Mechanisms for Rapid Evolution of Carbapenem Resistance in a Clinical Isolate of Pseudomonas aeruginosa

Abstract

Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic and acquired antibiotic resistance. Once colonized the human host, and thanks to antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which provide bacteria with antibiotic resistance as well as ability to better adapt to the host environment. Deciphering the evolutionary traits may provide important insights into the development of effective combinatory antibiotic therapy to treat P. aeruginosa infections. In this study, we investigated the molecular mechanisms by which a clinical isolate (ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA reference mapping were conducted to compare the transcriptional profiles and in vivo evolutionary trajectories between the two isolates. Our results demonstrated that oprD mutation together with ampC hyper-expression contributed to the increased resistance to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to negatively influence the ampC expression in P. aeruginosa.

Full text

fmicb-11-01390 June 18, 2020 Time: 17:19 # 1
ORIGINAL RESEARCH
published: 19 June 2020
doi: 10.3389/fmicb.2020.01390
Edited by:
Yuji Morita,
Meiji Pharmaceutical University, Japan
Reviewed by:
Carlos Juan,
Instituto de Investigación Sanitaria
de Palma (IdISPa), Spain
Erwin Bohn,
University Hospital Tübingen,
Germany
*Correspondence:
Fang Bai
baifang1122@nankai.edu.cn
Yongxin Jin
yxjin@nankai.edu.cn
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 31 January 2020
Accepted: 29 May 2020
Published: 19 June 2020
Citation:
Xu C, Wang D, Zhang X, Liu H,
Zhu G, Wang T, Cheng Z, Wu W,
Bai F and Jin Y (2020) Mechanisms
for Rapid Evolution of Carbapenem
Resistance in a Clinical Isolate
of Pseudomonas aeruginosa.
Front. Microbiol. 11:1390.
doi: 10.3389/fmicb.2020.01390
Mechanisms for Rapid Evolution of
Carbapenem Resistance in a Clinical
Isolate of Pseudomonas aeruginosa
Congjuan Xu1, Dan Wang1, Xinxin Zhang1, Huimin Liu2, Guangbo Zhu2, Tong Wang3,
Zhihui Cheng1, Weihui Wu1, Fang Bai1*and Yongxin Jin1*
1State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the
Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, China, 2Tianjin Union
Medical Center, Nankai University Affiliated Hospital, Tianjin, China, 3Department of Stomatology, Tianjin First Central
Hospital, Tianjin, China
Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic
and acquired antibiotic resistance. Once colonized the human host, and thanks to
antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which
provide bacteria with antibiotic resistance as well as ability to better adapt to the host
environment. Deciphering the evolutionary traits may provide important insights into the
development of effective combinatory antibiotic therapy to treat P. aeruginosa infections.
In this study, we investigated the molecular mechanisms by which a clinical isolate
(ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA
reference mapping were conducted to compare the transcriptional profiles and in vivo
evolutionary trajectories between the two isolates. Our results demonstrated that oprD
mutation together with ampC hyper-expression contributed to the increased resistance
to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding
murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to
negatively influence the ampC expression in P. aeruginosa.
Keywords: P. aeruginosa, carbapenem resistance, oprD,ampC,ldcA
INTRODUCTION
Pseudomonas aeruginosa, as an opportunistic human pathogen, is one of the leading causes of
nosocomial infections worldwide (Vincent et al., 1995). Infections by P. aeruginosa are difficult
to treat due to its intrinsic and acquired resistance to a wide range of antibiotics, leaving limited
number of effective antimicrobial agents. Carbapenems are used in clinical practice to treat
P. aeruginosa infections. However, carbapenem resistance of clinical P. aeruginosa isolates has been
increasingly reported (Davies et al., 2011). The mechanisms of carbapenem resistance are usually
multifactorial which include: (i) acquisition of carbapenemase encoding genes through horizontal
gene transfer (Poole, 2011;Potron et al., 2015), (ii) deficiency or repression of the porin (OprD)
for carbapenem (Davies et al., 2011;Poole, 2011), (iii) overexpression of mexAB-oprM efflux pump
(Poole, 2011;Liu et al., 2013;Choudhury et al., 2015), and (iv) overexpression of the chromosomal
gene (ampC) encoding the P. aeruginosa intrinsic cephalosporinase (Poole, 2011;Mirsalehian et al.,
2014). Although these and other studies have described the associated mechanisms of carbapenem
resistance among clinical isolates of P. aeruginosa, there is little information on the detailed
molecular mechanisms leading to the evolutionary dynamics of clinical P. aeruginosa isolates from
carbapenems susceptibility to resistance and the impact of each of these resistance mechanisms.
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In this study, we obtained two P. aeruginosa clinical isolates,
later demonstrated to belong to the same clone, from sputum
samples of the same patient with acute exacerbation of chronic
bronchitis before and after treatment with biapenem. The first
strain was obtained soon after the patient was admitted to the
hospital while the second strain was obtained 4 days after the
antibiotic treatment. The first isolate ISP50 was carbapenem
susceptible, whereas the second one IRP41 was carbapenem
resistant. Therefore, our goal was to decipher the molecular
mechanisms by which the carbapenem resistance had been
evolved so rapidly in the clinical setting. Our experimental results
demonstrated that an oprD null mutation combined with an
elevated ampC expression are the major contributory factors for
the conversion. Furthermore, we have shown for the first time
that LdcA functions as a repressor on the expression of ampC
in P. aeruginosa. These findings provide novel insights into the
regulatory mechanism of ampC expression in P. aeruginosa.
MATERIALS AND METHODS
Basic Characterization of the Bacterial
Strains
Bacterial strains and plasmids used in the study are listed in
Table 1. Carbapenem-susceptible (ISP50) and resistant (IRP41)
P. aeruginosa isolates characterized in this study were obtained
from sputum samples of the same patient with acute exacerbation
of chronic bronchitis before and after treatment with biapenem
for 4 days (dosage at 0.3 g ×2/day) at the Nankai University
Affiliated Hospital, Tianjin, China. The 16S rRNA encoding gene
was amplified (primers listed in Supplementary Table S1) and
sequenced to identify the species of these two isolates (Spilker
et al., 2004). Random amplified polymorphic DNA (RAPD)
typing was carried out using primer 272 as described previously
(Mahenthiralingam et al., 1996). Antibiotic susceptibility test
was conducted using the disk diffusion method according to
the manufacturer’s recommendations, and minimum inhibitory
concentration (MIC) of antibiotics was determined by the
two-fold serial dilution method. Susceptibility was interpreted
according to the Clinical and Laboratory Standards Institute
guidelines (CLSI 2011–2018).
Plasmid Construction
For overexpression of oprD, a 1,802 bp oprD-containing
fragment with its putative Shine-Dalgarno (SD) sequence
was PCR amplified using ISP50 and IRP41 genomic DNA as
templates (the used primers are displayed in Supplementary
Table S1). The PCR products were digested with BamHI
and HindIII, and then ligated into a shuttle vector pUCP24,
resulting in pUCP24-oprDISP50 and pUCP24-oprDIRP41,
respectively. Constructs of pUCP24-ampC, pMMB-ampRISP50,
pMMB-ampRIRP41, pUCP24-ldcAISP50 , pUCP24-ldcAIRP41 and
pUCP24-pbpC were all generated using similar procedures.
For deletion of the ldcA gene, a 836 bp fragment immediately
upstream of the ldcA start codon and a 913 bp fragment
downstream of the ldcA stop codon were PCR amplified,
digested with EcoRI-BamHI and BamHI-HindIII, respectively.
TABLE 1 | Bacterial strains and plasmids used in this study.
Strain or plasmid Description Source of
reference
Strains
DH5αF−φ80dlacZ1M15 endA1 recA1
hsdR17(rK−mK+)supE44 thi-1 relA1
1(lacZYA-argF)U169 gyrA96 deoR
TransGen
S17-1 RP4-2 Tc::Mu Km::Tn7TprSmrPro Res−
Mod+
Dr. Ramphal
ISP50 Clinical isolate from sputum samples of the
a female patient with acute exacerbations
of chronic bronchitis before treatment with
biapenem, IMPs
This study
IRP41 Clinical isolate from sputum samples of the
a female patient with acute exacerbations
of chronic bronchitis after treatment with
Biapenem for 4 days, IMPR
This study
Klebsiella
pneumoniae ATCC
700603
Klebsiella pneumoniae ATCC 700603 Fuxiang
Biotechnology
PA-NK41 KPC2 carbapenemase producing
P. aeruginosa
This study
PA14 Wild type P. aeruginosa strain Liberati et al.
(2006)
PA14pbpC::Tn PA14 with pbpC disrupted by insertion of
Tn
Liberati et al.
(2006)
PAO1 Wild type P. aeruginosa strain Kaufman et al.
(2000)
PAO11ldcA PAO1 with ldcA gene deleted This study
PA141ldcA PA14 with ldcA gene deleted This study
ISP501ldcA ISP50 with ldcA gene deleted This study
PAO11ampR PAO1 with ampR gene deleted This study
Plasmids
pUCP24 Shuttle vector between E. coli and
P. aeruginosa; Gmr
West et al.
(1994)
pMMB67EH-Gm Shuttle vector between E. coli and
P. aeruginosa; Gmr
Long et al.
(2019)
pEX18Tc Gene knockout vector, TcrHoang et al.
(1998)
pUCP24-oprDISP50 oprD gene from ISP50 in pUCP24, GmrThis study
pUCP24-oprDIRP41 oprD gene from IRP41 in pUCP24, GmrThis study
pUCP24-ampC ampC gene from ISP50 in pUCP24, GmrThis study
pUC18T-miniTn7T-
ampC-His
C-terminal His-tagged ampC in
pUC18T-miniTn7T, Tcr
This study
pUCP24-pbpC pbpC gene from ISP50 in pUCP24, GmrThis study
pUCP24-ldcAISP50 ldcA gene from ISP50 in pUCP24, GmrThis study
pUCP24-ldcAIRP41 ldcA gene from IRP41 in pUCP24, GmrThis study
pMMB-ampRISP50 ampR gene from ISP50 in
pMMB67EH-Gm, Gmr
This study
pMMB-ampRIRP41 ampR gene from IRP41 in
pMMB67EH-Gm, Gmr
This study
pEX18-ldcA ldcA gene deletion on pEX18Tc, TcrThis study
pEX18-ampR ampR gene deletion on pEX18Tc, TcrThis study
The two fragments were then ligated into pEX18Tc that was
digested with EcoRI and HindIII, resulting in pEX18-ldcA.
A pEX18-ampR was constructed by similar manipulations.
Gene knockouts in P. aeruginosa were carried out by conjugal
transfer of these plasmids followed by selection for single
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crossovers and then double crossovers as previously described
(Schweizer, 1992).
Multilocus Sequence Typing
Multilocus sequence typing (MLST) was performed following
a previous description (Curran et al., 2004) with minor
modifications to confirm the allelic profiles of the two isolates.
Briefly, genomic DNA was isolated from overnight bacterial
culture with a DNA purification kit (Tiangen Biotech, Beijing,
China) for use as PCR template. The internal fragments of
seven housekeeping genes (acsA,aroE,guaA,mutL,nuoD,ppsA
and trpE) were amplified by PCR and sequenced using primers
described previously (Supplementary Table S1) (Curran et al.,
2004). Gene sequences were then submitted to the P. aeruginosa
MLST database1for assignment of allelic numbers. A sequence
type (ST) was assigned to each isolate by combining the seven
allelic numbers.
PAE-MHT Assay for Carbapenemase
Test
The PAE-MHT assay was performed as described previously
(Pasteran et al., 2011) with minor modifications. Briefly, an
inoculum of Klebsiella pneumoniae ATCC 700603 was adjusted
to a 0.5 McFarland turbidity standard (Mcfarland, 1907) followed
by 10-fold dilution with sterile saline, and then inoculated onto
the surfaces of Mueller-Hinton agar (Oxoid) plates (diameter,
10 cm) by swabbing. The plates were allowed to stand at room
temperature. After 10 min, a disk of 10 µg imipenem/meropenem
(Oxoid, United Kingdom) was placed in the center of each
plate. Subsequently, one colony of each P. aeruginosa strain,
grown overnight on LB agar plate, was inoculated onto the
plate in a straight line from the edge of the disk to the
periphery of the plate using a 1-µl loop (BOOPU Biological
Technology, Changzhou, China). Presence of growth of the
K. pneumoniae ATCC 700603 toward imipenem/meropenem
disk was interpreted as carbapenemase positive.
Total RNA Isolation, RT-qPCR and
RNAseq Analysis
Bacterial overnight culture was inoculated into fresh LB medium
(1:50 dilution) and grown to an optical density of 1.0 (wavelength
of 600 nm). Total RNA was extracted using an RNAprep Pure
Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) and reverse
transcribed to cDNA with random primers and PrimeScript
Reverse Transcriptase (Takara). The cDNA was mixed with
indicated primers (Supplementary Table S1) and SYBR premix
Ex Taq II (Takara), and then quantitatively amplified in a
CFX Connect Real-Time system (Bio-Rad, United States).
A 30S ribosomal protein encoding gene rpsL was used as an
internal control.
For RNAseq, total RNA was extracted as described above,
quantified and qualified by Agilent 2100 Bioanalyzer (Agilent
Technologies, Palo Alto, CA, United States), NanoDrop (Thermo
Fisher Scientific Inc.) and 1% agarose gel. Then, the rRNA
1http://pubmlst.org/paeruginosa/
was depleted using Ribo-Zero rRNA Removal Kit (Bacteria,
Illumina). The mRNA was then fragmented and reverse-
transcribed. The double-strand cDNA was purified, ends repaired
and ligated with adaptors. After 11 cycles of PCR amplification,
the PCR products were cleaned, validated with an Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States),
and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad,
CA, United States). The resulting libraries were subjected to
sequencing using an Illumina HiSeq 2500 platform with a 2 ×150
paired-end configuration.
Sequence reads were mapped onto PAO1 reference genome
(NC_002516.2) via software Bowtie2 (v2.1.0). Gene expression
levels were analyzed with HTSeq (v0.6.1p1). Differentially
expressed genes were identified by the DESeq Bioconductor
package, with the fold-change larger than 2 and P-value no more
than 0.05 as cutoff values.
DNA Isolation and Reference Mapping
Bacterial genomic DNA was extracted with DNA purification
kit (Tiangen Biotech, Beijing, China). Fragments smaller than
500 bp were obtained from 200 ng genomic DNA by
sonication (Covaris S220), followed by end treatment and
adaptors ligation. Adaptor-ligated DNA fragments of about
470 bp were recovered using beads and then PCR amplified
for six cycles, and the PCR products were cleaned up using
beads, validated using a Qsep100 (Bioptic, Taiwan, China), and
quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA,
United States). Sequencing was carried out using a 2 ×150
paired-end (PE) configuration on an Illumina Hiseq instrument
according to manufacturer’s instructions (Illumina, San Diego,
CA, United States). The data were aligned to the PAO1 reference
genome (NC_002516.2) via software BW2 (version 0.7.12). Single
nucleotide variation (SNV) or InDel mutation were detected
using the software Samtools (version 1.1) and the Unified
Genotyper module from GATK.
PCR and Sequencing of oprD,ampR and
ldcA
The full-length oprD gene with its 105 bp upstream and 209 bp
downstream region was amplified and sequenced using primers
listed in Supplementary Table S1. The oprD gene sequence from
each isolate was aligned with the oprD sequence of the reference
strain PAO12. Analysis of ampR and ldcA were conducted with
similar procedures.
Western Blot Assay
Subcultured samples from equivalent number of bacterial cells
of an optical density of 1.0 (wavelength of 600 nm) were
separated on a 12% SDS-PAGE. The proteins were then
transferred onto a PVDF (polyvinylidene difluoride) membrane
and probed with a mouse monoclonal antibody against His-
tag (Cell Signaling Technology, United States) and the RNA
polymerase alpha subunit RpoA (Biolegend). Signals were
generated using the ECL-Plus kit (Millipore) and detected by a
Biorad imager (ChemiDocXRS).
2www.pseudomonas.com
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Quantification of Biofilm
Biofilm production was measured as described previously (Li
et al., 2013). Overnight culture of each bacterium was diluted
50-fold in LB broth and incubated in each well of a 96-well
plate at 37◦C. After 24 h, each well was washed with phosphate
buffered saline (PBS, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM
KCl, pH 7.4) for three times, stained with 0.1% crystal violet, and
then washed three times with PBS. Then, 0.2 ml of decolorizing
solution [methanol (4): acetic acid (1): water (5)] was added
into each well and incubated 10 min at room temperature. Each
sample was then subjected to measurement for OD590 using
Varioskan Flash (Thermo Scientific, Netherlands).
Statistical Analysis
Graphpad software was used to perform the statistical analyses.
The real-time qPCR results were analyzed by Student’s
t-test (two-tailed).
RESULTS
Clinical Isolates ISP50 and IRP41 Belong
to the Same Clone
Sputum samples from a patient with acute exacerbation of
chronic bronchitis were collected before and after treatment
with biapenem for 4 days (dosage at 0.3 g ×2/day). Two
mucoid isolates were selected, one before the biapenem treatment
which was sensitive to imipenem (ISP50), and the other one
after the biapenem treatment which showed resistance against
the imipenem (IRP41). PCR amplification of the 16S rDNAs
as well as their sequence analysis indicated that both of them
were P. aeruginosa (Supplementary Figure S1A). The MLST
analysis of both isolates revealed an allelic profile for acsA,aroE,
guaA,mutL,nuoD,ppsA and trpE as 17, 5, 12, 3, 14, 4, 7,
respectively, corresponding to the same sequence type, ST244.
RAPD typing was further performed on the IRP41 and ISP50
isolates, which also suggested that they were of the same clone
(Supplementary Figure S1B).
Isolate IRP41 Is Resistant to
Carbapenem
Imipenem susceptibilities of IRP41 and ISP50 were found
different based on the preliminary analysis with VITEK
automatic microbe analysis instrument (data not shown).
Therefore, we further examined the imipenem susceptibilities by
both MIC and disk diffusion methods, with the results shown
in Table 2 and Supplementary Figure S2. Initial isolate (ISP50)
was found to be susceptible to the imipenem (53.3 mm/diameter
of inhibition zone), while later isolate (IRP41) was resistant
to the imipenem (10.7 mm/diameter of inhibition zone),
with a 32-fold increase in MIC of imipenem. Although both
strains were susceptible to meropenem (another carbapenem
antibiotic) according to the Clinical and Laboratory Standards
Institute guidelines (CLSI 2011–2018), IRP41 displayed a smaller
diameter of inhibition zone (24 mm) compared to that of ISP50
(44 mm) (Supplementary Figures S2A–D). In addition, MIC
TABLE 2 | MICs (µg/ml) of indicated P. aeruginosa strains.
strains Imipenem
(µg/ml)a
Ampicillin
(µg/ml)
Meropenem
(µg/ml)a
Biapenem
(µg/ml)b
ISP50 0.3125 ND 3.125 0.78125
IRP41 10 ND 6.25 6.25
ISP50/pUCP24 0.3125 ND 3.125 0.78125
IRP41/pUCP24 10 ND 6.25 6.25
IRP41/pUCP24-oprDISP50 0.625 ND 3.125 3.125
IRP41/pUCP24-oprDIRP41 10 ND 6.25 6.25
IRP41/pUCP24-ldcAISP50 5 ND 3.125 3.125
IRP41/pUCP24-ldcAIRP41 10 ND 6.25 6.25
PAO1 1.5625 625 3.125 1.5625
PAO11ldcA 0.78125 1250 1.5625 0.78125
PA14 1.5625 312.5 1.5625 1.5625
PA141ldcA 0.78125 1250 0.3906 0.3906
ND, not determined; aClinical Laboratory Standards Institute (CLSI) susceptibility
breakpoints: imipenem, meropenem ≤2µg/ml, resistance breakpoints: imipenem,
meropenem ≥8µg/ml. bno CLSI breakpoint concentrations for biapenem have
been established, but previous studies suggested that MIC ≤4µg/ml is sensitive
and MIC ≥16 µg/ml is resistant (Hoban et al., 1993;Hang et al., 2018).
of meropenem/biapenem in IRP41 showed two-fold/eight-fold
increase compared to ISP50 (as displayed in Table 2).
Since the production of horizontally acquired carbapenem-
hydrolyzing enzymes has been defined as a contributory factor in
clinical P. aeruginosa isolates with carbapenem resistance (Potron
et al., 2015), we tested if the IRP41 produced carbapenemase
using a PAE-MHT assay. As the results shown in Supplementary
Figures S2E,F, compared to the PA-NK41, which produces a
KPC2 carbapenemase, no carbapenemase activity was observed
in the IRP41 or ISP50 strain, suggesting that the carbapenem
resistant phenotype of isolate IRP41 is not due to the presence
of any horizontally acquired carbapenemases.
Differential Expression of Genes
Relevant to β-Lactam Resistance
To elucidate the mechanism of reduced susceptibility to
carbapenem in isolate IRP41, we compared the global gene
expression profiles between strains ISP50 and IRP41. Expression
levels of 284 genes were altered comparing the two strains
(Table 3 and Supplementary Table S2). Among them, ampC
gene, encoding the β-lactamase, displayed a 285-fold higher
mRNA level in IRP41 than that in ISP50 (Table 3). To confirm
the up-regulation, we further examined the mRNA levels by real-
time qPCR. As shown in Figure 1A, consistent with the RNAseq
result, the mRNA level of ampC was 413-fold higher in the strain
IRP41 than that in ISP50. To further determine if the increased
ampC expression contributed to the reduced susceptibility to
carbapenem in IRP41, the ampC gene was overexpressed in
the ISP50 strain background and the MIC of carbapenems
was examined. As shown in Supplementary Table S3 and
Supplementary Figure S3A, overexpression of the ampC (1,239-
fold increase compared to that in ISP50/pUCP24) reduced
the susceptibility of ISP50 to imipenem/meropenem/biapenem,
with 16/2/4-fold increase in MIC. These results suggested
that increased expression of the ampC indeed contributed
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TABLE 3 | mRNA levels of genes related to β-lactam resistance and biofilm in
IRP41 compared to those in ISP50.
Gene ID Gene
name
Gene function Fold change
(IRP41/ISP50)
P-value
PA4110 ampC Beta-lactamase 285 6.42E−20
PA2233 pslC Biofilm formation protein PslC 30 4.71E−09
PA2272 pbpC Penicillin-binding protein 3A 8 1.49E−05
PA3719 armR MexR antirepressor ArmR 5 0.001828
PA2234 pslD Biofilm formation protein PslD 11 3.63E−06
PA2235 pslE Biofilm formation protein PslE 16 8.15E−08
PA2236 pslF Biofilm formation protein PslF 18 9.04E−08
PA2237 pslG Biofilm formation protein PslG 8 4.15E−05
PA2238 pslH Biofilm formation protein PslH 11 3.93E−06
PA2239 pslI Biofilm formation protein PslI 10 7.32E−06
PA2240 pslJ Biofilm formation protein PslJ 7 0.000124
PA2241 pslK Biofilm formation protein PslK 5 0.002139
PA2242 pslL Biofilm formation protein PslL 5 0.001121
to the decreased susceptibility of IRP41 to carbapenems, at
least partially.
Besides the ampC, a pbpC gene, encoding penicillin
binding protein 3a, displayed an eight-fold up-regulation in
IRP41 (Table 3). Real-time qPCR confirmed the increased
expression of the pbpC in IRP41 (Figure 1B). However,
overexpression of the pbpC (Supplementary Figure S3B)
in ISP50 (ISP50/pUCP24-pbpC) had no effect on the
bacterial MIC against imipenem/meropenem/biapenem
(Supplementary Table S3), although it restored the MIC of
carbenicillin in PA14pbpC::Tn to that of wild type PA14 strain
(Supplementary Table S3). These results indicated that the
elevated pbpC expression did not contribute to the carbapenem
resistance in IRP41.
Our RNAseq analysis also revealed that armR, encoding MexR
antirepressor (ArmR), was up-regulated five folds in IRP41. It
had been demonstrated that MexR represses the expression of
mexAB-oprM efflux pump (Daigle et al., 2007) whose over-
expression confers meropenem resistance in P. aeruginosa
(Okamoto et al., 2002). However, RNAseq results showed no
significant difference in the transcriptional levels of the mexAB-
oprM between the two strains, and this is confirmed by real-time
qPCR (Figures 1C,D). Further sequence analysis showed that no
mutation happened in the mexR,armR or mexR–mexA intergenic
region of the IRP41 and ISP50 strains (data not shown).
IRP41 Encodes an Inactive OprD
To further elucidate the molecular mechanisms of the decreased
susceptibility to carbapenem in IRP41, genome reference
mapping was performed to identify the accumulated mutations
in the genomes of IRP41 and ISP50 in comparison with PAO1
reference genome (see text footnote 2). Between strains ISP50
and IRP41, there are 84 frameshifts (deletion/insertion) and 377
nonsynonymous single nucleotide variations (SNV) (including
17 early stops) (Supplementary Table S4). Among them, a
G831A substitution in the oprD gene resulted in a premature
termination of the OprD in the IRP41 strain. To confirm the
result, oprD genes were PCR amplified from the genomic DNA
of strains ISP50 and IRP41. Sequence analysis of the amplicons
revealed that oprD in ISP50 was the same as that of PAO1,
while the oprD from IRP41 showed a G831A substitution, which
confirmed the reference mapping results. To assess if the G831A
substitution in oprD contributed to the reduced susceptibility of
the IRP41 to carbapenems, the oprD gene from both ISP50 and
IRP41 were expressed in the IRP41 strain background. As can
be observed in Table 2, introduction of the oprDISP50 reduced
the MIC of imipenem/meropenem/biapenem in IRP41, with
16/2/2-fold decrease, while the oprDIRP41 did not. These results
indicated that the G831A substitution in oprD indeed disrupted
its function and lead to the reduced susceptibility to carbapenem
in strain IRP41.
Mechanisms of ampC Hyper-Expression
in IRP41
AmpR is a transcriptional regulator encoded immediately
upstream of the ampC, regulating the ampC expression (Kong
et al., 2005). The genome reference mapping result revealed that
the ampR sequence of IRP41 is identical to that of PAO1, while
in strain ISP50 an “A” was substituted by “G” at 821th position of
the ampR gene, resulting in an E274G substitution. These results
were further confirmed by sequencing of the PCR amplicons. To
test if the nonsynonymous mutation in ampR of ISP50 resulted in
a lower level of ampC expression, both ampR genes from IRP41
and ISP50 were cloned and expressed in the background of ISP50.
As shown in Supplementary Figure S3C and Supplementary
Table S3, expression of the ampRISP50 conferred ISP50 strain a
higher ampC mRNA level and increased MIC against ampicillin
compared to that of ampRIRP41, although it had no observable
influence on the MIC of imipenem/meropenem/biapenem.
The ampRISP50 and ampRIRP41 were further introduced
into PAO11ampR, and their effects on MICs to imipenem,
meropenem, biapenem and ampicillin in PAO11ampR were
examined. As shown in Supplementary Table S3, both
ampRISP50 and ampRIRP41 restored the MIC against ampicillin
and biapenem in PAO11ampR to that in wild type PAO1
strain, while in contrast to ampRIRP41, the ampRISP50 conferred
PAO11ampR a repeatable two-fold increase in MIC against
imipenem compared to that of wild type PAO1. Unexpectedly, no
influence on MIC of meropenem was observed in PAO11ampR.
These results suggested that A821G SNV of ampR in fact confers
slightly higher ampC expression-activating capacity than the
wild type AmpR, thus resulting in higher, rather than lower
ampC in ISP50.
ldcA (PA5198) encodes a cytosolic LD-carboxypeptidase
which removes the terminal amino acid D-alanine from the
tetra peptide of the peptidoglycan (Korza and Bochtler, 2005).
Our genome reference mapping and PCR amplicons sequencing
results revealed that a C was deleted at 445 bp of the ldcA gene
in strain IRP41, leading to a frameshift mutation after R149 of
the LdcA. To test if this frameshift mutation in ldcA contributed
to the elevated expression of the ampC in IRP41, we expressed
the ldcAIRP41 or ldcAISP50 (same as ldcA of PAO1) in the IRP41
background. As shown in Figure 2A, the relative mRNA level of
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FIGURE 1 | Relative mRNA levels of indicated genes in indicated strains. (A) Relative mRNA levels of ampC in indicated strains. (B) Relative mRNA levels of pbpC in
indicated strains. (C) Relative mRNA levels of armR in ISP50 and IRP41. (D) Relative mRNA levels of mexB in ISP50 and IRP41. Total RNA was isolated from
indicated strains at OD600 of 1.0, and the relative mRNA levels of indicated genes were determined by real-time qPCR using rpsL as an internal control. ns, not
significant, ****P<0.0001, by Student’s t-test.
ampC in IRP41/pUCP24-ldcAISP50, but not in IRP41/pUCP24-
ldcAIRP41, was restored to that in ISP50/pUCP24, and the
MIC to imipenem/meropenem/biapenem in IRP41/pUCP24-
ldcAISP50 was decreased by two folds (Table 2). To confirm the
expression level of ampC gene, a C-terminal His-tagged ampC
driven by its native promoter was integrated into the genomes
of ISP50 and IRP41. As shown in Figure 2B, the AmpC-His level
was much higher in IRP41 than that in ISP50, and overexpression
of ldcAISP50, but not ldcAIRP41 , reduced the amount of AmpC-
His in the IRP41 to almost that of the ISP50. These results
demonstrated that frameshift-mutation of the ldcA in IRP41
contributes to the increased expression of the ampC in IRP41.
To further confirm the effect of ldcA on ampC repression,
the whole ldcA open reading frame was deleted in PA14 and
PAO1 backgrounds, resulting in PA141ldcA and PAO11ldcA,
respectively. Similar to previous report in E. coli (Templin et al.,
1999), both of the ldcA deletion mutants showed a reduced
growth rate and proneness to lysis during stationary growth
phase (Supplementary Figure S4). Interestingly, the expression
levels of ampC displayed 2.6-fold and 4.0-fold increase in
the 1ldcA mutants compared to their parental strains PAO1
and PA14, respectively (Figure 3A). Accordingly, the MIC of
ampicillin in PAO11ldcA and PA141ldcA displayed a two-fold
and four-fold increase compared to their respective parental
strains (Table 2). We further deleted the ldcA gene from the ISP50
strain. Consistent with the above observations, the expression
level of ampC in the ISP501ldcA increased 1.8-fold compared
to that in the ISP50 strain (Figure 3B). The difference in fold
increase of ampC mRNA levels between ISP50 and IRP41 (413
folds) vs. between ISP50 and ISP501ldcA (1.8 folds) suggested
that other factors also contributed to the increased expression of
the ampC in IRP41.
In addition, we also examined the effect of ldcA mutation on
MICs against piperacillin, ceftazidime, aztreonam, ciprofloxacin
and tobramycin. Except for a two-fold decrease against
ceftazidime and meropenem in PAO11ldcA, no MICs change
was observed for piperacillin, aztreonam, ciprofloxacin and
tobramycin in ldcA mutants (Table 4). However, we didn’t
obtain the IRP411ldcA, although deletion of ldcA in IRP41
strain had been tried for several times. We assume that it may
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FIGURE 2 | Mechanism of increased mRNA levels of ampC in IRP41. (A) Relative mRNA levels of ampC gene in indicated strains. Total RNA was isolated from
indicated strains at OD600 of 1.0, and the relative mRNA levels of ampC were determined by real-time qPCR using rpsL as an internal control. ns, not significant,
***P<0.001, by Student’s t-test. (B) Levels of AmpC-His in indicated strains detected with Western blot assay. Strains with an ampC-His in their chromosomes
were cultured to OD600 of 1.0. Samples from equal number of bacterial cells were loaded onto 12% SDS-PAGE gels and probed with an antibody against His-tag or
RpoA. The RNA polymerase alpha subunit RpoA served as a control.
FIGURE 3 | Relative mRNA levels of ampC gene in indicated strains (A,B). Total RNA was isolated from indicated strains at OD600 of 1.0, and the relative mRNA
levels of ampC gene were determined by real-time qPCR using rpsL as an internal control. **P<0.01, ***P<0.001, ****P<0.0001, by student’s t-test.
be due to presence of some unknown factors to prevent the
recombination in IRP41.
Since AmpR is the regulator of ampC, and its 274th
amino acid is different between IRP41 and ISP50, we
further compared the ampC expression levels and the MIC
to ampicillin/imipenem/meropenem/biapenem between
ISP501ldcA expressing AmpRISP50 and AmpRIRP41. As
shown in Supplementary Figure S3D, consistent with that
in the ISP50 strain, expression of AmpRISP50 in ISP501ldcA
(ISP501ldcA/pMMB-ampRISP50) resulted in a higher mRNA
level of ampC compared to that of ISP501ldcA/pMMB-
ampRIRP41, as well as a two-fold increase in MIC of ampicillin
(Supplementary Table S3). Similar to that in ISP50 strain,
overexpression of both AmpRIRP41 and AmpRISP50 showed no
influence on MIC of imipenem/meropenem/biapenem in the
ISP501ldcA strain. Again, these are consistent with the earlier
observation that the AmpR point mutation in ISP50 confers a
higher transcriptional activator activity on ampC.
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Furthermore, as the expression level of pbpC is much higher
in IRP41 than that in ISP50, we overexpressed pbpC in the
ISP501ldcA background to see if the elevated pbpC expression
had any effect on the ampC mRNA levels. However, the mRNA
level of ampC was not affected by the overexpression of pbpC
in ISP501ldcA (data not shown), consistent with such null effect
observed in the ISP50 background (Supplementary Table S3 and
Supplementary Figure S3E).
IRP41 Is a Better Biofilm Former Than
ISP50
Our RNA-seq result also revealed an increased expression
of pslD-pslL operon (Table 3) which is involved in biofilm
formation. Accordingly, we examined and compared the biofilm
formation between IRP41 and ISP50 strains. As the results
shown in Figure 4, the IRP41 strain produced more biofilm than
the ISP50 strain.
DISCUSSION
In this study, we examined MICs of imipenem, meropenem and
biapenem, three carbapenem antibiotics, for all the strains in
Table 2 and Supplementary Table S3. Compared to meropenem,
the contribution of AmpC overproduction and OprD deficiency
to biapenem susceptibility in IRP41 resembles to that of
imipenem. The difference among the susceptibility to imipenem,
meropenem and biapenem between ISP50 and IRP41 may reflect
their difference in penetration rate, stability to the hydrolysis
by β-lactamase, PBP protein binding profile (Yang et al.,
1995;Bonfiglio et al., 2002), β-lactamase deactivating capability
and export by the efflux pumps (Chen and Livermore, 1994;
Okamoto et al., 2002).
AmpC expression is regulated by AmpR, a LysR family
transcriptional regulator (Kong et al., 2005). Previous studies
reported that ampR gene point mutations were associated with
increased ampC expression in both Enterobacter cloacae and
P. aeruginosa clinical isolates (Kuga et al., 2000;Bagge et al.,
2002). Our study presents E274G substitution in AmpR actually
lead to a higher level of ampC expression. Besides the expression
of ampC, AmpR regulates expression of hundreds of genes
involved in diverse pathways such as physiological processes
and metabolism (Balasubramanian et al., 2015). It has been
demonstrated that ampR mutant displays an impaired growth
under iron-limiting conditions and sensitive to many agents
that affect cell growth (Balasubramanian et al., 2014, 2015).
Thus, it is possible that restoration to wild type AmpR from
E274G substituted AmpR of ISP50 provided the IRP41 strain
TABLE 4 | MICs (µg/ml) of indicated P. aeruginosa strains.
Strains TAZ (µg/ml) Cipro (µg/ml) Tob (µg/ml) Pip (µg/ml) AZT (µg/ml) Mem (µg/ml)
PAO1/pUCP24 3.125 0.3125 8 31.25 1.953125 3.125
PAO11ldcA/pUCP24 1.5625 0.3125 8 31.25 1.953125 1.5625
PAO11ldcA/pUCP24-ldcA 3.125 0.3125 8 31.25 1.953125 3.125
ISP50/pUCP24 3.125 0.625 16 62.5 7.8125 3.125
ISP501ldcA/pUCP24 3.125 0.625 16 62.5 7.8125 3.125
ISP501ldcA/pUCP24-ldcA 3.125 0.625 16 62.5 7.8125 3.125
TAZ, ceftazidime; Cipro, ciprofloxacin; Tob, tobramycin; Pip, piperacillin; AZT, aztreonam; Mem, meropenem.
FIGURE 4 | Biofilm formation by ISP50 and IRP41 strains. Overnight cultures of ISP50 and IRP41 were 50-fold diluted in LB and incubated in each well of a 96-well
plate for 24 h at 37◦C. Then the biofilm was stained with 0.1% crystal violet (A), dissolved by 0.2 ml of decolorizing solution and subjected to measurement at OD590
(B). ****P<0.0001 by Student’s t-test.
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a better adaption to the host environment. Also, an increased
expression of the pbpC is neither responsible for the higher
ampC expression nor the decreased susceptibility to carbapenem
in IRP41 strain. The regulatory mechanism for the elevated
pbpC expression in IRP41 and its functional roles remain to
be investigated.
AmpD, a cytosolic peptidoglycan amidase, cleaves the peptide
chain from N-acetyl-anhydromuramic acid peptides which are
the inducer molecules for the ampC expression via binding to
the AmpR (Langaee et al., 1998;Lister et al., 2009;Dhar et al.,
2018). Mutations in AmpD and its two homologous proteins,
AmpDh2 and AmpDh3, contribute to a stepwise de-repression
of the ampC in the wild-type strain PAO1 (Juan et al., 2006).
Similarly, ampD mutations leading to ampC de-repression have
been reported in clinical isolates (Schmidtke and Hanson, 2008).
However, some AmpC over-expressing P. aeruginosa strains do
not exhibit mutation in ampD,ampDh2,ampDh3,ampR, or the
ampR–ampC intergenic region (Wolter et al., 2007;Schmidtke
and Hanson, 2008), suggesting that other undiscovered factors
or pathways likely contribute to the upregulation of ampC
expression. Besides, mutational inactivation of dacB, encoding
penicillin-binding protein 4 (PBP4), has been reported to
trigger a stable AmpC overproduction in P. aeruginosa
(Moya et al., 2009). Mutation of the lytic transglycosylases
encoding genes sltB1 and mltB1 in PAO1 resulted in a stable
AmpC hyperproduction in the presence of β-lactam antibiotic
(Cavallari et al., 2013;Juan et al., 2017). Inactivation of
mpl (encoding cytosolic UDP-N-acetylmuramate:L-alanyl-γ-D-
glutamyl-meso-diaminopimelate ligase) and nuoN (encoding
NADH dehydrogenase I chain N) led to an increase in the
expression of ampC (Tsutsumi et al., 2013;Juan et al., 2017). In
the two isolates used in this study, no mutation was found in
the above genes, except for the AmpR with E274G substitution
in ISP50 which had no effect on the increased ampC expression
in IRP41. These suggested the presence of unknown molecular
basis driving AmpC hyperproduction in the IRP41 strain.
In the present study, inactivation of the ldcA contributes to
the elevated expression of the ampC in P. aeruginosa. LdcA,
encoding an LD carboxypeptidase, cleaves the D-alanine from
N-acetyl-anhydromuramic acid tetrapeptides (anhNAM-P4) in
P. aeruginosa (Korza and Bochtler, 2005), generating N-acetyl-
anhydromuramic acid tripeptide (anhNAM-P3). Functional loss
of the LdcA leads to the accumulation of anhNAM-P4, thus our
observation suggests that the anhNAM-P3 is less potent inducer
for the ampC expression than the anhNAM-P4. This is consistent
with a previous study in which the anhNAM-P4 was shown
to be a critical activator ligand for β-lactamase expression in
Stenotrophomonas maltophilia (Huang et al., 2017). Results from
previous studies implied anhNAM-P5 as the genuine AmpR-
binding signal (Dietz et al., 1997;Lee et al., 2016) and further
suggested that only muropeptides containing a terminal d-Ala-
d-Ala motif (i.e., muropentapeptides) is capable of binding to
the AmpR for ampC induction (Vadlamani et al., 2015;Dik
et al., 2017). However, in a most recent report, anhNAM-
P3 was demonstrated to function as an ampC inducer, albeit
much less potent than the anhNAM-P5 (Torrens et al., 2019).
As these studies were carried out under different conditions
(with or without antibiotic induction) using different mutant
backgrounds, it is possible that different signaling pathways may
have been involved in the AmpR mediated regulation of the
ampC in the clinical strains analyzed in this study.
This is the first report linking ldcA to the regulation of ampC
expression in P. aeruginosa. Interestingly, compared to the 400-
fold increase of ampC mRNA in IRP41, the 1ldcA mutants
of PA14, PAO1 and ISP50 showed only 1.8–4 folds increases
in the ampC mRNA compared to their parental strains. Such
minor changes in the ampC expression, in addition to the growth
defect of the ldcA mutant at stationary phase, may explain why
LdcA was not identified as a repressor for the ampC in previous
studies. One may argue that the observed 2–4 folds increase in
MIC to ampicillin may be due to the reduced growth of the
1ldcA mutant. However, we do not feel this is the case because
the proneness to lysis happens during the stationary growth
phase (OD600 about 2.0). In the case of IRP41, we postulate
that there are other factors contributing to the increased ampC
expression in addition to the ldcA. The mechanism underlying
this observation is yet to be elucidated.
OprD has been reported to be the most prevalent cause of
imipenem resistance among clinical isolates of P. aeruginosa.
Insertion of various IS elements and point mutations with
premature stops on the oprD gene had been associated
with the carbapenem resistance among clinical isolates of
P. aeruginosa (Hirabayashi et al., 2017). In this study, a G831A
substitution in oprD resulted in loss of the OprD function.
With the OprD inactivation and elevated ampC expression,
the clinical isolate IRP41 displayed resistance to carbapenem.
It has been demonstrated that derepression of AmpC in
PAO1 has no obvious impact on the MIC of imipenem (Juan
et al., 2006). While in another study, the loss of AmpC
from PAO1 displayed a four-fold increase in susceptibility to
imipenem (Masuda et al., 1999). Derepression of AmpC was
associated with 4–8/8–64-fold increase in MIC of imipenem in
an OprD+/OprD−background, respectively (Livermore, 1992;
Mushtaq et al., 2004). And also, mutational variants of the
AmpC cephalosporinase provide P. aeruginosa with imipenem
resistance (Rodriguez-Martinez et al., 2009). In our study,
overexpression of ampC conferred ISP50 a 16-fold increase in
MIC of imipenem. However, both AmpC and OprD are of PAO1-
type in ISP50, which suggests that other unknown factors may
contribute to the change in susceptibility to imipenem by the
overexpression of AmpC.
MexAB-OprM is able to expel a wide variety of antibiotics
(Kohler et al., 1999;Lister et al., 2009) and its expression is
repressed by MexR (Poole et al., 1996). A 53-amino-acid long
antirepressor, ArmR, could interact with and inhibit the MexR
function, upregulating the mexAB-oprM expression (Wilke et al.,
2008). However, in the present study, increased expression of
armR in IRP41 did not cause altered expression level of mexAB-
oprM. In fact, mexAB-oprM is expressed and contributes to
antimicrobial resistance in wild type Pseudomonas aeruginosa,
it is more difficult to detect mexAB-oprM overexpression in
mutant cells than the other efflux pump encoding genes (e.g.,
mexCD-oprJ,mexEF-oprN). In addition, previous study has
revealed MexR is a redox regulator which senses oxidative
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stress inside bacterial cells to regulate mexAB-oprM expression
(Chen et al., 2008). nalD encodes a second repressor of the
mexAB-oprM (Morita et al., 2006). Mutations in nalD resulted in
increased MexAB-OprM expression in lab and clinical isolates of
P. aeruginosa (Sobel et al., 2005). However, no mutation occurred
in the nalD gene of the IRP41 or ISP50 isolates. The mechanisms
underlying these observations are under active investigation.
It has been demonstrated that nalC (PA3721), encoding a
TetR family repressor, negatively regulated armR, and S127P
substitution in NalC impaired its armR-repressing capacity (Cao
et al., 2004). Compared to ISP50, no mutations were observed
in the intergenic region between nalC and PA3720, while a
S127P substitution in NalC happened in IRP41 (Supplementary
Table S4), which may be the cause to result in the increased armR
transcriptional level in IRP41.
IRP41 is a better biofilm former than ISP50. However, the
MICs against ciprofloxacin and levofloxacin in IRP41 are the
same as ISP50 (data not shown), and the MICs against gentamicin
and tobramycin display a two-fold decrease in IRP41 than that
in ISP50 (data not shown). A previous study had shown that the
Psl did not affect bacterial MIC to biapenem for planktonic cells
(Murakami et al., 2017). Therefore, we assumed that the increased
biofilm production did not contribute to the increased MIC to
carbapenem in IRP41.
It is not necessary for clinical isolates processing a major
resistance determinant against a certain antibiotic category
to accumulate the other resistance mechanisms. However,
the concomitant presence of multiple carbapenem resistance
mechanisms has been observed in P. aeruginosa (Meletis et al.,
2014). In our study, the overproduction of AmpC β-lactamase
together with OprD deficiency leads imipenem-susceptible ISP50
to imipenem-resistant IRP41 derivative, as well as a reduced
susceptibility to meropenem and biapenem.
DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in NCBI, under
accession PRJNA635437.
ETHICS STATEMENT
We have a waiver from the ethics committee, exempting this
study from the requirement to have ethics approval and written
informed as the clinical strains used in the study come from
the routine procedures of the clinical laboratory rather than the
clinical trials.
AUTHOR CONTRIBUTIONS
YJ and FB conceived and designed the experiments. CX, DW,
XZ, HL, GZ, and TW performed the experiments. WW, FB, ZC,
and YJ analyzed the data. YJ wrote the manuscript. All authors
contributed to the article and approved the submitted version.
FUNDING
This work was supported by the National Science Foundation
of China (31600110, 31870130, 31670130, 81670766, and
31970680), Science and Technology Committee of Tianjin
(17JCQNJC09200 and 19JCYBJC24700), National Key Research
and Development Project of China (2017YFE0125600), Science
and Technology Program of Sichuan Province (2018JZ0069), and
the Fundamental Research Funds for the Central Universities,
Nankai University (63191121, 63191117, and 63191521).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2020.01390/full#supplementary-material
FIGURE S1 | PCR results of ISP50 and IRP41 strains. (A) 16S rDNA gene
amplification; (B) RAPD typing.
FIGURE S2 | Carbapenem susceptibility and carbapenemase production of
indicated strains. (A,B) Imipenem inhibition zones of indicated strains on a 6-cm
disk. (C,D) Meropenem inhibition zones of indicated strains on a 6-cm disk. (E,F)
PAE-MHT assay using K. pneumoniae ATCC 700603 as indicator.
Carbapenemase production test on imipenem (E) and meropenem (F) on a 10-cm
disk, 0: ISP50, 1: IRP41, 2: PA-NK41, a producer of KPC2 carbapenemase.
FIGURE S3 | Relative mRNA levels of indicated genes in indicated strains.
(A,C–E) Relative mRNA levels of ampC in indicated strains. (B) Relative mRNA
levels of pbpC in indicated strains. Total RNA was isolated from indicated strains
at OD600 of 1.0, and the relative mRNA levels of ampC or pbpC gene were
determined by real-time qPCR using rpsL as an internal control. ns, not significant,
∗P<0.05, ∗∗ P<0.01, ∗ ∗ ∗ P<0.001, ∗ ∗ ∗ ∗ P<0.0001, by Student’s t-test.
FIGURE S4 | Growth curves of indicated strains.
TABLE S1 | Primers used in this study.
TABLE S2 | Transcriptome analysis: differentially expressed genes by RNAseq
without those listed in Table 2.
TABLE S3 | MICs (µg/ml) of indicated P. aeruginosa strains.
TABLE S4 | SNVs identified by genome reference mapping with elimination of
SNVs with synonymous mutation and the same mutation between
ISP50 and IRP41.
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