APPENDICES TO BODY OF REPORT OD2010

Because this work was carried out at three different institutions, different abbreviations have been used for some of the different disinfectants and for the bacterial mutants derived from growth with the disinfectants.

The five biocides used were:-

  • A compound disinfectant containing a mix of Quaternary ammonium compounds, formaldehyde and glutaraldehyde. The abbreviations used for this disinfectant are QACFG,SK and ABD (aldehyde based disinfectant).
  • An oxidising compound based disinfectant with the abbreviations OXC and VS.
  • A tar oil phenol disinfectant with the abbreviations TOP and FFS.
  • A dairy sterilizing disinfectant which comprised a quaternary ammonium biocide, non-ionic surfactant and excipients. The abbreviations used this disinfectant are DSD.
  • Triclosan has the abbreviation T or TRIC.

A.1

Table 1. Strains used in this study to select mutants and control strains.

Designation / Description / Reference
Parent strains / L354 / SL1344 / 20
L108 / SL1344 (tolC::aph) / 21
L357 / DT104 / 21
L358 / DT104 (penta resistant, GyrA Phe83) / 21
L378 / Ciprofloxacin resistant field isolate, GyrA Phe83 / This study
L643 / SL1344 (acrB::aph) / 14
L696 / SL1344 (GyrA Asp87) / 10
L699 / SL1344 (cyclohexane resistant) / 23
Control strains / AG100 / E. coli K12 / 24
AG102 / AG100 (cyclohexane tolerant, ∆marR) / 24

Table 2. Susceptibility of mutants to antibiotics and disinfectants and times for a 5-log reduction in viable numbers.

Antibiotic susceptibility /
Disinfectant susceptibility
Strain / Selective agent / MIC (µg/ml) /
Time (min) for 5-log kill
/
MIC (%, v/v)
Cip / Chl / Tet / EtBr / AF / Kan / OXC (0.6%, v/v) / QACFG (0.025%, v/v) / DSD (0.2%, v/v) / TOP (0.075%, v/v) / OXC / QACFG / DSD / TOP
L357 / 0.015 / 2 / 1 / 512 / 128 / 2 / 14.5 +/- 0.5 / 0.4 / 0.025 / 0.1 / 0.2
TOP1 / TOP / 0.015 / 4 / 1 / 1024 / 128 / 2 / 10.6 +/- 0.3 / 0.2
TOP1 / TOP / 0.015 / 4 / 1 / 1024 / 128 / 8 / 6.2 +/- 0.3 / 0.2
L378 / 4 / >256 / 256 / 2048 / 256 / 2 / 38 +/- 3.2 / 0.4 / 0.025 / 1.6 / 0.2
DSD1 / DSD / 4 / >256 / 128 / 1024 / 256 / 2 / 34 +/- 2.1 / >3.2
L108 / <0.015 / 1 / 0.5 / 16 / 8 / >32 / 15 +/- 0.8 / 0.4 / 0.006 / <0.003 / 0.025
DSD2 / DSD / <0.015 / 1 / 0.5 / 16 / 8 / >32 / 25 +/- 1.2 / 0.12
L358 / 0.5 / >256 / 128 / 1024 / 256 / 2 / 12.1 +/- 0.2 / 0.8 / 0.025 / 0.8 / 0.2
QACFG1 / QACFG / 2 / >256 / 128 / 1024 / 256 / 2 / 13.5 +/- 0.7 / 0.025
QACFG2 / QACFG / 2 / >256 / 256 / 1024 / 256 / 2 / 10.2 +/- 0.5 / 0.025
QACFG3 / QACFG / 2 / >256 / 256 / 1024 / 256 / 2 / 14.2 +/- 0.7 / 0.025
L354 / 0.015 / 4 / 1 / 1024 / 128 / 2 / 8.2 +/- 0.8 / 0.4 / 0.025 / 0.1 / 0.2
OXC1 / OXC / 0.015 / 4 / 0.5 / 1024 / 128 / 2 / 13 +/- 2.1 / 0.8
OXC2 / OXC / 0.015 / 8 / 0.5 / 1024 / 128 / 2 / 11.5 +/- 1 / 0.8

Values in bold indicate changes in tolerance to antibiotics or disinfectants of mutants compared to their respective parent strains.

Cip = ciprofloxacin, Chl = chloramphenicol, Tet = tetracycline, EtBr = ethidium bromide, AF = acriflavine, Kan = kanamycin, OXC = oxidizing compound, QACFG = Quaternary ammonium compound + aldehyde + formaldehyde farm disinfectant, DSD = dairy sanitizer disinfectant, TOP = Tar oil Phenol. +/- values indicate standard deviation from the mean. OXC1, TOP1 etc are mutants derived from growth with specific compounds

Table 3. Summary of protein changes after disinfectant exposure.

Exposure / Number of proteins (mean + s.d.)
Control / Treated / Control only / Treatment only / Common to treatment and control /

Increased expression

(P<0.05) /

Decreased expression

(P<0.05)
0.04% TOP / 437
(696 + 129) / 429
(682 + 69) / 79 / 70 / 360 / 12 / 23
0.15 w/v OXC / 596
(927 + 91) / 511
(861 + 45) / 173 / 88 / 424 / 32 / 67

OXC = oxidizing compound, TOP = Tar oil phenol disinfectant.

Table 4. Proteins with statistically significantly altered expression after treatment with TOP

Group / Description / Protein / Ratio
Efflux
RND family, multidrug efflux pump / AcrB / 2.8**
Multidrug efflux pump / EmrA / 2.7**
Membrane fusion protein of AcrAB-TolC / AcrA / 1.9**
Outer membrane efflux channel / TolC / 1.8***
OmpF assembly / AsmA / 0.5*
ATP synthesis
Membrane-bound ATP synthase, F1 sector, beta-subunit / AtpD / 0.9*
Membrane-bound ATP synthase, F0 sector, subunit b / AtpF / 0.8*
Chemotaxis
Methyl-accepting chemotaxis protein I / TsR / 0.4**
Methyl-accepting transmembrane citrate/phenol chemoreceptor / TcP / 0.3***
Methyl accepting chemotaxis protein II / CheM / 0.2***
Hydrogenase/dehydrogenase
Pyruvate dehydrogenase, dihydrolipoyltransacetylase component / AceF / 2.2**
Pyruvate dehydrogenase, decarboxylase component / AceE / 2.0***
Putative hydrogenase, membrane component / OmpA / 1.3*
2-oxoglutarate dehydrogenase / SucA / 0.5*
Plasma membrane proline dehydrogenase/ pyrroline-5-carboxylate dehydrogenase / PutA / 0.3**
Miscellaneous
Sigma D (sigma 70) factor of RNA polymerase / RpoD / 2.5*
Alkyl hydroperoxide reductase / AhpC / 2.2**
Fructose-bisphosphatase / Fbp / 1.7*
50S ribosomal subunit protein L6 / RplF / 1.6*
Pyruvate kinase I / PykF / 1.4*
Transcriptional activator of ntrl gene / OsmE / 0.8*
DNA-binding protein HU-alpha / HupA / 0.7*
Succinyl-coa synthetase, beta subunit / SucC / 0.7***
Part of modulator for protease specific for ftsh phage lambda cii repressor / HflC / 0.6**
Glycoprotein/polysaccharide metabolism / YbaY / 0.6*
ATP-dependent protease, Hsp 100 / ClpB / 0.6*
Phosphoenolpyruvate carboxykinase / PckA / 0.5*
Periplasmic L-asparaginase II / AnsB / 0.5*
Glycerophosphodiester phosphodiesterase / GlpQ / 0.5*
Mannose-specific enzyme IID / ManZ / 0.4*
Putative outer membrane lipoprotein / STM1607 / 0.4*
Glutathione oxidoreductase / Gor / 0.4*
Stringent starvation protein A, transcriptional regulator / SspA / 0.4**
Aspartate ammonia-lyase (aspartase) / AspA / 0.4*
Scaffolding protein for murein-synthesizing holoenzyme / MipA / 0.4*

Ratio indicates protein expression after disinfectant treatment divided by expression in untreated control. * = P <0.05, ** = P <0.01, *** = P <0.001.

TOP = Tar oil phenol disinfectant.

Table 5. Ten most statistically significantly over-expressed and repressed proteins after treatment with OXC

Description / Protein / Ratio
30S ribosomal subunit protein S3 / RpsC / 8.0***
uptake of enterochelin; tonB-dependent uptake of B colicins / ExbB / 6.5***
membrane-bound lytic murein transglycosylase A / MltA / 3.8***
subunit of cysteine synthase A and O-acetylserine sulfhydrolase A / CysK / 3.6***
ABC superfamily (atp&memb), cytochrome-related transporter / CydC / 3.5**
periplasmic serine protease Do, heat shock protein / HtrA / 3.5**
glucose dehydrogenase / Gcd / 3.3*
outer membrane protein receptor / transporter for ferrichrome, colicin M, and phages T1, T5, and phi80 / FhuA / 3.3***
putative outer membrane lipoprotein / YiaD / 2.6**
putative helicase / RhlB / 2.4*
phosphoenolpyruvate synthase / Pps / 0.2*
glycine cleavage complex protein P, glycine decarboxylase / GcvP / 0.2*
putative methyl-accepting chemotaxis protein / STM3138 / 0.1**
methyl-accepting chemotaxis protein III, ribose and galactose sensor receptor / Trg / 0.1**
L-lactate dehydrogenase / LldD / 0.1*
periplasmic L-asparaginase II / AnsB / 0.1**
phosphoenolpyruvate carboxykinase / PckA / 0.1***
fumarate reductase, anaerobic, flavoprotein subunit / FrdA / 0.1*
flagellar biosynthesis; flagellin, filament structural protein / FliC / 0.1***
putative methyl-accepting chemotaxis protein / STM3216 / 0.1***

Ratio indicates protein expression after disinfectant treatment divided by expression in untreated control. * = P <0.05, ** = P <0.01, *** = P <0.001

OXC = oxidizing compound

A.2

FIGURE LEGENDS

Figure 1. Survival of SL1344 (open bars), OXC-1 (grey bars) and OXC-2 (black bars) after exposure to OXC (0.6%).

P>0.05 relative to parent strain at the same time point.

OXC = oxidizing compound, OXC-1 and 2 a mutants derived from growth with OXC

Figure 2. Norfloxacin accumulation.

Open bars = - CCCP, hatched bars = + CCCP. * = P>0.05 relative to parent strain at the same time point. QACFG = Quaternary ammonium compound + aldehyde + formaldehyde farm disinfectant.

Figure 3. Growth of L108 and DSD-2 with and without exposure to DSD.

Each point is the average of 9 values, error bars are omitted for clarity. The arrow indicates the time at which DSD was added to each well. Grey symbols = L108, open symbols = DSD-2, triangles = no disinfectant, squares = 0.1% DSD, circles = 0.2% DSD.

DSD = Dairy sanitizing disinfectant, DSD-1 is a mutant derived from growth with DSD.

Figure 4 Colonisation and persistence of L700 (LoT), L701 (MeT), and L702 (HiT) in chicks for up to 27 day post inoculation.

The error bars represent +/- 1 standard deviation. * P<0.05, ** P<0.01 and *** P<0.001. Values in parentheses indicate the number of birds in which the had no detectable cfu following plating.

A.3

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  2. Randall, L. P., C. S. Clouting, K. O. Gradel, F. A. Clifton-Hadley, R. D. Davies, and M. J. Woodward. 2005. Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli. J. Appl. Microbiol.98: 556-563.
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  4. Randall, L. P., S. W. Cooles, A. R. Sayers, and M. J. Woodward. 2001. Cyclohexane resistance in Salmonella of different serovars is associated with increased resistance to multiple antibiotics, disinfectants and dyes. J. Med. Microbiol. 50: 1-6.
  5. Randall, L. P., and M. J. Woodward. 2002. The multiple antibiotic resistance (mar) locus and its significance. Res. Vet. Sci.72: 87-93.
  6. Russell, A. D. 2000. Do biocides select for antibiotic resistance? J. Pharm. Pharmacol. 52: 227-233.
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  14. Eaves, D. J., V. Ricci, and L. J. Piddock. 2004. Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance. Antimicrob. Agents Chemother. 48: 1145-50.
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B.1

TABLE 1. MICs of populations selected following sequential growth experiments a

Population / MICs of selected antimicrobial agents
CIP / CHL / TET / KAN / AMP / TRIC / OXC / TOP / QACFG / DSD
OXC 7d / <0.03 / 8-16 / 2-4 / 2-4 / 2-8 / 0.25 / 0.20 / 0.40 / <0.05 / 0.40
TOP 7d / <0.03 / 8 / 2 / 2-4 / 2 / 0.12 / 0.20 / 0.40 / <0.05 / 0.2-0.4
QACFG 7d / <0.03 / 16 / 2-4 / 2-4 / 16 / 1.00 / 0.20 / 0.40 / <0.05 / 0.40
16TRIC 16d / <0.03 / 16 / 2-4 / 4 / 8-16 / 64.00 / 0.20 / 0.40 / <0.05 / 0.2-0.4
LB 7d / <0.03 / 8 / 2 / 2 / 2-4 / 0.25 / 0.20 / 0.40 / <0.05 / 0.2-0.4
LB 1d / <0.03 / 8 / 2 / 2 / 2-4 / 0.06 / 0.20 / 0.2-0.4 / <0.05 / 0.20
L696 OXC 7d / 0.12-0.25 / 8 / 1-2 / 2-4 / 2 / 0.06 / 0.20 / 0.40 / <0.05 / 0.40
L696 TOP 7d / 0.12 / 8 / 1-2 / 2-4 / 2-4 / 0.25 / 0.20 / 0.40 / <0.05 / 0.2-0.4
L696 QACFG 7d / 0.25 / 16 / 2-8 / 2 / 16 / 0.25 / 0.20 / 0.40 / <0.05 / 0.20
L696 16TRIC 16d / 0.25 / 16 / 2-4 / 2 / 8-16 / >128 / 0.20 / 0.40 / <0.05 / 0.20
L696 LB 7d / 0.06-0.12 / 8 / 2-4 / 2-4 / 2-4 / 0.25 / 0.20 / 0.40 / <0.05 / 0.20
L696 LB 1d / 0.12-0.25 / 8 / 2-4 / 2 / 2 / 0.06 / 0.20 / 0.40 / <0.05 / 0.2-0.4

a MICs of ciprofloxacin (CIP), chloramphenicol (chl), tetracycline (tet), kanamycin (kan) ampicillin (amp) andtriclosan (tric) are presented in μg ml-1, while those of OXC, TOP, QACFG and DSD are presented in %. Populations are named as follows (parent strain, if not mentioned it is the wild type) (disinfectant and concentration if it is not close to the MIC, from which population was isolated) (days of sequential growth in disinfectant). For example, population OXC 7d is derived from wild type, was isolated from LB broth containing OXC and was sequentially grown for 7 days etc.

TABLE 2. MICs of isolated strains a

Isolate / MICs of selected antimicrobial agents
CIP / CHL / TET / KAN / AMP / TRIC / OXC / TOP / QACFG / DSD
OXC R1 / 0.06 / 32 / 2 / 1 / 8 / 0.25 / 0.20 / 0.40 / <0.05 / 0.40
OXC R2 / <0.03 / 8 / 2 / 2 / 2 / 0.12 / 0.20 / 0.40 / <0.05 / 0.20
QACFG R1 / <0.03 / 8 / 2 / 1 / 8 / 0.25 / 0.20 / 0.20 / <0.05 / 0.20
QACFG R2 / <0.03 / 32 / 16 / 2 / 8 / 0.25 / 0.20 / <0.05 / <0.05 / 0.1
TOP R1 / <0.03 / 8 / 2 / 2 / 4 / 0.12 / 0.20 / 0.40 / <0.05 / 0.2
TOP R2 / <0.03 / 16 / 16 / 2 / 4 / 0.25 / 0.20 / 0.10 / <0.05 / 0.10
TRIC R1 / <0.03 / 8-16 / 2-4 / 2 / 8 / 64 / 0.20 / 0.40 / <0.05 / 0.40
TRIC R2 / <0.03 / 16 / 2-4 / 2 / 8 / 64 / 0.20 / 0.40 / <0.05 / 0.2-0.4
WT / <0.03 / 8 / 2 / 2 / 2-4 / 0.06 / 0.20 / 0.2-0.4 / <0.05 / 0.20

a MICs of ciprofloxacin (CIP), chloramphenicol (chl), tetracycline (tet), kanamycin (kan) ampicillin (amp) andtriclosan (tric) are presented in μg ml-1, while those of OXC, TOP, QACFG and DSD are presented in %.

B.2

FIG. 1. Frequency of variants tolerant to 0.05 μg ml-1 triclosan (1A), 400 μg ml-1 acriflavine (1B), 1.2 μg ml-1 tetracycline (1C), 6 μg ml-1 chloramphenicol (1D) and 2 μg ml-1 ampicillin (1E) within L354 populations obtained following overnight growth in LB broth (LB1d) or sequential growth in LB (LB 7d) without biocide or supplemented with 0.2% VS (VS 7d), 0.025% FFS (FFS 7d), 0.006% SK (SK 7d) or 0.06 μg ml-1 triclosan (16T 16d). Sequential growth at rising concentrations, as described in text was for seven days in all cases, except with Triclosan, which was for 16 days.

FIG. 2. mRNA levels of transcripts of acrB gene in logarithmic phase cultures of OXCR1, QACFG R2, TOP R2, TRIC R1 and TRIC R2.

See top of appendices for disinfectant abbreviations

OXCR1, QACFGR2 etc are mutants from growth with respective disinfectants.

B.3

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  14. Moken, M.C., McMurry, L.M. and Levy, S.B. (1997)Selection of multiple-antibiotic-resistant (Mar) mutants of Escherichia coli by using the disinfectant pine oil: Roles of the mar and acrAB loci. Antimicrobial Agents and Chemotherapy 41(12) 2770-2772.
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C.1

Table 1. S. Typhimurium parent strains used in this study

Strain / Comment / Origin/Reference
L354 / SL1344 / Wray and Soijka 1978
L643 / SL1344 acrB::aph / Eaves et al., 2004
L108 / SL1344 tolC::aph / Eaves et al., 2004
L696 / SL1344 GyrA Asp 87 / Ricci et al., 2006
L699 / SL1344 cyclohexane tolerant / Webber et al., 2006
L357 / DT104 fully antibiotic sensitive strain / Randall et al., 2005
L358 / DT104 penta resistant veterinary isolate, GyrA Phe 83 / Randall et al., 2005
L359 / DT104 multiply antibiotic resistant derivative of L357 / Randall et al., 2005
L378 / S. Typhimuriumveterinary isolate, GyrA Phe 83 / Veterinary laboratories agency, U.K

Table 2. Mutation selection frequency and phenotype of selected triclosan resistant mutants.

Strain / Parent/Mutant / Origin / Selection frequency / MIC (mg/L)a / FabI
Tric / Cip / EtBr
L354 / Parent / SL1344 / N/A / 0.06 / 0.03 / 1024 / WT
L707 / Mutant Low / L354 / 7.0 X 10-9 / 4 (32) / 0.03 / 1024 / WT
L709 / Mutant Low / L354 / 7.0 X 10-9 / 4 (32) / 0.03 / 1024 / G93S

L378

/ Parent / CipR field isolate / N/A / 0.25 / 1 / 1024 / WT
L691 / Mutant Low / L378 / 5.8 X 10-8 / 4 (16) / 1 / 1024 / WT
L690 / Mutant High / L378 / 6.0 X 10-8 / >128 / 1 / 1024 / G93V
L696 / Parent / CipR mutant of L354 / N/A / 0.25 / 0.25 / 1024 / WT
L700 / Mutant Low / L696 / 9.6 X 10-8 / 4 (16)b / 0.25 / 1024 / WT
L701 / Mutant Int / L696 / 9.2 X 10-8 / 32 (128) / 0.25 / 1024 / G93V
L702 / Mutant High / L696 / 9.4 X 10-8 / 128 (512) / 0.25 / 1024 / G93V
L699 / Parent / Cyclohexane tolerant / N/A / 0.5 / 0.12 / 1024 / WT
L703 / Mutant Int / L699 / 8.6 X 10-7 / 32 (64) / 0.12 / 1024 / G93V
L705 / Mutant High / L699 / 9.0 X 10-7 / 64 (128) / 0.12 / 1024 / WT

a Tric = triclosan, Cip = ciprofloxacin, EtBr = ethidium bromide.

b Numbers in brackets are fold increase in triclosan resistance compared to parent.

Table 3. Effect of gene disruptions and pBAD-fabI over-expression on triclosan susceptibility.

MIC triclosan (g/ml)
Strain / Wild-type / tolC::aph / acrB::aph / marA::aph / ramA::aph / +pBADfabIa
SL1344 / 0.06 / 0.015 (4) / 0.06 (0) / 0.06 (0) / 0.06 (0) / 0.06 (0)
L696 / 0.25 / 0.06 (4) / 0.25 (0) / 0.25 (0) / 0.25 (0) / 1 (4)
L700 / 4 / 0.12 (33) / 2 (2) / 2 (2) / 2 (2) / 8 (2)
L701 / 32 / 8 (4) / 1 (32) / 16 (2) / 2 (16) / 32 (0)
L702 / 128 / 0.5 (256) / 4 (32) / 2 (64) / 4 (32) / 16 (8)

a Experiments performed in the presence of 0.002% arabinose.

Numbers in brackets refer to fold change in triclosan susceptibility relative to parent.

Table 4. Proteins with significantly altered expression in all three triclosan resistant mutants.

Expression ratioa
Function / Protein / Description / L700 (Lot) / L701 (Met) / L702 (Hit) / Average
Generation of pyruvate/fatty acid / ArcA / Putative arginine deiminase (arcA) / 7.0 / 3.0 / 3.3 / 4.4
GcvP / Glycine cleavage complex protein P / 2.2 / 5.6 / 4.6 / 4.1
PpS / Phosphoenolpyruvate synthase / 1.5 / 3.7 / 2.8 / 2.7
FadB / 3-hydroxyacyl-coA dehydrogenase / 3.0 / 2.0 / 2.0 / 2.3
MaeB / Malate transferase / 2.1 / 2.3 / 1.7 / 2.0
GltA / Citrate synthase / 1.3 / 1.7 / 2.3 / 1.8
GapA / Glyceraldehyde-3-phosphate dehydrogenase A / 1.3 / 1.7 / 1.9 / 1.6
GlpK / Glycerol kinase / 1.5 / 1.7 / 1.5 / 1.6
MdH / Malate dehydrogenase / 1.4 / 1.5 / 1.8 / 1.6
Stress response/regulation / HtrA / Periplasmic serine protease, heat shock protein / 2.3 / 3.6 / 6.2 / 4.0
HNS / DNA-binding protein, pleiotropic regulator / 2.4 / 2.0 / 2.3 / 2.2
CspC / Cold shock protein, putative regulator / 1.6 / 1.8 / 1.5 / 1.6
Porphyrin biosynthesis / HemL / Glutamate-1-semialdehyde aminotransferase / 3.3 / 3.0 / 4.0 / 3.4
HemX / Uroporphyrinogen methylase / 1.4 / 2.1 / 2.3 / 1.9
Miscellaneous / AhpC / Alkyl hydroperoxide reductase / 1.6 / 2.5 / 2.3 / 2.1
TktA / Transketolase 1 isozyme / 1.4 / 1.6 / 1.8 / 1.6
YbhC / Putative pectinesterase / 1.5 / 1.5 / 1.8 / 1.6
RpsA / 30S ribosomal subunit protein S1 / 1.5 / 1.7 / 1.5 / 1.6
TufA / Protein chain elongation factor / 1.3 / 1.6 / 1.6 / 1.5
WzzB / Regulator of length of O-antigen of LPS / 0.5 / 1.6 / 1.9 / 1.3
LamB / Phage lambda receptor protein / 0.5 / 1.5 / 0.5 / 0.8
STM3152 / Putative methyl-accepting chemotaxis protein / 0.3 / 0.6 / 0.3 / 0.4
NupC / Nucleoside transport / 0.5 / 0.5 / 0.2 / 0.4
YjiJ / Putative carbon starvation protein / 0.5 / 0.3 / 0.4 / 0.4
CheA / Sensory histidine protein kinase / 0.2 / 0.6 / 0.2 / 0.3

a Expression ratio relative to L696.