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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code OD2011

2. Project title

Development of proteomic targeted test(s) for microbial multiple antibiotic resistant zoonotic food borne pathogens.

3. Contractororganisation(s)

Veterinary Laboratories Agency (Weybridge),Woodham Lane,New Haw, Addlestone,Surrey.KT15 3NB.     

54. Total Defra project costs £ 349,992.00

5. Project: start date................ 01 October 2003

end date................. 31 March 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domainThe journals in which we wish to publish will view the final report as prior publication and we will be unable to publish our data. As soon as the manuscripts are accepted for publication we will inform Defra so that the final report can be released in to the public domain.

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work. The multiple antibiotic resistance (MAR) phenotype is characterised by reduced susceptibility (MICs

increased by 2-8 fold) to structurally unrelated antibiotics including tetracycline, chloramphenicol, some

penicillins and quinolones and certain biocides. This phenotype is considered to be of considerable

biological significance as it is an important intermediate in the acquisition of genes, often on mobile

genetic elements or gene mutations, which confer a level of resistance to antibiotics associated with

clinical disease. Previous genetic studies have indicated that this phenotype is facilitated by the

expression of efflux pump and porin proteins which respectively export and reduce the uptake of

antibiotics by the bacterial cell. Currently, there is no single test for the MAR phenotype. The objective of

the current project was to develop a protein based test for MAR in Salmonella Typhimurium. The key

protein effectors of MAR were determined by exposure to antibiotics and disinfectants, in mutants where

specific genes had been deleted and in MAR mutants themselves.

A range of mutants of Salmonella enterica serovar Typhimurium (strain SL1344) were prepared with

deletions in genes associated with the MAR phenotype, as determined by previous micro-array studies, for

subsequent phenotypic (MIC estimation) and proteomic analysis. The efflux pump acrB, acrB + acrF, tolC

and the outer membrane protein genes ompC and ompF and acrR (transcriptional regulator of acrA and

acrB) were inactivated in S. Typhimurium SL1344 (L354) for subsequent proteomic analysis using the

one-step technique previously described for E. coli (Datsenko and Wanner 2000) and adapted for S.

Typhimurium (Eaves et al 2004).

Automated methods for the analysis of the S. Typhimurium proteome (complement of cellular proteins)

were set up and validated. Key aims were to develop a method for proteome analysis that was of generic

applicability, automatable and provided superior proteome coverage, particularly of outer membrane

proteins. This was to enable identification of the key proteins associated with MAR. Proteins were

digested with the enzyme trypsin, resultant peptides separated by 2-dimensional liquid chromatography

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and identified by mass spectrometry (2D-HPLC-MSn). The data was processed using specialist

(bioinformatics) software from The Scripps Research Institute, USA to determine the proteins expressed.

Further analysis of these very large data sets was accomplished with Microsoft Excel and Access and

enabled detection of proteins with significant changes in protein synthesis and identification of biomarkers

of MAR. Proteome analysis was validated for reproducibility (inter and intra assay coefficients of

variation), sensitivity and comparison of proteomes between replicates. Typically, a total of 816 + 11

individual proteins were identified which included 371 + 33, 565 + 15 and 262 + 5 from the cytosolic, cell

envelope and cell wall fractions respectively. A range of proteins (n=20) previously associated with the

mar locus in E. coli (Barbosa & Levy, 2000) were detected including the effector proteins AcrA, AcrB, TolC

and OmpF (Coldham and Woodward, 2004). The analysis of proteomes by 2D-HPLC-MSn was validated

by comparison with the more traditional (but labour intensive) gel based methods (Coldham et al., 2006)

and analysis of gene deletion mutants where the target protein should be absent (Coldham et al., 2007).

These proteomic methods have been widely used in a range of Defra funded projects, particularly OD2010

(use and abuse of disinfectants) but also those for the analysis of Brucella and Mycobacterium bovis

proteomes. Further opportunities exist to exploit this technology in a wide range of areas including

antibiotic drug resistance.

The expression of proteins was evaluated in S. Typhimurium proteomes by 2-dimensional HPLC mass

spectrometry following treatment with fluoroquinolone antibiotics (Coldham et al., 2006), the disinfectants

Farm Fluid S and Virkon S (Randall et al., 2007) and triclosan (Webber et al., 2007). A common theme

was increased expression of stress response proteins, particularly those of the efflux pump AcrAB/TolC.

Analysis of the cell envelope proteomes from the knockout mutants revealed that deletion of acrB and tolC

was associated with increased expression of the AcrE/AcrF and AcrD efflux pumps, respectively,

confirming the coordinated expression observed previously by RT-PCR at the transcript level (Ricci et al.,

2006). In the OmpC/OmpF knockout mutants increased expression of the proteins HtrA, FepA, IbpA,

IbpB, OsmY and OmpR (regulates expression of OmpC/OmpF) was observed indicating the activation of

compensatory mechanism in outer membrane components. In four MAR mutants there were also

changes in the expression of proteins which mediate molecular flux. The expression of the efflux pump

proteins AcrA, AcrB and TolC relative to controls (100%), were increased in MAR mutants by 197% + 51,

182% + 30 & 216% + 31 (P<0.0.1) respectively where as the outer membrane protein OmpF was

significantly reduced (P<0.01) in the MAR mutants. These studies with the knockout and MAR mutants

clearly demonstrated the central role of efflux pump and outer membrane proteins in the control of solute

accumulation and efflux and their likely role in MAR.

The data from the proteomic studies were reviewed in silico for the discovery of biomarkers of MAR.

Data sets arising from the proteomic studies are very large and were reduced and summarised using

Microsoft Access and Excel. These analyses revealed that changes in the expression of proteins, such

as AcrAB/TolC, OmpF and OmpC, which control solute accumulation and export, showed the most

consistent changes and most likely to provide biomarkers for MAR.

A range of fluorescent probes were evaluated for use in the detection of the MAR phenotype using an

automated standard laboratory 96 well plate reader. This type of assay is based on the principle of a

change in fluorescence following entry into and accumulation by the bacterial cell, usually as a

consequence of binding to DNA. The assay was developed and validated using the parent strain

(SL1344), MAR mutants (positive controls), heat inactivated cells (negative control) and the gene deletion

mutants (positive and negative controls as considered appropriate). Positive controls had reduced, while

negative controls had increased accumulation of fluorescent probe. Ethidium bromide and Hoescht

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333342 (bis benzimide) proved the best fluorescent probes on the basis of the data obtained using the

control strains. Further validation studies continued with H33342, as this probe is less toxic than ethidium

bromide with is a possible mutagen. These studies focused on the sensitivity to inhibitors and the analysis

of strains resistant to antibiotics and biocides/disinfectants from Defra funded project OD2010. The

accumulation of H33342 was increased in the parent strain following exposure of cells to the efflux pump

inhibitor phenylalanine-arginine--naphthylamide and carbonyl cyanide-m-chlorophenylhydrazone (CCCP)

which collapses the proton gradient. The effect of these inhibitors was reduced in a range of MAR

mutants thereby effectively providing amplification of the test. Strains with reduced susceptibility to

disinfectants and certain antibiotics (with MAR phenotype) over expressed the AcrAB/TolC efflux pump

and accumulated less H33342 whereas those which were not MAR did not. A panel of veterinary isolates

of Salmonella enterica (Webber et al., 2006) were analysed which further supported the association

between MAR and H33342 accumulation. The H33342 uptake assay has also introduced at Birmingham

university and similar data were obtained using positive and negative control bacterial strains.

In conclusion the current project has delivered a validated assay for the detection of the MAR phenotype

in S. enterica. The H33342 fluorescence assay has already been used for the phenotypic analysis of

strains (from OD 2010; Webber et al., 2007; Bagnall et al., 2007) and will be introduced at both the VLA

(under the surveillance contract) and at Birmingham university for the detection of the MAR phenotype.

This project has also provided automated methods for proteome analysis which has facilitated a unique

insight and new understanding of the complex web of adaptive changes in strains resistant to antibiotics

and disinfectants from Defra project OD2010.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Objectives:1. Production and characterisation of S. Typhimurium knock-out mutants in chromosomal regulatory loci

and outer membrane proteins that are associated with innate resistance to multiple antibiotics

characteristic of the Mar phenotype.

2. Procedures will be developed for the identification and analysis of expression levels of cell envelope

proteins by 2-dimensional HPLC and nanospray mass spectrometry using an appropriate

bioinformatics package.

3. Cell envelope proteomes prepared from S. Typhimurium following treatment with fluoroquinolone and

disinfectant will be analysed for protein identity and levels of expression by 2-dimensional HPLC-

mass spectrometry.

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4. Cell envelope proteomes prepared from the mutants produced in objective 01 will be analysed for

protein identity and levels of expression by 2-dimensional HPLC-mass spectrometry.

5. The data from sections 01 to 04 will be reviewed for selection of target proteins for a Mar test or tests.

6. Assays will designed for detection of proteins identified as potential targets in 05 for the Mar

phenotype.

7. The test for the Mar phenotype will be validated using isolates in the VLA collection.

ALL OBJECTIVES WERE MET ON TIME AND WITHIN BUDGET

IntroductionRecent UK surveillance data for the occurrence of antimicrobial resistance in Salmonella Typhimurium isolated

from animals and their environment demonstrated a marked decrease in sensitivity to all antimicrobial agents

tested (Jones et al., 2002). These organisms present a pool for the exchange of antibiotic resistance between

themselves and for transfer, via the food chain, to humans (Teale, 2002). Clearly, this presents a significant

hazard since Salmonella is one of the most common food borne zoonoses in the UK and transmission of resistant

Salmonella Typhimurium DT104 to from pigs to the humans has been demonstrated (Molbak et al., 1999).

Fluoroquinolones, including ciprofloxacin, are widely used in veterinary and human medicine for the treatment of

enteric infections, particularly those caused by Salmonella (Cherubin et al., 1991). Early studies, predating the

introduction of fluoroquinolones, suggested that antibiotic resistance was due to mechanisms encompassing gene

mutation and the acquisition of genes via mobile elements including plasmids and transposons (Saunders, 1984;

Hall and Collins, 1995). More recent studies have revealed that innate chromosomal multi-drug resistance

systems, such as the multiple antibiotic resistance (mar) locus of E. coli and Salmonella, also play a key role

(Randall and Woodward, 2002; Levy, 2002), including the development of resistance to fluoroquinolones.

Identification of functional proteins associated with MAR will provide targets for a specific test for this phenotype.

Application of such a test will enable discrimination between isolates with the MAR-like phenotype with a single

rapid test and those which are sensitive to antibiotics. This will provide an early marker for the likely development

of higher level resistance by acquisition of further mutation and provide reassurance that changes in practice,

such as the use of disinfectants, are effective in reducing the development of antibiotic resistance. This approach

may also be applicable for other genera for which other specific markers may be identified and applied similarly.

01/ Preparation and characterisation of specific knock out MAR associated mutants of Salmonella Typhimurium.Mutants of SL1344 with the acrB, acrR, tolC, ompC or ompF genes specifically inactivated by insertion of a

kanamycin resistance cassette within the coding region of the gene were created using established methodology

as previously described (Eaves et al, 2004; Buckley et al, 2006). Correct insertion of the aph resistance allele was

verified using combinations of PCR and sequencing. Each mutant was phenotypically characterised with respect

to growth kinetics by automated determination of optical density using a FluoSTAR optima (BMG Labtech) and

susceptibility to a panel of antimicrobial agents was determined using the agar dilution method as described by

the British Society for Antimicrobial Chemotherapy.

02/ Proteomic procedures for the analysis of microbial cell envelope proteins.All of the project work for this objective has been published in peer reviewed journals and abstracts of these

publications are provided below.

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The first paper describes the analysis of the S. Typhimurium proteome using semi-automated procedures capable

of detecting both outer membranes proteins and those previously associated with the mar locus and MAR

phenotype from micro-array transcriptomic studies. Outer membrane proteins are considered important as they

provide the interface with the environment and have proved difficult to detect by the more traditional 2-

dimensional gel electrophoresis. The relative abundance of the proteins may also be readily compared using the

spectrum count (Liu et al., 2004) method following published guidelines (Gao et al., 2004) and denotes the

number of peptide counts (‘hits’) detected for each protein.

Coldham and Woodward (2004) Characterization of the Salmonella Typhimurium Proteome by semi-automated

two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance. J.

Proteome Research 3, 595-603.

Abstract The proteome of Salmonella enterica serovar Typhimurium was characterised by 2-dimensional HPLC

mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic

resistance (MAR). Bacteria (2.15 + 0.23 x1010 c.f.u.; mean + s.d.) were harvested from liquid culture and proteins

differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope

and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides

separated into fractions (n=20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX

fraction were further separated by reverse phase chromatography and detected by mass spectrometry. Peptides

were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 + 11 individual

proteins were identified which included 371 + 33, 565 + 15 and 262 + 5 from the cytosolic, cell envelope and

OMP preparations respectively. A significant correlation was observed (r2=0.62 + 0.10; P<0.0001) between

consensus rank position for duplicate cell preparations and an average of 74 + 5 % of proteins were common to

both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A

range of proteins (n=20) previously associated with the mar locus in E. coli were detected including the key Mar

effectors AcrA, TolC and OmpF.

A key issue with all post-genomic research activities concerns the validation of such analyses particularly with

respect to rates of false positive and negative identifications. Proteomes from the gene deletion mutants

prepared under objective 01/ were analysed by 2D-HPLC-MSn for the target proteins which should of course be

absent. This approach enabled the bioinformatics of the proteomic assay to be calibrated to provide a false

positive rate of 1% (generally accepted standard level). An abstract summarising details of this section of work

are presented below.

Coldham, N.G., Webber, M., Piddock L.J.V., Carter, B. and Woodward (2007) Estimation of false positive and

false negative SEQUEST peptide identifications at defined loci using gene deletion mutants of Salmonella

Typhimurium (this manuscript has been resubmitted to the Journal of Proteome Research following an editorial

request for revision).

Abstract False positive and false negative SEQUEST tryptic peptide identifications were determined by

comparison of proteomes prepared from Salmonella enterica serovar Typhimurium and isogenic mutants

constructed with defined deletions in genes encoding the proteins AcrB, AcrF, TolC and OmpC. Proteomes were

prepared in triplicate from all strains and analysed by 2D-LC-MSn. False positive peptide identifications were

detected by the analysis of proteomes from mutants for the deleted protein. The SEQUEST cross correlation

(Xcorr) scores of the false positives (n=89; unfiltered) were (mean + sd; range max-min) 1.33 + 0.32; 2.22-0.54

and had upper tolerance limits (99% of population, P< 0.05) of 2.3, 2.1 and 2.5 for [M+H] +, [M+2H]2+ and [M+3H]3+

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ions respectively. False negative peptides (n=410) were estimated at each deleted locus in the unfiltered parent

strain by subtraction of true negative identifications determined in the gene deletion mutant and true and false

positives in the parent. Thus, Xcorr filtering produced far more false negative peptide identifications per locus

(30.3 + 15.4%) than false positives were eliminated (14.6 + 18.6%), and reduced proteome coverage from 3975 +

14 to 919 + 13 proteins. Threshold filter values that provided a false positive rate of 1% using gene deletion

mutants were much higher at 1.7 to 2.0% with decoy database searches. The principle advantage and potential

application for the gene deletion approach was empirical validation of threshold filter values against genuine false

positive identifications, comparison with decoy database searches and automated detection of false negatives.

Another important goal for this objective was to develop robust procedures which could be widely applied to

proteome analyses. The chromatography of tryptic peptides by nanospray HPLC-MSn using capillary columns is

perhaps the greatest point of weakness and procedures for preparing such HPLC columns and their quality

control assessment during routine proteome analysis has been published and the abstract is provided below.

Howells, L.C., Whelan, A.O., Woodward, M.J. and Coldham, N.G. (2005) Simple and effective technical

procedures for the packing and application of nanospray PicoFrit HPLC columns for proteomic analyses.

Proteomics 2005 5 3864-3867.

A simple procedure was developed for packing PicoFrit HPLC columns with chromatographic stationary phase

using a reservoir fabricated from standard laboratory HPLC fittings. Packed columns were mounted in a stainless

steel ultra-low volume precolumn filter assembly containing a 0.5 m pore size steel frit. This format provided a

conduit for the application of the nanospray voltage and protected the column from obstruction by sample

material. The system was characterised and operational performance assessed by analysis of a range of peptide

standards (n=9).

This method for the analysis of microbial proteomes by 2D-HPLC-MSn developed under objective 02/ has been

widely applied to a range of Defra funded projects at the VLA providing considerable added value. A range of

mutants with reduced susceptibility to biocides, disinfectants and certain antibiotics prepared under project 0D

2010 were analysed using these proteomic procedures. This methodology has provided a unique insight into

adaptive changes following disinfectant/biocide exposure and details of the publications arising from these studies

are provide in section 9 (Karatzas et al., 2007, Webber et al., 2007, Randall et al., 2007). The 2D-HPLC-MSn

method has also been applied to the analysis of proteomes from Brucella (various species), Mycobacterium bovis

and M. tuberculosis. Further studies are also using this method to detect serum biomarkers for Mycobacterium

bovis infection in cattle under a VLA seed corn funded initiative.

03/ Comparative analysis of cell envelope proteomes following treatment with fluoroquinolone and disinfectant. The coordinated regulation of protein effector expression is a key feature of innate reduced susceptibility to

multiple antibiotics. The chromosomal multiple antibiotic resistance locus (mar) of E. coli, in cooperation with

other regulatory loci, plays a pivotal role in innate reduced susceptibility (circa 4 fold) to some unrelated antibiotics

and certain disinfectants. Over-expression of AcrAB-TolC efflux pump contributes to MAR in E coli, and has also

been associated in conjunction with mutations in gyrA with resistance to fluoroquinolones in Salmonella enterica.

The AcrAB efflux pump of E. coli and S. enterica belongs to the Resistance/Nodulation/Cell division (RND) family

and consists of a proton anti-porter (AcrB) and a membrane fusion protein (AcrA). These two proteins associate

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with an outer membrane channel protein, such as TolC, to form a functional efflux pump unit providing selective

molecular translocation of solutes from the periplasm to the external environment. Reduced expression of porin

proteins located in the outer cell membrane may act synergistically with efflux pumps to reduce penetration of

antibiotic into the bacterial periplasm. Protein expression was investigated initially by 2DGE, and subsequently by

2-dimensional HPLC-MSn (2D-HPLC-MSn) for wider proteome coverage, to determine the physiological response

and potential protein effectors of innate resistance. An abstract summarising details of this section of work and

tables illustrating the relative expression of certain proteins are presented below.

Coldham, N.G., Randall L.P., Piddock, L.J.V. and Woodward, M.J. (2006) Effect of fluoroquinolone treatment on

the proteome of Salmonella enterica serovar Typhimurium analysed by 2D-gel electrophoresis and 2D-HPLC-

MSn. J. Antimicrob. Chemother. 58 1145-1153.

Abstract Aims: The physiological response of Salmonella enterica serovar Typhimurium to fluoroquinolone

antibiotics was investigated using proteomic methods.

Methods: Proteomes were prepared from strain SL1344 following treatment of broth cultures with ciprofloxacin

(0.03 and 0.08 mg/L; 2 and 0.5 x MIC) and enrofloxacin (0.03 mg/L) and from a multiple antibiotic resistant (MAR)

mutant. Protein expression was determined by 2-dimensional-HPLC-MSn and also after exposure to

ciprofloxacin by 2-dimensional gel electrophoresis.

Results: The number of proteins (mean + s.d ) detected by 2-dimensional gel electrophoresis derived from

control cultures of the wild-type strain was significantly (P<0.05) reduced from 296 + 77 to 153 + 36 following

treatment with ciprofloxacin (0.03 mg/L). Raised expression (P<0.05) of 17 proteins was also detected, and

increases of up to 8 fold (P<0.0001) were observed for sub-units of F1F0-ATP synthase, TolC and Imp. Analysis

by 2-dimensional-HPLC-MSn provided for higher proteome coverage with 787 + 50 proteins detected which was

reduced (P<0.005) to 560 + 14 by ciprofloxacin (0.03 mg/L). Increased expression of 43 proteins was observed

which included those detected by 2-dimensional gel electrophoresis and additionally the efflux pump protein AcrB.

The basal expression of the AcrAB/TolC efflux pump was elevated in the MAR mutant compared to the untreated

wild-type and augmented following treatment with ciprofloxacin (0.03 mg/L). F1F0-ATP synthase and Imp were

only elevated in the mutant when treated with ciprofloxacin.

Conclusions: These studies suggest that increased expression of AcrAB/TolC was associated with resistance

while others, such as F1F0-ATP synthase and Imp, were a response to fluoroquinolone.

The key finding of this study was increased expression of the AcrAB/TolC efflux pump proteins. A table of

proteins which showed the greatest increase in response to fluoroquinolone exposure are provided below in table

1. These studies suggested that efflux pump proteins, particularly AcrAB/TolC were likely to provide biomarkers

for the MAR phenotype.

Table 1 Percentage increase in selected protein expression in response to treatment with fluoroquinolone.

Protein Functional annotation Percentage increase in protein expression

WT Cip0.03 mg/L

WT Cip

0.008 mg/L

WT Enro0.03 mg/L

MAR1 cip 0.03

mg/L

AcrA acridine efflux pump 11 21 40 15AcrB RND family, acridine efflux

pump90* 27 114*** 42**

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TolC outer membrane channel; specific tolerance to colicin E1; segregation of daughter chromosomes, role in organic solvent tolerance

113** 96* 218*** 66***

Imp Organic solvent tolerance protein

58* 63* 113* 80**

AtpA membrane-bound ATP synthase, F1 sector, alpha-subunit

63** 61* 79* 62**

AtpC membrane-bound ATP synthase, F1 sector, epsilon-subunit

100* 93** 66** 43

AtpD membrane-bound ATP synthase, F1 sector, beta-subunit

98* 63* 142** 62**

AtpF membrane-bound ATP synthase, F0 sector, subunit b

80** 71* 115** 74**

AtpG membrane-bound ATP synthase, F1 sector, gamma-subunit

100** 106** 147** 21

AtpH membrane-bound ATP synthase, F1 sector, delta-subunit

46 83 136** 67*

OmpC outer membrane protein 1b (ib;c), porin

ND 266** 2266* 128*

OmpA putative hydrogenase, membrane component

18.2 50* 96* 35

WT indicates wild type parent strain, MAR1 indicates a MAR mutant strain 1, * P<0.05; ** P<0.01; *** P<0.001, ND indicated not detected.

The effect of ciprofloxacin (0.03 mg/L) on the expression of the AcrAB/TolC efflux pump following exposure of a

MAR mutant (strain MAR1) for 90 minutes was also investigated. Increased expression of the AcrAB/TolC efflux

pump was observed in the MAR mutant compared to the parent (wild type) which was further augmented in both

strains following treatment with ciprofloxacin (figure 1).

The expression of the AcrAB/TolC efflux pump system and further stimulation by exposure to ciprofloxacin is

shown in figure 1

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Further studies considered the effect of exposure for 90 minutes to a tar oil phenol (TAP; 0.04 % v/v) or an

oxidising compound (OXC; 0.15% w/v) disinfectant. Analysis of proteomes by 2D-LC-MSn revealed significantly

(P<0.05) increased expression of 13 and 31 proteins following exposure to TAP and OXC respectively. The

pattern of increased protein expression was quite different between the TAP and OXC disinfectants (table 2) with

increased expression of the AcrA/B-TolC efflux pump observed only after exposure to the former agent. The

findings clearly demonstrated common adaptive responses to the stress of exposure to fluoroquinolones and

certain disinfectants such as increased expression of the AcrA/B-TolC efflux pump.

Table 2 Comparison of selected proteins with increased expression following exposure to TAP and OXC

disinfectants

Protein Functional annotation Expressionratio

TAP OXCAcrB RND family, acridine efflux pump 2.79** 2.19EmrA multidrug resistance secretion protein 2.67** 2.25RpoD sigma D (sigma 70) factor of RNA polymerase, major

sigma factor during exponential growth2.5* 1.03

AhpC alkyl hydroperoxide reductase, C22 subunit; detoxification of hydroperoxides

2.22** 0.75

AceF pyruvate dehydrogenase, dihydrolipoyltransacetylase component

2.22** 1.16

AceE pyruvate dehydrogenase, decarboxylase component 2.04*** 1.06GrxA glutaredoxin1 redox coenzyme for glutathione-

dependent ribonucleotide reductase2.00*** ND

AcrA acridine efflux pump 1.86** 1.22TolC outer membrane channel; specific tolerance to colicin

E1; segregation of daughter chromosomes, role in organic solvent tolerance

1.78*** 1.14

Fbp fructose-bisphosphatase 1.70* 0.57*RplF 50S ribosomal subunit protein L6 1.56* 2.00*

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PykF pyruvate kinase I (formerly F), fructose stimulated 1.42* 1.02RpsC 30S ribosomal subunit protein S3 ND 8.00***

ExbBuptake of enterochelin; tonB-dependent uptake of B colicins

0.75 6.50***

MltA membrane-bound lytic murein transglycosylase A 0.75 3.80**

CysKsubunit of cysteine synthase A and O-acetylserine sulfhydrolase A

ND 3.60**

CydC ABC superfamily (atp&memb), cytochrome-related transporter

ND 3.50*

HtrA periplasmic serine protease Do, heat shock protein 1.80 3.50*Gcd glucose dehydrogenase ND 3.33**FhuA outer membrane protein receptor / transporter for

ferrichrome, colicin M, and phages T1, T5, and phi80 0.65 3.27***

YiaD putative outer membrane lipoprotein 1.08 2.63**RhlB putative helicase 1 2.37*** indicates P<0.05; ** P<0.01; *** P<0.001, ND indicated not detected.

The effect of the biocide triclosan (0.02 ug/ml) on protein expression following exposure for 90 minutes was also

investigated. Although changes in protein expression were less clear cut than effects seen with fluoroquinolones,

the efflux pump protein AcrB was raised but in contrast that of TolC was significantly reduced. Micro-array

transcriptomic studies at Birmingham provided a good correlation with this proteomic data, and 20 out of 27

proteins whose corresponding gene was on the array were similarly up- or down-expressed. Further studies with

mutant strains (from OD2010) with low, medium, and high resistance to triclosan suggested adaptation to

triclosan through mechanisms (e.g. mutation in the gene of the target enzyme FabI) other than changes in the

expression of the AcrAB/TolC efflux pump.

04/ Comparative analysis of cell envelope proteomes from mutants. The effectors of MAR were investigated in Salmonella Typhimurium (strain SL1344) by analysis of protein

expression in isogenic MAR mutants (n=4; selected following exposure to tetracycline, Levy et al., 1983) and

following exposure for 90 minutes to the mar locus inducer salicylate (5 mM).

These MAR mutants (MAR1-4) were characterised by MICs of ampicillin, chloramphenicol, nalidixic acid,

ciprofloxacin, tetracycline and triclosan which were typically elevated by 2-4 doubling dilutions in the MAR

mutants compared to the parent strain (table 3). Unlike the parent, the MAR mutants were all tolerant of

cyclohexane. These phenotypic characteristics were consistent with that of MAR mutants (Randall and

Woodward, 2002).

Table 3 Susceptibility of strains to antibiotics, biocide and solvents.

Strain MIC mg/L or solvent tolerance

Amp Chlor Nal Cip Tet Tric Hexane Cyclohexane

WT 1 4 4 0.015 1 0.13 + -

MAR1 8 16 8 0.060 4 0.5 + +

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MAR2 8 16 8 0.130 4 0.5 + +

MAR3 4 16 8 0.060 4 0.5 + +

MAR4 2 16 8 0.060 4 0.5 + +

Proteomes were extracted with urea and detergents, digested with trypsin and peptides analysed by 2-D-HPLC-

MSn. The expression of select proteins in the urea and boiled SDS extracts of MAR mutant proteomes are

presented in tables 4 and 5 respectively. Once again a common theme was evident with increased expression of

efflux pump proteins. The expression of the efflux pump proteins AcrA, AcrB and TolC was increased in the MAR

mutants by 86.8% + 31.6, 82.5% + 26.8 and 115% + 61.8 (P<0.0.1) respectively and following treatment with

salicylate by 26%, 38% & 100% (P<0.05). Similarly, expression of the outer membrane protein OmpF was

significantly reduced (P<0.01) in the MAR mutants, and with salicylate by 71.0% + 21.9 and 76.2% respectively,

compared to the parent. In contrast, certain significant differences in protein expression were observed between

the MAR mutants and treatment with salicylate. The expression of OmpX was increased with salicylate by

2038% but reduced in MAR mutants 79.3% + 22.7. Also, the putative omp COG3203 (16764875) was only

expressed after treatment with salicylate. This novel proteomic data supports and extends published micro-array

data and reveals that other stress response loci, in addition to mar, also contribute to the MAR phenotype in

Salmonella Typhimurium. Particularly, these studies clearly demonstrated a key role for increased expression of

efflux pump proteins and reduced elaboration of porins as a common mechanism across the MAR mutants and

following treatment with salicylate for reducing the accumulation of antibiotics.

Table 4 Percentage change in selected protein expression in MAR mutants and following treatment with salicyclate in urea proteome extracts.

Protein Functional annotation Strain/treatmentMAR1 MAR2 MAR3 MAR4 Sal

Efflux pump proteinsAcrA acridine efflux pump 129*** 91*** 71* 56** 26*AcrB RND family, acridine efflux pump 88** 116*** 74*** 52*** 38*TolC outer membrane channel; specific tolerance to

colicin E1; segregation of daughter chromosomes, role in organic solvent tolerance

56** 201** 114*** 91** 100***

Hydrogen peroxide protectionTpx thiol peroxidase 28 ‡ ‡ ‡ ‡AphF alkyl hydroperoxide reductase, F52a subunit;

detoxification of hydroperoxidesND ND ND ND 233*

AhpC alkyl hydroperoxide reductase, C22 subunit; detoxification of hydroperoxides

-47 17 0 0 -6

Dps stress response DNA-binding protein; starvation induced resistance to H2O2

0 37 41* 31* -33

SodA superoxide dismutase, manganese -11 -6 13 37 42SodB † 50 -33 16 -58*Drug/solvent resistanceYhcQ putative membrane located multidrug resistance

proteinND ND ND ND ‡

PqiB paraquat-inducible protein B 267** ‡ ND ‡ NDImp Organic solvent tolerance protein -14* -10 -14 -15 -6Heat shock/proteaseHtrA periplasmic serine protease Do, heat shock protein 12.9 1300** 1100*** 475** -44*UspA putative Universal stress protein UspA and related

nucleotide-binding protein75 200* 267** 233 -20

IbpA small heat shock protein ‡ † 100 133* †IbpB small heat shock protein ‡ 20 160 200** -53*ClpX specificity component of clpA-clpP ATP-dependent

serine protease, chaperone‡ 127** 93* 66* -55

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Lon DNA-binding, ATP-dependent protease la; cleaves RcsA and SulA, heat shock k-protein

-44 158 133* 92* -22

LonH putative protease ND -8 -16 -58* -25OsmY hyperosmotically inducible periplasmic protein,

RpoS-dependent stationary phase gene100 158*** 142** 125** -13

MetabolismGlgA glycogen synthase 0 167*** 71 116*** -61*GlgB 1,4-alpha-glucan branching enzyme ‡ ‡ ‡ ‡ NDDeoA thymidine phosphorylase ND 2000*** -20 40 ‡GcvP glycine cleavage complex protein P, glycine

decarboxylase‡ 238* 225** 137* -10

GcvT glycine cleavage complex protein P, glycine decarboxylase

ND ‡ ‡ ‡ -33

COG3959 putative transketolase ND 175 175** 150* -63**NfnB dihydropteridine reductase/oxygen-insensitive

NAD(P)H nitroreductase‡ ‡ ‡ ‡ 38

mdaB NADPH specific quinone oxidoreductase ND ‡ ND ‡ 166**MiscellaneousRecA DNA strand exchange and recombination protein

with proteinase and nuclease activity‡ 0 0 21.4 -12

ElaB putative inner membrane protein 100 450** 275* 400* 17YgaM putative inner membrane protein ND 200* 100* 225** -20Outer membrane proteinsCOG0845 membrane permeases, predicted cation efflux pump ‡ 14.3 -28 0 ‡OmpW outer membrane protein W; colicin S4 receptor;

putative transporterND ‡ ‡ ‡ ND

FepA outer membrane porin, receptor for ferric enterobactin (enterochelin) and colicins B and D

ND 100 † † ‡

CirA outer membrane porin, receptor for colicin I, requires TonB

ND 100 -40 † ‡

COG3203(16763889)

putative outer membrane protein ‡ 66 † 0 ‡

COG3203(16764875)

putative outer membrane protein ND ND ND ND ‡

Table 5 Percentage change in selected protein expression in MAR mutants and following treatment with salicyclate in boiled SDS proteomes.

Protein Functional annotation MAR1 MAR2 MAR4 SalAcrA acridine efflux pump ND ND ND NDAcrB RND family, acridine efflux pump 450*** 228* 284* -2.9TolC outer membrane channel; specific tolerance

to colicin E1; segregation of daughter chromosomes, role in organic solvent tolerance

250*** 18 30 -10

OmpF outer membrane protein 1a (ia;b;f), porin -47** -76** -90*** -76***OmpX outer membrane protease, receptor for phage

OX2-55* -83** † 2038**

COG0840 putative methyl-accepting chemotaxis protein † † † -42**COG320216763889

putative outer membrane protein ND ND ND ‡

COG320216764875

putative outer membrane protein ND ND ND ‡

EmrA multidrug resistance secretion protein ND ND ND ‡OmpC outer membrane protein 1b (ib;c), porin 66 -70* -63* 45

* indicates P<0.05; ** P<0.01; *** P<0.001, ND indicated not detected. . † indicates that this protein was only found in the proteome of the parent strain. 100‡ indicates that this protein was only found in the proteome of the MAR or salicylate exposed strain.

SID 5 (2/05) Page 14 of 23

Further studies concerned the analysis of the cell envelope proteomes from the acrB, tolC, ompC, ompF and

acrR gene deletion mutants. These studies were designed to probe the inter relationship of efflux pumps and

porins and provide comparison with both phenotypic data (MICs and antibiotic/fluorescent probe accumulation

studies). The deletion of acrB and tolC was associated with increased expression of the AcrE/AcrF and AcrD

efflux pumps (Figure 2), respectively, confirming the coordinated and possibly compensatory expression of

alternative efflux pumps observed previously by RT-PCR. In the OmpC/OmpF knockout mutants increased

expression of the proteins HtrA, FepA, IbpA, IbpB, OsmY and OmpR (regulates expression of OmpC/OmpF) was

observed indicating the activation of compensatory mechanism in outer membrane components. However the

expression of OmpC and OmpF was reduced following deletion of both acrB and tolC (Figure 3). In the acrR

gene deletion mutant the expression of the AcrA and AcrB protein were increased (Figure 4) but not TolC or the

other proteins associated with the MAR mutants (tables 4 and 5). Moreover the acrR gene deletion mutant was

not cyclohexane resistant and did not have a MAR phenotype. These studies revealed changed expression of

multiple proteins following the deletion of single genes and a complex framework of protein expression

regulation /interdependence.

Figure 2 Expression of efflux pump proteins in gene deletion mutants.

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Figure 3 Expression of outer membrane proteins in gene deletion mutants

Figure 4 Expression of the efflux pump proteins AcrAB/TolC in the acrR gene deletion mutant

05/ Review of data from 01/ to 04/ for selection of protein(s) for MAR test.

A key issue in the analysis of data from post-genomic studies is the handling and interrogation of such sets with

appropriately robust statistical analysis. Data sets arising from the proteomic studies are very large and were

compiled (replicates) reduced (common proteins to all replicates) and summarised (major differences) using

SID 5 (2/05) Page 16 of 23

Microsoft Access. This enabled compilation of data between replicates (n=3) and identification of proteins

common to control and MAR mutants and those found in only controls or MAR mutants. Typically, analysis of a

Salmonella proteome generated some 25,000 peptide identifications (from each replicate and typically 6

replicates/experiment) using proprietary software (SEQUEST) which was filtered (DTASELECT) to a false

positive peptide identification rate of 1%. This was established using the gene deletion mutants as the deleted

proteins should not be present in the proteome (Coldham et al., 2007). The outputs from DTASELECT were in

the format of text files (*.txt) which contained information in separate fields describing the protein identification

number, spectrum count (the number of tryptic peptides of that protein identified) and functional annotation. The

text files were read into Microsoft Access where the proteins common to all replicates (n=3) could summarised

and matched with the gene/ protein name (e.g. tolC). Microsoft Access also enabled ‘queries’ to be run for the

interrogation of such large data sets to find proteins differentially express. The outputs of such analyses were in

the form of Excel files which enabled the data to be further ranked and statistically compared in Microsoft Excel.

Each Excel workbook (file) covered a single experiment, such as the effect of a fluoroquinolone by comparing

control untreated cultures against exposed, and yielded five groups which were each presented in a separate

Excel work sheet; group 1 - proteins found in controls (flasks 1-3), group 2 - proteins found following

fluoroquinolone treatment (flask 4-6), group 3 - proteins found in controls only (flasks 1-3), group 4 - proteins

found after fluoroquinolone treatment only (flasks 4-6), and finally, group 5 - those common to controls and

fluoroquinolone treatment (flasks 1-6). The statistical significance of percentage changes in protein expression in

group 5 were determined using a two tailed Student’s t-test. This approach to the analysis of such large and

complex data sets has been published elsewhere (Coldham et al., 2006) and can be viewed on line [available as

Supplementary data at JAC online (http://jac.oxfordjournals.org/].

The experimental data from the studies under objective 02-04/ were reviewed in this fashion for the discovery of

biomarkers of MAR. These analyses revealed that changes in the expression of proteins, such as AcrAB/TolC,

OmpF and OmpC, which control solute accumulation and export, showed the most consistent changes and most

likely to provide biomarkers for MAR.

06/ Development of test for MAR phenotype.A test for reduced cellular uptake and increased efflux of antibiotics consistent with the MAR phenotype was

developed using fluorescent probes. The principle of these tests is to evaluate the functional activity of the AcrAB/TolC efflux pump and porin expression using the cellular accumulation of fluorescent probe. The hypothesis under investigation is that increased expression of the AcrAB/TolC efflux pump and or reduced expression of porins (e.g. OmpF) is associated with reduced accumulation of probe. In the following experiments this hypothesis is tested using the gene deletion and MAR mutants and selective efflux pump inhibitors. The probes evaluated included ethidium bromide, acridine orange, bis benzimides (H33342 & 33258) and rhodamine 6G. These are all cations and bind to DNA which induces a change in the emission spectrum and enables the essential discrimination between free (in the medium) and that bound to DNA with in the bacterial cell. Using this principle a rapid assay system, in 96 well plate format (also 384 well plate compatible), was developed for use with an automated fluorescence detection plate scanner (Fluostar). This approach is favoured since multiple fluorescent probes may be used to determine the functional activity of protein biomarkers (such as the AcrAB/Tolc efflux pump or OmpF) involved in antibiotic uptake and export. Such scanners are now standard items of laboratory equipment, and for example, are

installed at the VLA (n=3), Birmingham and Bristol university laboratories for a wide range of applications. A

SID 5 (2/05) Page 17 of 23

diagram illustrating the simplicity of the work flow for this type of fluorescence accumulation assay is presented in

figure 5. The data from the plate reader is exported via Excel and may be copied and pasted into pro-forma

spread sheets for data reduction and graphical presentation. Initial studies focussed on the accumulation of

ethidium bromide (excitation 510 emission 610 nm) by comparison of the parent Salmonella Typhimurium, with

isogenic MAR mutants (positive controls), gene deletion mutants (positive and negative controls as appropriate)

and heat inactivated parent or in the presence of inhibitors (negative controls). The effect of substrate

concentration and density of bacterial suspension were also determined. Preliminary studies indicated that a

concentration of 100 M ethidium bromide provided for the greatest difference in accumulation between the

parent and MAR mutants (n=2). The accumulation of ethidium bromide by the parent strain (SL1344) and a MAR

mutant at different cell densities (0.2, 0.1 and 0.05 – OD 600 nm) and in the presence of the efflux pump inhibitor

(collapses the proton gradient) with incubation time is shown in figure 6. Each data point was derived from 8

replicates with coefficients of variation typically <3%. The statistical difference between strains was evaluated using a paired (two tailed) Students T test. Statistical significances are indicated on the

graphs 8, 9 and 10 for the data sets (strains tested) where appropriate.

Figure 5 Diagram illustrating the fluorescence based 96 well plate assay for MAR mutants

Figure 6 Accumulation of ethidium bromide by parent and MAR mutant strains

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Further studies investigated the accumulation of ethidium bromide (100 M) by the positive and negative control

strains described above (Figure 7). The accumulation of ethidium bromides was reduced in MAR mutants and in

strains where either the porins OmpC or OmpF had been deleted. Deletion of the efflux pump protein TolC

significantly increased accumulation, as anticipated, but removal of AcrB had little effect which may reflect

induction of the AcrEF efflux pump (see data from the analysis of gene deletion mutants figure 2). Heat

inactivation of the parent strain (90o C for 10 mins) caused greatly enhance accumulation of ethidium bromide

due to denaturation of outermembrane porin and efflux pump proteins.

Figure 7 Accumulation of ethidium bromide by different Salmonella Typhimurium strains.

These studies (figure 6) and others (not shown) demonstrated an apparent maxima followed by reduced

fluorescence by the parent strain during the incubation period. This phenomena has been described previously

for ethidium bromide accumulation by P. aeruginosa cells and represents quenching of fluorescence as the

intracellular concentration increases (Babayan and Nikaido, 2004) rather than up regulation of efflux pump activity

and export of the substrate. This quenching of the fluorescent signal may be manually corrected by the analysis

of appropriate control wells or the use of polarised fluorescence analysis.

Further studies were continued with Hoeshct bis benzimide probes H33342 and H33258 (excitation 350 emission

460 nm). These compounds were evaluated as they are less toxic (not mutagens) and have higher quantum

SID 5 (2/05) Page 19 of 23

yields than ethidium bromide. Parallel studies with H33342 and H33258 at different substrate concentrations

demonstrated that the optimum concentration of H33342 was 2.5 M. The accumulation of H33258 was very low

as this compound is relatively polar with a phenolic substituent which may prevent uptake by the bacterial cell.

The uptake of H33342 is presented in figure 8 and is essentially similar to that obtained with ethidium bromide but

less quenching of fluorescence was observed.

Figure 8 Accumulation of bis benzimide by different Salmonella Typhimurium strains.

The effect of the inhibitors CCCP and Phe-Arg-β-naphthylamide on H33342 accumulation by the parent S.

Typhimurium was investigated to validate measurement of active efflux. CCCP inhibits active efflux by collapsing

the proton gradient across the cytoplasmic membrane thereby removing the motive force for RND type efflux

pumps such as AcrAB/TolC. Phe-Arg-β-naphthylamide has been shown to be a competitive inhibitor of the AcrB

and prevents ligand binding and efflux. Dose response curves illustrating the effects of CCCP and Phe-Arg-β-

naphthylamide on bis benzimide accumulation after 30 minutes incubation are presented in figure 9.

Figure 9 Dose response curves for the effect of efflux pump inhibitors on bis benzimide accumulation

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07/ Validation of test for MAR phenotype.

The fluorescence assay using the H33342 probe (bis benzimide) was validated as a test for MAR by the analysis

of a range of well characterised MAR mutants from project OD2010 (Use and abuse of disinfectants), a panel of

Salmonella strains previously analysed using the cyclohexane test (Webber et al., 2006) and finally, to

demonstrate transferability, by analysis of similar strains at VLA and Birmingham University.

A variety of mutants arising from studies investigating the development of mutants with reduced susceptibility to

disinfectants were analysed. The first series comprised three mutant strains produced following daily passage for

7 days in media containing a tar oil phenol disinfectant (FFS), an oxidising compound based disinfectant (VS) a

compound disinfectant containing a mix of quaternary ammonium compounds, formaldehyde and glutaraldehyde

(SK) were isolated. The SK and VS mutants had a MAR phenotype with increased expression of AcrAB/TolC,

reduced expression of OmpF and were cyclohexane tolerant. The FFS mutant was not MAR (and cyclohexane

sensitive) but had reduced expression of OmpF. The accumulation of H33342 by these strains is shown in Figure

10 and was consistent with their efflux/porin protein expression profiles. The accumulation of bis benzimide was

significantly (P<0.05) reduced by all three mutants but least in the FFS strain. Further details on the strains are

provided in the final report for OD2010 and a manuscript has been prepared describing this work (Karatzas et al.,

2007).

Figure 10 Uptake H33342 by mutants with reduced susceptibility to disinfectants

A second series of S. Typhimurium mutant strains were produced following a single passage with overnight

exposure on media containing a tar oil phenol disinfectant (FFS), an oxidising compound based disinfectant (VS)

a compound disinfectant containing a mix of quaternary ammonium compounds, formaldehyde and

glutaraldehyde (SK). These strains were produced from parent strains of Salmonella Typhimurium DT104, a

multi-drug resistant Salmonella Typhimurium DT104 and Salmonella Typhimurium SL1344. Compared to their

parents small decreases in sensitivities to certain antibiotics were observed but none became MAR (the MDR

parent was already MAR) and in all cases the uptake of H33342 was the same of that of the parent. Significantly

(P<0.0001) reduced accumulation of H33342 was observed by the multidrug resistant Salmonella Typhimurium

DT104 compared to either SL1344 or Salmonella Typhimurium DT104 (data not shown). A manuscript including

this work has been submitted for publication (Randall et al., 2007) and a poster presented at ECCMID 2007

(Bagnall et al., 2007; see section 9).

The final series were of experiments with three mutant strains derived from a gyrA mutant of SL1344 with low,

medium and high resistance to triclosan requiring 4-8 g/ml, 16-32 g/ml and >32 g/ml of triclosan for inhibition.

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Proteomic analysis revealed that none of these strains had changed expression of AcrAB/TolC, OmpF or OmpC.

Their accumulation of H33342 was not significantly reduced compared to both SL1344 or their immediate gyrA

parent. Further details of these strains are provide in the final report for OD2010 (Use and abuse of disinfectants)

and two papers including this work are in the process (accepted/in press) of publication (Randall et al., 2007A;

Randall et al., 2007B).

A panel of Salmonella strains (n=43 Webber et al., 2006) comprising 14 different serotypes isolated from

livestock in Great Britain by the VLA were assay using the H33342 accumulation assay. This include 26

ciprofloxacin resistant isolates, and 19 cyclohexane tolerant strains. The MIC to Nal, Cip, Chl, Tet, acridine

orange, ethidium bromide and triclosan was determined for all strains. A good correlation was obtained between

cyclohexane tolerant strains and reduced accumulation of H33342 (16 of the 19). Three strains had reduced

accumulation of H33342 but were not cyclohexane tolerant and the mechanism for this required further

investigation. Statistical analysis of this data was performed using analysis of covariance (similar to Anova but with covariate embedded) and with a general linear model which examined the difference between

intercepts for the strains following log transformation of the data.

The fluorescence assay using the H33342 probe (bis benzimide) was further validated by the analysis of identical

strains at Birmingham University including SL1344, 4 MAR mutants and mutants with deletions in the genes

encoding acrB, tolC, ompC and ompF. A similar pattern of data was obtained with reduced accumulation of

H33342 by the MAR mutants and following deletion of OmpC and OmpF. Increased accumulation was observed

following deletion of tolC and acrB, the latter was divergent from findings at VLA and will be further investigated.

Uptake and implementationThe fluorescence assays described have several positive attributes which include:-

The sensitivity to detect reduced expression of outer membrane porins (such as OmpC and OmpF) in

addition to increased expression of certain efflux pumps such as AcrAB/TolC. This conclusion is supported by reduced uptake of H33342 in the porin gene deletion mutants and in MAR mutants where there was increased expression of the AcrAB/TolC efflux pump. Conversely where this efflux pump was disrupted, by deletion of AcrB or TolC increased uptake of H33342 was observed. The precise mechanism for the MAR phenotype of the 43 field isolates can not be assigned but was highly consistent with either increase efflux pump activity (such as AcrAB/TolC) or reduced porin expression or both.

The report format from the assay is a numerically scaled enabling comparison of accumulation rates,

unlike the cyclohexane test which provides a binary output (tolerant or sensitive).

The test is also relatively rapid, providing data in under 6 hours, and can be further enhanced by the use

of selective inhibitors.

The fluorescence assay using primarily accumulation of H33342 (ethidium bromide also available) will be applied

to the analysis of Salmonella strains under the surveillance contract at the VLA. This will enable the detection of

and changes in the prevalence of MAR strains for comparison with epidemiological data and changes in

cleansing and disinfection procedures.

References to published material

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9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.The following papers/manuscripts have been produced directly from project OD2011:

1. Coldham, N.G. Webber, M., Piddock, L.J.V and Woodward, M.J. Comparative proteomics between Salmonella Typhimurium MAR mutants and following exposure to salicylate. (manuscript in final preparation)

2. Coldham, N.G. Webber, M., Piddock, L.J.V. Carter, B. and Woodward, M.J. Estimation of false positive and false negative SEQUEST peptide identifications at defined loci using gene deletion mutants of Salmonella Typhimurium. (Status: revised manuscript re-submitted to Proteomics Sept. 07).

3. Coldham NG, Randall LP, Piddock LJV Woodward MJ. Effect of fluoroquinolone treatment on the proteome of Salmonella enterica serovar Typhimurium analysed by 2D-gel electrophoresis and 2D-HPLC-MSn. (2006) J. Antimicrob. Chemother. 58 1145-1153.

4. Coldham, N.G. and Woodward, M.J. Characterization of the Salmonella Typhimurium Proteome by semi-automated HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance. (2004) J. Proteome Research 3 595-603.

5. Ricci, V., Tzakas, P., Buckley, A., Coldham, N.G., Piddock, L.J.V. Ciprofloxacin-resistant Salmonella enterica serovar Typhimurium strains are difficult to select in the absence of AcrB and TolC. (2006) Antimicrobial Agent and Chemotherapy 50 38-42.

6. Howells LC, Whelan AO, Woodward MJ, Coldham NG. Simple and effective technical procedures for the packing and application of nanospray PicoFrit HPLC columns for proteomic analyses. (2005) Proteomics 5 3864-3867.

There has also been considerable overlap with Defra funded project OD 2010 (Use and abuse of non-antibiotic antimicrobials as major contributors toward the development of antibiotic resistance). The proteomic methods and H33342 accumulation assay developed under project OD2011 have been applied to various Salmonella strains from OD2010 (with the agreement of the Defra project officer) and the following manuscripts have been produced:

1. Randall LP, Cooles SW, Coldham NG, Penuela EG, Mott AC, Woodward MJ, Piddock LJV and Webber MA. Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics. J. Antimicrob. Chemother. Published on line ahead of print Sept 07.

2. Karatzas KAG, Randall LP, Webber M, Piddock LJV, Humphrey TJ, Woodward MJ, Coldham NG. Phenotypic and molecular characterization of MAR isolates of Salmonella enterica ser. Typhimurium selected following prolonged treatment with commercial farm disinfectants (Status: submitted to Appl. Environ. Microbiol and corrected post reviewers comments).

3. Webber MA, Randall LP, Cooles SW, Coldham NG, Woodward MJ, and Piddock LJV. Distinct mechanisms contribute to triclosan resistance in Salmonella enterica serovar Typhimurium (Status: submitted to PNAS April 07).

4. Randall LP. Bagnall M, Karatzas A, Coldham NG Piddock, LJV.; Woodward MJ Fitness and dissemination of disinfectant selected multiple antibiotic resistant (MAR) strains of Salmonella enterica serovar Typhimurium in chickens. Accepted by J. Antimicrob. Chemother

Poster presentations (the abstracts for these were peer reviewed)1. Coldham, N.G., Randall, L.P., Piddock, L.J.V* and Woodward, M.J. Development of a protein

based test for Multiple Antibiotic Resistant Salmonella Typhimurium. Proceedings of the 15th European Congress of Clinical Microbiology and Infectious Disease. Abstract and poster. Copenhagen, Denmark.

2. Coldham, N. G1*., Randall, L. P1., Piddock, LJV2 , Woodward, M. J1. Comparative analysis of Salmonella Typhimurium proteomes from MAR mutants and following treatment with salicylate. Proceedings of the 16th European Congress of Clinical Microbiology and Infectious Disease. Abstract and poster. Nice, France.

3. Coldham, N. G1*., Randall, L. P1., Piddock, L.J.V.2 , Woodward, M. J1. Development of a rapid fluorescence assay in 96 well plate assay for multiple antibiotic resistant (MAR) Salmonella Typhimurium. Proceedings of the 17th European Congress of Clinical Microbiology and Infectious Disease. Abstract and poster. Munich, Germany.

4. M.C. Bagnall, L.P. Randall, N.G. Coldham, A. Karatzas, T. Humphrey, L.J.V. Piddock and M.J.

Woodward. 2007. Effect of multiple passages of Salmonella Typhimurium in the presence of disinfectants on susceptibility to antimicrobials, on persistence in the one-day-old chick model and efflux systems. 2007. In Proceedings of the 17th European Congress of Clinical Microbiology and

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