR isolates. However, eight different PCR fingerprinting patterns were generated for the remaining 75 non-MDR isolates. Based on the differences in the banding patterns, a distinct cluster (A) was identified among the 6 MDR isolates which contain thebpeB,amrB, andBPSS1119 andpenA genes, and among the 75 non-MDR isolates, only a single distinct cluster (B) was found (Table 2 and Figure 2).Figure 2
Random amplified polymorphic DNA (RAPD) polymorphisms ofB. pseudomallei clinical isolates amplified by primer 272. The representative fingerprint patterns of two clusters RAPD were shown (i) RAPD cluster A (MDR) and (ii) RAPD cluster B (non-MDR).


## 3.1. Antibiotic Sensitivity Testing

### 3.1.1. Disk Diffusion

The results indicated that, of the 81B. pseudomallei isolates tested, six were found to be MDR (resistant to MERO, IMP, and CAZ). However, the remaining 75 isolates were resistant to at least one of the antimicrobial agents (non-MDR). The MDR isolates had susceptibility results that were distinct from other isolates tested (Table 2). The MERO- and IMP-resistant isolates had smaller inhibition zone (15.7 mm) compared to the sensitive isolates (20 to 25 mm). These isolates were also persistently mucoid and the inhibition zone diameter was not distinct. Among the 81B. pseudomallei isolates, only 4.94% were CAZR and 2.47% showed intermediate susceptibility to CAZ, which is among the drug of choice for treatment of melioidosis. The CAZR isolates had no inhibition zone compared to the CAZ-sensitive isolates (24 to 29 mm).Table 2
Origin, resistogram (disk diffusion), and RAPD of 81B. pseudomallei isolates and a B. thailandensis isolate.
StrainID
Origin of isolationa
Antimicrobial susceptibility testing
PCR detection
CL
CLA
DOXY
IMP
MERO
TGC
TS
CAZ
AMC
PenA, bpeB, amrB & BPSS1119
RAPD
Bp1
Blood
I
S
S
S
S
R
S
S
S
Negative
B
Bp2
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp3
Blood
I
I
R
R
S
R
S
S
I
Negative
B
Bp4
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp5
Blood
R
R
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp6
Blood
S
S
S
S
S
R
S
S
I
Negative
B
Bp7
Blood
I
S
S
S
S
R
S
S
R
Negative
B
Bp8
Blood
S
S
S
S
S
I
S
S
S
Negative
B
Bp9
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp10
Blood
S
S
S
S
S
R
S
S
R
Negative
B
Bp11
Urine
S
S
S
S
S
R
S
S
R
Negative
B
Bp12
Blood
I
S
S
S
S
S
S
S
I
Negative
B
Bp13
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp14
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp15
Blood
S
I
S
S
S
S
S
S
I
Negative
B
Bp16
Sputum
I
I
S
S
S
R
S
S
I
Negative
B
Bp17
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp18
Pus
R
I
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp19
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp20
Blood
S
I
S
S
S
R
S
S
I
Negative
B
Bp21
Blood
I
S
S
S
S
R
R
S
R
Negative
B
Bp22
Blood
S
S
S
S
S
R
R
S
S
Negative
B
Bp23
Blood
I
I
S
S
S
R
I
S
I
Negative
B
Bp24
Lung
S
S
S
S
S
R
I
S
I
Ngative
B
Bp25
Blood
I
R
R
R
R
R
R
I
R
PenA, bpeB, amrB & BPSS1119
A
Bp26
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp27
Blood
S
S
S
S
S
S
R
S
I
Negative
B
Bp28
Blood
S
I
S
S
S
S
I
S
I
Negative
B
Bp29
Blood
I
S
S
S
S
S
R
S
I
Negative
B
Bp30
Spleen
I
S
S
S
S
S
S
S
I
Negative
B
Bp31
Blood
S
S
S
S
S
R
R
S
I
Negative
B
Bp32
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp33
Blood
I
I
S
S
S
S
R
S
I
Negative
B
Bp34
Blood
S
I
S
S
S
S
I
S
S
Negative
B
Bp35
Blood
R
R
R
R
R
S
R
I
S
PenA, bpeB, amrB&BPSS1119
A
Bp36
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp37
Blood
R
I
R
R
R
R
R
R
I
PenA, bpeB, amrB, & BPSS1119
A
Bp38
Blood
I
I
S
S
S
R
I
S
S
Negative
B
Bp39
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp40
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp41
Sputum
I
S
S
S
S
R
S
S
R
Negative
B
Bp42
Blood
I
I
S
S
S
S
I
S
I
Negative
B
Bp43
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp44
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp45
Blood
I
I
S
S
S
S
S
S
S
Negative
B
Bp46
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp47
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp48
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp49
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp50
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp51
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp52
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp53
Blood
I
R
S
S
S
R
I
S
I
Negative
B
Bp54
Blood
I
R
S
S
S
S
S
S
R
Negative
B
Bp55
Blood
R
S
S
S
S
S
I
S
I
Negative
B
Bp56
Blood
I
I
S
S
S
S
I
S
R
Negative
B
Bp57
Pus
I
R
S
S
S
S
I
S
R
Negative
B
Bp58
Sputum
S
S
S
S
S
S
R
S
R
Negative
B
Bp59
Sputum
S
I
S
S
S
R
I
S
I
Negative
B
Bp60
Sputum
S
S
S
R
R
R
R
S
R
Negative
B
Bp61
Animal
S
I
S
S
S
S
I
S
S
Negative
B
Bp62
Animal
I
S
S
S
S
I
I
S
R
Negative
B
Bp63
Animal
S
S
S
S
S
S
I
S
R
Negative
B
Bp64
Animal
S
S
S
S
S
S
S
S
S
Negative
B
Bp65
Animal
I
S
S
S
S
I
S
S
R
Negative
B
Bp66
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bp67
Animal
S
S
S
S
S
S
S
S
I
Negative
B
Bp68
Animal
I
S
S
S
S
R
S
S
R
Negative
B
Bp69
Animal
I
S
S
S
S
S
S
S
S
Negative
B
Bp70
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bt71
Soil
I
I
S
S
S
R
I
S
R
Negative
B
Bp72
Blood
I
I
S
S
S
R
S
S
R
Negative
B
Bp73
Blood
R
I
S
S
S
R
S
S
R
Negative
B
Bp74
Blood
I
R
S
S
S
R
I
S
R
Negative
B
Bp75
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp77
Blood
R
R
S
S
S
R
S
S
S
Negative
B
Bp78
Blood
R
I
S
S
S
I
S
S
S
Negative
B
Bp79
Blood
R
R
I
S
S
R
I
R
R
PenA, bpeB, amrB&BPSS1119
A
Bp80
Blood
R
R
I
S
S
R
I
S
I
Negative
B
Bp81
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp82
Soil
I
S
S
S
S
R
S
S
R
Negative
B
Foot note: R: resistant; I: intermediate; S: sensitive; CL: chloramphenicol; AMC: amoxiclin/clavulanic acid; DOXY: doxycycline; MERO: meropenem; IMP: imipenem; CAZ: ceftazidime; TGC: tigecycline; CLA: clarithromycin; TS: trimethoprim/sulfamethoxazole.
Intermediate is counted as resistant.
aBlood, pus, sputum, urine, spleen, and lungs are of human origin.
Bp:B. pseudomallei; Bt: B. thailandensis.


### 3.1.2. MIC Etest

The highest reading for the Etest results (0.125–128 mg/L) was demonstrated for the MICs of CAZ, which also correlated with the most resistant isolate identified by disk diffusion (Tables2 and 3). The Etest results also demonstrated that 100% of the CAZR isolates were shown to be resistant to MERO. The isolate with the highest measurable CAZ Etest result was also found to confer highest resistance towards MERO.Table 3
Broth microdilution and Etest MICs of 81B. pseudomallei isolates.
Antimicrobial agent
MIC (mg/L)
Broth dilution
Etest
Range
50%
90%
Range
50%
90%
Chloramphenicol
4–16
8
16
0.25–24
3
8
Amoxiclin/clavulanic acid
4–8
8
8
0.125–2
0.25
0.25
Doxycycline
0.5–1
0.5
1
0.19–16
0.19
16
Meropenem
3-4
2
4a
1–4
1
1.5
Imipenem
0.5–1
0.5
1a
0.094–8
0.094
0.125
Ceftazidime
2–64
2
4a
0.125–128
0.250
0.5
Tigecycline
3-4
2
6
0.5–32
3
6
Clarithromycin
4–16
4
16
1.5–48
1
32
Trimethoprim/sulfamethoxazole
4–64
16
64
0.003–0.25
0.032
0.125
Foot note:amultidrug resistant isolates.


### 3.1.3. MIC Broth Microdilution

Conventional broth microdilution MIC results for CAZ, AMC, MERO, IMP, and TS were found to correlate with the Etest MIC results (Table3). However, there was no significant correlation between MICs of MERO Etest and MICs determined by the disc diffusion and Etest.


### 3.1.4. PCR Amplification

PCR results demonstrated that the efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) were present in all the six MDR isolates found in this study (Table 2, Figure 1). We also observed that isolates which showed the presence ofpenA also demonstrated the presence ofbpeB,amrB, andBPSS1119.Figure 1
PCR results demonstrating the agarose gel electrophoretic analysis of PCR products generated from amplification of efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) fromB. pseudomallei. The presence of a 594 bp, 303 bp, 356 bp, and 124 bp fragments in the agarose gel indicated a positive result forbpeB,amrB,BPSS1119, andpenA, respectively. Ladder: 100 bp ladder (MBI Fermentas, USA).


### 3.1.5. Molecular Typing of Antibiotic-ResistantB. pseudomallei Strains Using RAPD

A total of six different PCR fingerprinting patterns were generated from the six MDR isolates. However, eight different PCR fingerprinting patterns were generated for the remaining 75 non-MDR isolates. Based on the differences in the banding patterns, a distinct cluster (A) was identified among the 6 MDR isolates which contain thebpeB,amrB, andBPSS1119 andpenA genes, and among the 75 non-MDR isolates, only a single distinct cluster (B) was found (Table 2 and Figure 2).Figure 2
Random amplified polymorphic DNA (RAPD) polymorphisms ofB. pseudomallei clinical isolates amplified by primer 272. The representative fingerprint patterns of two clusters RAPD were shown (i) RAPD cluster A (MDR) and (ii) RAPD cluster B (non-MDR).


## 3.1.1. Disk Diffusion

The results indicated that, of the 81B. pseudomallei isolates tested, six were found to be MDR (resistant to MERO, IMP, and CAZ). However, the remaining 75 isolates were resistant to at least one of the antimicrobial agents (non-MDR). The MDR isolates had susceptibility results that were distinct from other isolates tested (Table 2). The MERO- and IMP-resistant isolates had smaller inhibition zone (15.7 mm) compared to the sensitive isolates (20 to 25 mm). These isolates were also persistently mucoid and the inhibition zone diameter was not distinct. Among the 81B. pseudomallei isolates, only 4.94% were CAZR and 2.47% showed intermediate susceptibility to CAZ, which is among the drug of choice for treatment of melioidosis. The CAZR isolates had no inhibition zone compared to the CAZ-sensitive isolates (24 to 29 mm).Table 2
Origin, resistogram (disk diffusion), and RAPD of 81B. pseudomallei isolates and a B. thailandensis isolate.
StrainID
Origin of isolationa
Antimicrobial susceptibility testing
PCR detection
CL
CLA
DOXY
IMP
MERO
TGC
TS
CAZ
AMC
PenA, bpeB, amrB & BPSS1119
RAPD
Bp1
Blood
I
S
S
S
S
R
S
S
S
Negative
B
Bp2
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp3
Blood
I
I
R
R
S
R
S
S
I
Negative
B
Bp4
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp5
Blood
R
R
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp6
Blood
S
S
S
S
S
R
S
S
I
Negative
B
Bp7
Blood
I
S
S
S
S
R
S
S
R
Negative
B
Bp8
Blood
S
S
S
S
S
I
S
S
S
Negative
B
Bp9
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp10
Blood
S
S
S
S
S
R
S
S
R
Negative
B
Bp11
Urine
S
S
S
S
S
R
S
S
R
Negative
B
Bp12
Blood
I
S
S
S
S
S
S
S
I
Negative
B
Bp13
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp14
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp15
Blood
S
I
S
S
S
S
S
S
I
Negative
B
Bp16
Sputum
I
I
S
S
S
R
S
S
I
Negative
B
Bp17
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp18
Pus
R
I
R
R
R
R
R
R
R
PenA, bpeB, amrB & BPSS1119
A
Bp19
Blood
I
I
S
S
S
R
S
S
S
Negative
B
Bp20
Blood
S
I
S
S
S
R
S
S
I
Negative
B
Bp21
Blood
I
S
S
S
S
R
R
S
R
Negative
B
Bp22
Blood
S
S
S
S
S
R
R
S
S
Negative
B
Bp23
Blood
I
I
S
S
S
R
I
S
I
Negative
B
Bp24
Lung
S
S
S
S
S
R
I
S
I
Ngative
B
Bp25
Blood
I
R
R
R
R
R
R
I
R
PenA, bpeB, amrB & BPSS1119
A
Bp26
Blood
S
S
S
S
S
S
S
S
I
Negative
B
Bp27
Blood
S
S
S
S
S
S
R
S
I
Negative
B
Bp28
Blood
S
I
S
S
S
S
I
S
I
Negative
B
Bp29
Blood
I
S
S
S
S
S
R
S
I
Negative
B
Bp30
Spleen
I
S
S
S
S
S
S
S
I
Negative
B
Bp31
Blood
S
S
S
S
S
R
R
S
I
Negative
B
Bp32
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp33
Blood
I
I
S
S
S
S
R
S
I
Negative
B
Bp34
Blood
S
I
S
S
S
S
I
S
S
Negative
B
Bp35
Blood
R
R
R
R
R
S
R
I
S
PenA, bpeB, amrB&BPSS1119
A
Bp36
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp37
Blood
R
I
R
R
R
R
R
R
I
PenA, bpeB, amrB, & BPSS1119
A
Bp38
Blood
I
I
S
S
S
R
I
S
S
Negative
B
Bp39
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp40
Blood
S
S
S
S
S
R
S
S
S
Negative
B
Bp41
Sputum
I
S
S
S
S
R
S
S
R
Negative
B
Bp42
Blood
I
I
S
S
S
S
I
S
I
Negative
B
Bp43
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp44
Blood
I
I
S
S
S
S
S
S
I
Negative
B
Bp45
Blood
I
I
S
S
S
S
S
S
S
Negative
B
Bp46
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp47
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp48
Blood
I
R
S
S
S
S
S
S
I
Negative
B
Bp49
Blood
S
I
S
S
S
R
S
S
R
Negative
B
Bp50
Blood
I
I
S
S
S
R
I
S
R
Negative
B
Bp51
Blood
S
S
S
S
S
R
I
S
R
Negative
B
Bp52
Blood
R
R
S
S
S
R
S
S
R
Negative
B
Bp53
Blood
I
R
S
S
S
R
I
S
I
Negative
B
Bp54
Blood
I
R
S
S
S
S
S
S
R
Negative
B
Bp55
Blood
R
S
S
S
S
S
I
S
I
Negative
B
Bp56
Blood
I
I
S
S
S
S
I
S
R
Negative
B
Bp57
Pus
I
R
S
S
S
S
I
S
R
Negative
B
Bp58
Sputum
S
S
S
S
S
S
R
S
R
Negative
B
Bp59
Sputum
S
I
S
S
S
R
I
S
I
Negative
B
Bp60
Sputum
S
S
S
R
R
R
R
S
R
Negative
B
Bp61
Animal
S
I
S
S
S
S
I
S
S
Negative
B
Bp62
Animal
I
S
S
S
S
I
I
S
R
Negative
B
Bp63
Animal
S
S
S
S
S
S
I
S
R
Negative
B
Bp64
Animal
S
S
S
S
S
S
S
S
S
Negative
B
Bp65
Animal
I
S
S
S
S
I
S
S
R
Negative
B
Bp66
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bp67
Animal
S
S
S
S
S
S
S
S
I
Negative
B
Bp68
Animal
I
S
S
S
S
R
S
S
R
Negative
B
Bp69
Animal
I
S
S
S
S
S
S
S
S
Negative
B
Bp70
Animal
I
S
S
S
S
I
S
S
S
Negative
B
Bt71
Soil
I
I
S
S
S
R
I
S
R
Negative
B
Bp72
Blood
I
I
S
S
S
R
S
S
R
Negative
B
Bp73
Blood
R
I
S
S
S
R
S
S
R
Negative
B
Bp74
Blood
I
R
S
S
S
R
I
S
R
Negative
B
Bp75
Blood
S
S
S
S
S
S
S
S
S
Negative
B
Bp77
Blood
R
R
S
S
S
R
S
S
S
Negative
B
Bp78
Blood
R
I
S
S
S
I
S
S
S
Negative
B
Bp79
Blood
R
R
I
S
S
R
I
R
R
PenA, bpeB, amrB&BPSS1119
A
Bp80
Blood
R
R
I
S
S
R
I
S
I
Negative
B
Bp81
Blood
I
S
S
S
S
R
S
S
I
Negative
B
Bp82
Soil
I
S
S
S
S
R
S
S
R
Negative
B
Foot note: R: resistant; I: intermediate; S: sensitive; CL: chloramphenicol; AMC: amoxiclin/clavulanic acid; DOXY: doxycycline; MERO: meropenem; IMP: imipenem; CAZ: ceftazidime; TGC: tigecycline; CLA: clarithromycin; TS: trimethoprim/sulfamethoxazole.
Intermediate is counted as resistant.
aBlood, pus, sputum, urine, spleen, and lungs are of human origin.
Bp:B. pseudomallei; Bt: B. thailandensis.


## 3.1.2. MIC Etest

The highest reading for the Etest results (0.125–128 mg/L) was demonstrated for the MICs of CAZ, which also correlated with the most resistant isolate identified by disk diffusion (Tables2 and 3). The Etest results also demonstrated that 100% of the CAZR isolates were shown to be resistant to MERO. The isolate with the highest measurable CAZ Etest result was also found to confer highest resistance towards MERO.Table 3
Broth microdilution and Etest MICs of 81B. pseudomallei isolates.
Antimicrobial agent
MIC (mg/L)
Broth dilution
Etest
Range
50%
90%
Range
50%
90%
Chloramphenicol
4–16
8
16
0.25–24
3
8
Amoxiclin/clavulanic acid
4–8
8
8
0.125–2
0.25
0.25
Doxycycline
0.5–1
0.5
1
0.19–16
0.19
16
Meropenem
3-4
2
4a
1–4
1
1.5
Imipenem
0.5–1
0.5
1a
0.094–8
0.094
0.125
Ceftazidime
2–64
2
4a
0.125–128
0.250
0.5
Tigecycline
3-4
2
6
0.5–32
3
6
Clarithromycin
4–16
4
16
1.5–48
1
32
Trimethoprim/sulfamethoxazole
4–64
16
64
0.003–0.25
0.032
0.125
Foot note:amultidrug resistant isolates.


## 3.1.3. MIC Broth Microdilution

Conventional broth microdilution MIC results for CAZ, AMC, MERO, IMP, and TS were found to correlate with the Etest MIC results (Table3). However, there was no significant correlation between MICs of MERO Etest and MICs determined by the disc diffusion and Etest.


## 3.1.4. PCR Amplification

PCR results demonstrated that the efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) were present in all the six MDR isolates found in this study (Table 2, Figure 1). We also observed that isolates which showed the presence ofpenA also demonstrated the presence ofbpeB,amrB, andBPSS1119.Figure 1
PCR results demonstrating the agarose gel electrophoretic analysis of PCR products generated from amplification of efflux pump genes (bpeB,amrB, andBPSS1119) and class A β-lactams gene (penA) fromB. pseudomallei. The presence of a 594 bp, 303 bp, 356 bp, and 124 bp fragments in the agarose gel indicated a positive result forbpeB,amrB,BPSS1119, andpenA, respectively. Ladder: 100 bp ladder (MBI Fermentas, USA).


## 3.1.5. Molecular Typing of Antibiotic-ResistantB. pseudomallei Strains Using RAPD

A total of six different PCR fingerprinting patterns were generated from the six MDR isolates. However, eight different PCR fingerprinting patterns were generated for the remaining 75 non-MDR isolates. Based on the differences in the banding patterns, a distinct cluster (A) was identified among the 6 MDR isolates which contain thebpeB,amrB, andBPSS1119 andpenA genes, and among the 75 non-MDR isolates, only a single distinct cluster (B) was found (Table 2 and Figure 2).Figure 2
Random amplified polymorphic DNA (RAPD) polymorphisms ofB. pseudomallei clinical isolates amplified by primer 272. The representative fingerprint patterns of two clusters RAPD were shown (i) RAPD cluster A (MDR) and (ii) RAPD cluster B (non-MDR).


## 4. Discussion

In the current scenario, melioidosis is considered as a fatal disease with no effective vaccine available. Treatment also requires prolonged and high dosage antibiotic therapy to accomplish complete eradication. Nevertheless, prolonged therapy can lead to the development of resistance and to make matters worse, the causative agent,B. pseudomallei, is intrinsically resistant to a wide range of antibiotics. Thus, treatment options for melioidosis are limited to a small number of antimicrobial agents such as CAZ (primary treatment) and TS, DOXY, or AMC (secondary treatment) [21]. Despite many trials, CAZ remains as the drug of choice for treatment of severe melioidosis [22]. Carbapanems have also been shown to be highly active againstB. pseudomallei [23]. As a result, resistance developed towards these antibiotics can pose a significant challenge in treatment of melioidosis.In our study, comparison of Etest and broth microdilution against disk diffusion test for 81B. pseudomallei strains and aB. thailandensis strain showed broad range of MICs and zone inhibition, respectively. Our findings may also be indicative of the increased antibiotic resistance inB. pseudomallei clinical strains. In short, it appears that either disc diffusion or Etest could be applied for susceptibility testing in the case of CL, AMC, DOXY, MERO, IMP, CAZ, TGC, CLA, and TS inB. pseudomallei isolates (Table 2).Additionally, in general, the Etest based antimicrobial susceptibility testing has the advantage of providing quantitative MICs results, which may be useful for clinicians to select appropriate treatments while the disc diffusion is a more reliable and cost effective technique for determining the prevalence of resistance amongB. pseudomallei isolates in the more routine monitoring of melioidosis. A study by Piliouras et al., on the comparison of antibiotic susceptibility testing methods for TS withB. pseudomallei, also reported that the disc diffusion test was inappropriate for assessing the susceptibility ofB. pseudomallei to TS. The study suggested that MIC based methods such as Etest may be a better choice for the determination of susceptibility for TS [24]. Difficulty in interpretation of disc diffusion results have also been implicated in previous studies which demonstrate varying susceptibility test results for TS withB. pseudomallei [25, 26]. This may be attributed to a few reasons including the imprecise endpoints for this combination of antimicrobial agent and organism as well as NCCLS guidelines being based on control standards ofPseudomonas aeruginosa and notB. pseudomallei [27, 28]. Thus, it is also evident that further creation of suitable internationally accepted MIC/zone inhibition breakpoints needs to be established through multilaboratory quality control. Several studies have been proposed for determination of the MICs, since these methods are cost effective and still able to produce reliable results [15, 29].We also performed molecular level investigation to detect the presence of efflux pump and class Aβ-lactamase genes, which were found to be present in all the MDRB. pseudomallei isolates. Previous studies have associated the mutations in class A β-lactamase gene ofB. pseudomallei to the resistance towards some cephalosporins and β-lactamase inhibitors [15]. It has been previously demonstrated that mutations in theB. pseudomallei class A β-lactamase coded bypenA may confer resistance of the organism to CAZ. Both the low and high level of CAZR in the study ofB. pseudomallei clinical isolates were confirmed by the detection ofpenA gene [15]. The presence ofpenA inB. pseudomallei used in our study matched the disc diffusion and MICs results.Additionally, the necessity to characterise genes encoding for MDR efflux pump arises from the increased resistance ofB. pseudomallei to a number of antibiotics. The presence of resistant nodulation division (RND) efflux pumps may contribute to the acquired resistance to fluoroquinolones and cross-resistant to unrelated antimicrobials [30]. This is not surprising as the efflux pumps can be associated with MDR isolates since they may be specific for one substrate or transport a wide range of antibiotics of multiple classes [31]. PrimersMxBs,MxYs, andMxFs were used to amplify genes that encode proteins which are parts of the RND efflux pump [30]. The role of this efflux pump is associated with resistance towards a wide range of antibiotics including pefloxacin, ofloxacin, and CAZ inB. pseudomallei and accompanied by an increased resistance to aminoglycosides, β-lactams, macrolides, and CL [30, 31]. In this study, isolates that contained these genes (bpeB,amrB, andBPSS1119) were shown to be resistant to AMC, CLA, and CAZ as reported using the disc diffusion and MICs. ThebpeB,amrB, andBPSS1119 genes used in our study focus on chromosomally encoded MDR efflux pumps, which have been the best developed and the most widely used for demonstrated efflux pump genes using primers [31].In conclusion, our study demonstrated the antimicrobial susceptibility profiles ofB. pseudomallei against antibiotics that are commonly used for treatment including CAZ, AMC, DOXY, and TS as well as other antimicrobials such as CL, MERO, IMP, TGC, and CLA. The antimicrobial susceptibility profiles did not show any significant correlation with the origin of the isolates as similarly reported in a previous study [32]. We also highlighted that the CAZR is due to the presence ofpenA,bpeB,amrB, andBPSS1119 inB. pseudomallei. However, except for the six blood isolates, none of the animal, soil, sputum, pus, lung, or spleen isolates were tested positive with the PCR detection assays forpenA,bpeB,amrB, andBPSS1119. Thus, it may be possible that the resistant blood isolates were from relapse patients or patients who did not comply with antibiotic treatment. However, this remains to be elucidated. Horizontal dissemination of these genes may contribute to further emergence of CAZR in various other Gram-negative bacteria. Therefore, appropriate surveillance and control measures are essential to prevent further spread of the CAZR organisms. Further study using PCR-single-stranded conformational polymorphism (PCR-SSCP) to identify the point mutations which take place in theB. pseudomallei isolates will be used to compliment and facilitate better understanding of the impact of CAZR in clinical practice.



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*Source: 132971-2014-10-14.xml*