Mutations in Neisseria gonorrhoeae grown in sub-lethal concentrations of monocaprin do not confer resistance
Mutations in Neisseria gonorrhoeae grown in sub-lethal concentrations of monocaprin do not confer resistance
Colin P. Churchward¤ 0 1 2
Alan Calder 0 1 2
Lori A. S. Snyder 0 1 2
0 Current address: National Heart and Lung Institute, Imperial College London , London , United Kingdom
1 School of Life Sciences , Pharmacy, and Chemistry , Kingston University , Kingston upon Thames , United Kingdom
2 Editor: William M. Shafer, Emory University School of Medicine , UNITED STATES
Neisseria gonorrhoeae, due to its short lipooligosaccharide structure, is generally more sensitive to the antimicrobial effects of some fatty acids than most other Gram negative bacteria. This supports recent development of a fatty acid-based potential treatment for gonococcal infections, particularly ophthalmia neonatorum. The N. gonorrhoeae genome contains genes for fatty acid resistance. In this study, the potential for genomic mutations that could lead to resistance to this potential new treatment were investigated. N. gonorrhoeae strain NCCP11945 was repeatedly passaged on growth media containing a sub-lethal concentration of fatty acid myristic acid and monoglyceride monocaprin. Cultures were resequenced and assessed for changes in minimum inhibitory concentration. Of note, monocaprin grown cultures developed a mutation in transcription factor gene dksA, which suppresses molecular chaperone DnaK and may be involved in the stress response. The minimum inhibitory concentration after exposure to monocaprin showed a modest two-fold change. The results of this study suggest that N. gonorrhoeae cannot readily evolve resistance that will impact treatment of ophthalmia neonatorum with monocaprin.
Funding: This work was supported by the Sparks
Children's Medical Research charity (www.sparks.
org.uk), which has provided for this work with
Project Grants 11KIN01 and 13KIN01 to support
Colin Churchward and this research. The funder
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
Monocaprin is a powerful fast-acting bactericidal agent against Neisseria gonorrhoeae [
has recently been proposed as a candidate for treatment of gonococcal eye infections, such as
ophthalmia neonatorum, where topical treatment would rapidly kill the bacteria without
irritating the eye . As with any antimicrobial, attention must be given to possible resistance
mechanisms. There are many ways that bacteria can acquire resistance to antimicrobials and
some of these can be induced in the laboratory experimentally. Growth of the bacteria on
media containing a sub-lethal concentration of the antimicrobial will select for genomic
mutations that confer an addition level of fitness in this environment to out-compete non-mutated
cells. Previously, N. gonorrhoeae has been passaged on media containing increasing amounts
of the fluoroquinolone ciprofloxacin, resulting in an isolate that had 10,000 times greater
resistance than the parental isolate [
]. Mutations in the gyrA and parC genes were identified;
mutations in the same genes also developed naturally in clinical isolates [
]. By 2006, the US
Centres for Disease Control and Prevention no longer recommended fluoroquinolones for
treatment of gonococcal infections . Trying to predict how an organism will change
genetically and / or phenotypically given a set of conditions or given a certain stimuli in the
laboratory is known as experimental evolution. Bacteria are commonly studied in this way as they
have short generation times which make them ideal for this method of study. The now low
cost of next generation sequencing means that resequencing whole genomes following
experimental evolution is feasible. This ªevolve and re-sequenceº strategy [
] can identify
mutations in unexpected regions of the genome.
Although the lipopolysaccharide (LPS) of most Gram negative bacteria provides some
intrinsic resistance to fatty acids, N. gonorrhoeae have short lipooligosaccharide (LOS)
structures and are generally more sensitive to fatty acids [
]. N. gonorrhoeae possesses other
mechanisms to protect itself from the antimicrobial action of fatty acids. It has previously been
shown that isolates from men that have sex with men have reduced permeability of
hydrophobic agents [
]. The FarA-FarB-MtrE efflux pump has been demonstrated to confer decreased
sensitivity to certain fatty acids [
]. Transcription of farAB is controlled by the FarR protein
and integration host factor [
]. Therefore, mutations in farA, farB, mtrE, their promoters,
or any of their regulators could have an effect on the resistance profile of the bacteria.
The purpose of this study was to identify genomic mutations that resulted from passage of
N. gonorrhoeae on media containing sub-lethal concentrations of the monoglyceride
monocaprin and to determine any changes in the minimum inhibitory concentration (MIC). Parallel
cultures grown in a sub-lethal concentration of the saturated fatty acid myristic acid (C14:0).
Myristic acid was chosen as a positive control for selection of genomic mutations and increase
of MIC because it has very good bacteriostatic properties against N. gonorrhoeae [
] but also
has a known mechanism of resistance in the farAB-mtrE encoded efflux pump system [10±12].
An ocular formulation of monocaprin is a promising candidate for the treatment of
ophthalmia neonatorum [
], particularly in cases of antibiotic resistant gonococcal infections,
therefore understanding the potential to develop resistance and the mechanisms involved are
Results and discussion
The MIC values before experimental evolution were 125 μM for myristic acid and 250 μM for
monocaprin. Therefore, sub-lethal concentrations of 62.5 and 125 μM were used in the
experimental evolution for myristic acid and monocaprin, respectively. The non-selective, myristic
acid-, and monocaprin-containing cultures were successfully grown for a total of twenty
passages each. Growth on plates containing myristic acid and monocaprin was observed to be
slower than those on non-selective plates, especially the first few passages on the myristic
acidcontaining plates. The MICs were unchanged between the starting culture and the
non-selective cultures after 20 passages (Table 1).
It is known that N. gonorrhoeae have an efflux pump-based mechanism of resistance against
myristic acid [10±12]. Loss of the FarR regulator of expression of the farAB efflux pump genes
results in an increase in the MIC for those fatty acids that are substrates of the efflux pump,
including myristic acid [
]. After 20 passages, the bacteria grown on media with sub-lethal
myristic acid had a sixteen-fold increase in MIC compared to the starting inoculum (Table 1).
One myristic acid grown replicate (14:0±1) appeared to have a growth rate advantage over the
other replicate, although the MICs were the same. The MICs of the bacteria grown on
sublethal monocaprin increased eight-fold for myristic acid in both duplicate samples. It appears
that growth in the presence of sub-lethal monocaprin has conditioned the N. gonorrhoeae for
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Monocaprin MIC (μM)
Myristic acid MIC (μM)
growth on myristic acid resulting in an increase in the myristic acid MIC. From these results,
it is evident that resistance to myristic acid can readily develop, which is not unexpected
considering previous studies [10±12].
After experimental evolution, the monocaprin MIC for the samples passaged on plates
containing myristic acid or monocaprin were both 500 μM (Table 1). Unlike the eight and sixteen
fold change for the myristic acid MIC, the MIC for monocaprin has simply doubled that of the
starting inoculum (Table 1). This suggests that passage on sub-lethal concentrations of both
myristic acid and monocaprin prepares the bacterial cells for growth on a modest increase in
monocaprin. Mechanisms contributing to such an increase may be related to a general stress
The paired sequencing reads from MicrobesNG were successfully mapped to the GenBank
N. gonorrhoeae strain NCCP11945 reference genome [
]. Twelve mutations within coding
sequences (CDSs) were present in all the sequenced samples in comparison to the reference
sequence (Table 2). These differences are either the result of sequencing errors from the
original sequencing, sequencing errors in our data, or mutations that have occurred in the N.
gonorrhoeae strain NCCP11945 isolate during the few passages between it being sequenced
] and reaching our laboratory. The original sequencing was done by Sanger sequencing
with an eight-fold coverage and predicted error rate of 0.15 per 10,000 bases [
], which would
equate to 33 errors in the 2,232,025 bp genome. These sequencing differences identified here
were either in pseudogenes or are predicted by SNAP2 [
] not to cause a functional effect in
the encoded protein.
GAC ! GCC
GCC ! GGC
ACG ! GCG
AAC ! AGC
GGT ! G-T
GGT ! G-T
ATC ! AGC
TTT ! TCT
GAT ! GAA
ATA ! AAA
GAC ! CTT
CTG ! ATC
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Ten unique mutations were found in CDSs of the non-selectively passage cells (Table 3),
although none of these were present in both replicates (Table 3). These changes are indicative
of the general rate at which mutations can occur and become part of the bacterial population.
Four of the mutations are present in all of the sequencing reads from the isolate, whilst the
remainder are present in the majority of reads. Each of these CDS mutations would result in a
nonsynonymous change, including two that generate premature termination codons, albeit
late in the gene for NGK_RS05395 at 351/400, and one that alters the initiation codon to the
less favoured TTG (Table 3). These cultures have been continuously maintained on standard
gonococcal media and passaged to fresh media every two to three days. This is often standard
practice in research laboratories where cultures can be kept growing in incubators for
extended periods of time. However as evidenced here, mutations can arise via repeated passage
that may impact downstream experimental use of the bacterial culture. Care must be taken to
use minimally passaged isolates from well-maintained freezer stocks particularly when
comparing parent and experimentally generated mutants. When comparing results to the reference
genome, the potential for mutations to have arisen in the course of experiments must be
Six sequence differences were found in CDSs from the samples passaged on sub-lethal
myristic acid (Table 4). A SNP mutation was identified in the 24th codon of farR that changed
it from a glutamine (Q) codon to an ochre stop codon in one isolate (Fig 1). This mutation is
present in all sequence reads from this sample, however it is not present in any of the sequence
reads of the duplicate myristic acid sample. This mutation would prevent expression of FarR,
resulting in overexpression of the farAB encoded fatty acid efflux pump system [10±12]. This
sample was observed to have a growth advantage over its other replicate but did not show a
greater MIC compared to the other replicate (Table 1). The FarR transcriptional regulator also
activates glnA responsible for glutamine biosynthesis [
] in N. gonorrhoeae and nadA in N.
]. This mutation is not surprising as mutations in the promoter region and
open reading frame of another transcription regulator of an efflux pump system, mtrR, has
mutation identified in either the non-selective isolate 1 (NS-1) or the non-selective isolate 2 (NS-2) culture.
^ number of sequencing reads containing the mutation identified.
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mutation identified in either the myristic acid passaged isolate 1 (14:0±1) or the myristic acid passaged isolate 2 (14:0±2) culture.
^ number of sequencing reads containing the mutation identified.
been demonstrated to confer decreased susceptibility to some common antimicrobials [
]. It is interesting to note that the mutation does not confer a higher level resistance to
myristic acid compared to the parallel culture.
The monocaprin passaged isolates had two non-synonymous SNP mutations (Table 5),
both of which were predicted by SNAP2 to have a functional effect. The mutation in the
phosphate permease protein was only present in one isolate. This is a membrane protein which is
responsible for passively allowing entry of phosphate ions into the cell. It is not clear what
effect this mutation would have on the cells ability to protect it from monocaprin. The
mutation in dksA (NGK_RS106025) is present in all of the sequencing reads from both monocaprin
grown cultures. The main role of DksA, a zinc-containing multi-functional protein, is as a
transcription factor that binds directly to RNA polymerase and negatively regulates rRNA
expression by destabilizing rRNA::promoter complexes [
], positively regulates several amino
acid biosynthesis genes [
], and regulates fis expression [
]. An important gene regulator,
DksA also inhibits transcript elongation, exonucleolytic RNA cleavage, pyrophosphorolysis,
and increases intrinsic termination (http://www.uniprot.org/uniprot/P0ABS1), as well as
being involved with RecN in repair of DNA double-strand breaks [
] and suppression of
]. The mutation causes a substitution of the 75th amino acid from a threonine to an
isoleucine (Fig 2), predicted by SNAP2 to have a functional effect on the protein with a change
in the predicted secondary structure. The D71, D74, or A76 residues of the E. coli homolog
form part of the coiled-coil tip that is responsible for the DksA-specific effects on open
complex formation [
]. These amino acids are conserved in N. gonorrhoeae and the T75I
change is 12 amino acids away from these conserved residues (Fig 2). The protein appears to
be highly conserved within N. gonorrhoeae; at the time of writing there were 322 DksA protein
sequences in the GenPept database and of these 319 (99%) have an amino acid sequence
Fig 1. Mutations identified in N. gonorrhoeae grown with sub-lethal myristic acid. The SNP in myristic acid grown culture 14:0±1 is present at the 70th
nucleotide into farR (black highlight) and results in the formation of a premature stop codon after 23 amino acids ( ). The full length FarR is 146 amino
5 / 10
mutation identified in either the monocaprin passaged isolate 1 (MG10-1) or the monocaprin passaged isolate 2 (MG10-2) culture.
^ number of sequencing reads containing the mutation identified.
' identified via pBLAST analysis.
identical to N. gonorrhoeae strain NCCP11945. The remaining three have separate single
amino acid substitutions, none of which were at residue 75. DksA is involved in the `stringent
response' which is the stress response that occurs when bacteria are exposed to heat shock,
experience stress conditions, or are starved of essential cellular components such as amino
acids, fatty acids, or iron .
All four samples passaged on media containing sub-lethal antimicrobials showed an
increase in MIC for both antimicrobials, suggesting that mechanisms of survival in the
presence of one confer cross-resistance to the other. However, no common mutations in CDSs
across these four sets of sequencing data were present. The increases in MICs may therefore be
a result of general adaptation to stressful growth conditions, rather than being due to specific
resistance mechanisms against monocaprin or myristic acid. A modest increase in MIC such
as those observed here for monocaprin (Table 1) could arise due to an adaptation of the
bacterial cell to the stresses experienced by exposure to the antimicrobials, due to selection for a
portion of the population expressing a different phase variable repertoire of outer membrane
proteins or LOS with different permeability, or other general changes that are not specific to
The promoters of farR, farAB, mtrR, and mtrCDE were examined for any signs of mutation
in the genome sequence data. In N. gonorrhoeae, modified promoter regions can enhance the
expression of efflux pumps; a mutation in the sequence upstream of mtrC acts as an alternative
promoter region enabling transcription of mtrCDE without MtrR control [
]. No mutations
in these regions were observed in the experimental evolution sequencing data. Of note, N.
gonorrhoeae strain NCCP11945 used in this study has a single base deletion in the promoter
located inverted repeat within the mtrR mtrC promoter region. This single base mutation
results in a loss of expression of MtrR and increased expression of the MtrCDE efflux pump in
N. gonorrhoeae strain FA19 [
], which suggests that overexpression of the MtrCDE efflux
pump is insufficient to confer resistance to monocaprin. Further, the FarR mutant here would
Fig 2. Mutations identified in N. gonorrhoeae grown with sub-lethal monocaprin. An alignment of a portion of DksA from E. coli (DksA E.
coli) and the monocaprin culture mutation of N. gonorrhoeae (DksA N. gonorrhoeae C10). The grey highlighting indicates residues important
in RNA polymerase binding in E. coli, conserved in N. gonorrhoeae. The black highlight indicates the mutation in N. gonorrhoeae grown with
6 / 10
be overexpressing both MtrCDE and FarAB-MtrE, yet the MIC of monocaprin in this isolate
(14:0±1, Table 4) remains 500 μM (Table 1).
Ophthalmia neonatorum, infection by N. gonorrhoeae of the eyes of newborns, is due to
transmission of the bacteria from the infected birth canal of the mother to the eyes of the infant
during birth. This is a `dead-end' infection; the bacteria cannot transmit to a new host from the
infant eye infection. As a result, any treatment applied to eliminate ophthalmia neonatorum that
is not used to also treat reproductive tract infections is isolated to the patient, with no chance of
resistant organisms evolving in the host and being transmitted to others. Monocaprin-based
formulations have been shown to rapidly kill N. gonorrhoeae without causing irritation [
]. For this
reason, we did not apply the strategy used previously by Belland et al. [
] of subjecting the
bacteria to increasingly higher concentrations of monocaprin over time. For this particular clinical
manifestation and this particular proposed application, given the rapid killing time of
monocaprin and `dead-end' nature of the infection, there is no scope for resistance to evolve within the
host. Therefore, our experimental design here did not include a progressive increase in
monocaprin concentration as had previous resistance studies [
]. Additional evidence presented here
demonstrates that the opportunity for evolution of resistance to such a treatment is minimal
within the patient and within the timeframe of treatment, therefore further supporting the
development of these candidates as treatments for gonococcal eye infections.
Our results parallel those of previous researchers investigating the potential for resistance
to emerge to monoglycerides. In Staphylococcus aureus, continuous passage on media
containing sublethal concentrations of monolaurin for a year did not generate resistance [
]. It is
hypothesized that resistance to monoglycerides is not seen and are less likely to develop than
resistance to antibiotics because there are multiple modes of antibacterial action that would all
need to be overcome [
In conclusion, N. gonorrhoeae strain NCCP11945 adapted to growth in media containing
sub-lethal concentrations of the fatty acid myristic acid and the monoglyceride monocaprin.
Passage on either of these two antimicrobials resulted in a doubling of the MIC for
monocaprin, likely due to general stress adaptation. Passage on monocaprin or myristic acid resulted
in eight- and sixteen-fold increases, respectively, in the myristic acid MIC. N. gonorrhoeae is
known to have mechanisms of resistance to myristic acid, such as efflux pumps [10±12]. It
appears that the bacteria were able to increase their ability to withstand the presence of
myristic acid just by being in an environment surrounded by a similar hydrophobic agent. However,
the MIC for monocaprin showed only a modest two-fold increase in MIC, suggesting that it
would be a suitable candidate for treatment of gonococcal infections, such as ophthalmia
]. Genome sequencing revealed that the mutations from the
monocaprin-containing culture are of a more general nature than those from the myristic acid cultures. It is likely
that the mutations in dksA, present in both monocaprin cultures, have compensated for stress
upon the cell due to the presence of monocaprin in the culture and that the small MIC increase
observed here is the extent of what can be achieved by general adaptive mutations. The
likelihood that monoglycerides such as monocaprin have multiple modes of antimicrobial action
and that resistance have not emerged after a year of passage in other species [
encouraging for their application as antimicrobials. Monocaprin is therefore a promising candidate
for the treatment of gonococcal infections such as ophthalmia neonatorum.
Bacterial growth and experimental evolution
N. gonorrhoeae strain NCCP11945 was grown in three conditions: on standard GC agar (GC
base (Oxoid) with Kellogg's supplements [
]); on GC agar with sub-lethal monocaprin
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(Sigma-Aldrich; C10:0MG; 125 μM); and on GC agar with sub-lethal myristic acid
(SigmaAldrich; C14:0; 62.5 μM). Duplicate cultures were grown for each condition. Minimally
passaged N. gonorrhoeae strain NCCP11945 received from the sequencing project [
incubated at 37ÊC, 5% CO2 for 48 hours. Bacterial cells were removed into 1 ml GC broth to a
turbidity of 0.5 McFarland standard and used to create a continuous streak over a whole GC
agar plate using a sterile cotton swab. For the first passage, six GC plates were inoculated: two
GC agar; two GC agar with 125 μM monocaprin; and two GC agar with 62.5 μM myristic acid.
Cultures were passaged every 48±72 hours for 20 passages.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was determined by agar dilution. GC agar
plates were made containing 500 mM, 250 mM, 125 mM, 62.5 mM, 32.25 mM, and 16.125
mM of either myristic acid or monocaprin. Bacterial cells were suspended in 1 ml GC broth to
a turbidity of 0.5 McFarland standard and used to create a continuous streak over the whole
plate with a sterile cotton swab. Plates were incubated at 37ÊC, 5% CO2 for 48 hours. The
lowest concentration which prevented growth was deemed the MIC. The sub-lethal concentration
was the highest concentration which did not prevent growth.
DNA extraction and genome sequencing
A Qiagen Gentra Puregene Yeast/Bac kit was used to extract DNA from 500 μl of a bacterial
suspension in GC broth equivalent to a 0.5 McFarland standard. DNA from the 20th passage of
the six experimental evolution cultures and DNA from the starting inoculum were sent to
MicrobesNG (University of Birmingham, Birmingham, UK, supported by the BBSRC, grant
number BB/L024209/1) for whole genome Illumina HiSeq 2x250 bp paired-end sequencing.
Genome sequence data quality control, assembly, and analysis
FastQ files of trimmed paired read data, were downloaded from the MicobesNG server.
Read files were checked using FastQC version 0.11.2 (http://www.bioinformatics.babraham.ac.
uk/projects/fastqc) and aligned using UGENE version 1.20.0 [
] Bowtie2 plug-in 
against the published sequence of N. gonorrhoeae strain NCCP11945 (accession number
]. Assemblies in UGENE were checked manually for identified variant
positions. Genes containing sequence variants were identified and predicted consequences
determined using SNAP2 [
]. The promoter regions of farR, farAB, mtrR, and mtrCDE were
subject to in-depth analysis.
We would like to acknowledge the contribution of the MicrobesNG sequencing centre at the
University of Birmingham, Birmingham, UK in generating genomic data.
Conceptualization: Colin P. Churchward, Lori A. S. Snyder.
Formal analysis: Colin P. Churchward.
Funding acquisition: Lori A. S. Snyder.
Investigation: Colin P. Churchward, Alan Calder.
Methodology: Colin P. Churchward, Alan Calder.
8 / 10
Project administration: Lori A. S. Snyder.
Supervision: Lori A. S. Snyder.
Writing ± original draft: Colin P. Churchward, Lori A. S. Snyder.
Writing ± review & editing: Colin P. Churchward, Alan Calder, Lori A. S. Snyder.
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