International Journal of Food Microbiology 113 (2007) 315 – 320

www.elsevier.com/locate/ijfoodmicro

Efflux pump activity in fluoroquinolone and tetracycline resistant

Salmonella and E. coli implicated in reduced susceptibility
to household antimicrobial cleaning agents
C.A. Thorrold a , M.E. Letsoalo b , A.G. Dusé a , E. Marais a,⁎
a
Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand Medical School and National Health Laboratory Services,
P.O. Box 2115, Houghton 2041, Johannesburg, South Africa
b
Biostatistics and Epidemiology Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham, 2131, South Africa
Received 6 August 2005; received in revised form 12 November 2005; accepted 24 August 2006

Abstract

It has been shown that the inappropriate use of antimicrobial household agents selects for organisms with resistance mechanisms (e.g. efflux
pumps), which could lead to the development of antibiotic resistance. The reverse hypothesis, that antibiotic-resistant organisms become tolerant
to other antibacterial agents (e.g. disinfectants) due to the action of efflux pumps, has however not been extensively examined. The objective of
this study was to establish whether there is a link between antibiotic resistance in potential gastrointestinal pathogens and reduced sensitivity of
these organisms to commonly used household antimicrobial agents. In this study, tetracycline and ofloxacin sensitive and resistant Escherichia
coli (9 strains) and Salmonella spp. (8 strains) were isolated from poultry and clinical samples. In order to assess whether these bacteria had active
efflux pumps, ethidium bromide accumulation assays were performed. Extrusion of the active components of three commercial household agents
(triclosan, sodium salicylate, and ortho-phenylphenol) by efflux pumps was tested using spectrophotometric accumulation assays. In order to
simulate the kitchen environment, in-use disinfectant testing using the commercial household agents was performed to determine changes in their
efficacy due to antibiotic resistance. Active efflux pump activity and extrusion of all three active ingredients was observed only in the antibiotic
resistant organisms. The antibiotic sensitive bacteria were also more susceptible than the resistant isolates to the household antimicrobial agents at
concentrations below that recommended by the manufacturer. These resistant bacteria could potentially be selected for and result in hard to treat
infections.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Household antimicrobials; Eff lux pump; Food pathogens

1. Introduction already been described in a number of clinically important bac-

The occurrence of bacteria resistant to multiple antibiotics
presents a serious problem in the treatment of bacterial infec-
tions (Bolhuis et al., 1995). One of the underlying mechanisms
for this multidrug resistance phenotype is the expression and
increased activity of efflux pumps, which facilitate the transfer
of toxic substances from within cells to the external environ-
ment (Bohn and Bouloc, 1998; Webber and Piddock, 2003).
Efflux systems that contribute to antibiotic resistance have
⁎ Corresponding author. Tel.: +27 11 489 8587; fax: +27 11 489 8530.
E-mail address: maraise@pathology.wits.ac.za (E. Marais).
0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2006.08.008
teria, including Salmonella typhimurium (Webber and Piddock,
2003), and Escherichia coli (Poole, 2000). Efflux pumps can
transport a variety of structurally dissimilar compounds or may
be specific for a single substrate (Poole, 2000; Webber and
Piddock, 2003).
There is little doubt that the inappropriate clinical application
of antimicrobial agents has led to the increased rates of multi-
drug resistant bacteria (Cruchaga et al., 2001), and the use of
antibiotics in the veterinary and farming arena has recently
come under scrutiny (Singer et al., 2003). The introduction of
antibiotics in livestock rearing has resulted in a variety of
developments in animal husbandry practices (Manie et al.,
316 C.A. Thorrold et al. / International Journal of Food Microbiology 113 (2007) 315–320

1998; Butaye et al., 2003). These include the breeding of ani- Table 2
mals in batteries/pens that are filled to capacity at optimal Zone diameters (including disk) of the eight Salmonella strains and nine E. coli
strains at time of study and after serial passage (brackets)
temperature and low light intensity to enhance growth rates and
increase body mass (Manie et al., 1998). Many antibiotics are Bacterial Zone diameter (mm) Result (R /S)
species
administered therapeutically for disease prevention, but sub- Tetracycline Of loxacin Tetracycline Of loxacin
therapeutic doses are also administered to improve feed effi- (30 μg) (5 μg) (30 μg) (5 μg)
ciency and accelerate weight gain in poultry, swine, sheep and Salmonella # 1 19 (20) 29 (29) S (S) S (S)
cattle (Manie et al., 1998; Chopra and Roberts, 2001; Geornaras Salmonella # 2 20 (19) 28 (32) S (S) S (S)
Salmonella # 3 21 (20) 28 (26) S (S) S (S)
et al., 2001; White et al., 2001; McEwen and Fedorka-Cray,
Salmonella # 4 21 (19) 30 (31) S (S) S (S)
2002; Butaye et al., 2003). Salmonella # 5 6a 30 a R S
Although not everyone is in agreement as to whether sub- Salmonella # 6 8 (9) 29 (33) R (R) S (S)
therapeutic doses lead to the development of antibiotic resis- Salmonella # 7 13 (14) 39 (36) R (R) S (S)
tance in bacteria, there is extensive evidence that they result in Salmonella # 8 8 (9) 46 (37) R (R) S (S)
E. coli # 1 28 (26) 36 (37) S (S) S (S)
significant selection pressures on animal pathogens and com-
E. coli # 2 26 (25) 33 (32) S (S) S (S)
mensals (Quednau et al., 1998; McEwen and Fedorka-Cray, E. coli # 3 29 a 42 a S S
2002). The antibiotic resistance that has arisen in zoonotic E. coli # 4 28 (27) 38 (37) S (S) S (S)
organisms such as Salmonella, Listeria and E. coli poses a E. coli # 5 6a 11 a R R
threat to both animal and human health (Chopra and Roberts, E. coli # 6 6 (7) 6 (7) R (R) R (R)
E. coli # 7 6 (6) 5 (8) R (R) R (R)
2001). Since potential pathogens can be transferred from
E. coli # 8 6 (6) 8 (9) R (R) R (R)
animals to humans via the food chain (Parsonnet and Kass, E. coli # 9 14 (18) 6 (6) R (R) R (R)
1987), they could result in diseases that are difficult to treat or
A zone size of ≥14 and ≥12 is considered as resistant for tetracycline and
transfer their resistance genes to other bacteria (Chopra and of loxacin, respectively (NCCLS, 2001).
Roberts, 2001). a
Serial passage not performed as strains no longer viable.
Several brands of household disinfectant products are mar-
keted as being superior to others due to the addition of one or
more antibacterial compounds, since it is claimed that these shown in Table 2. Ethics clearance was obtained from the
products kill or inactivate bacteria more efficiently than the University of the Witwatersrand for the use of patient isolates.
regular items (Kusumaningrum et al., 2002). Researchers have
recently started to express concern regarding the use of anti- 2.2. Media
microbial chemicals in the domestic setting due to possible
selection of resistant organisms (McDonnell and Russell, All media components were purchased from Diagnostic
1999). Media Products (Rietfontein, Johannesburg, South Africa), un-
This study was undertaken to establish whether there is a less otherwise indicated, and all chemicals used were of
relationship between antibiotic resistant organisms and reduced analytical grade.
susceptibility to the antimicrobial agents found in commonly
used household disinfectant products, and whether this could be 2.3. Isolation of bacteria from poultry and clinical samples
due to the action of efflux pumps.
Twenty five grams of the poultry sample was processed by
2. Materials and methods stomaching in 225 ml sterile buffered peptone water. E. coli and
salmonellae were isolated using standard microbiological cul-
2.1. Bacterial strains ture techniques. In order to isolate tetracycline or ofloxacin
resistant bacteria, 160 μg of tetracycline (Oxoid, Basingstoke,
E. coli and Salmonella species were isolated from fresh and England), or 80 μg of ofloxacin (Oxoid) was added to 10 ml of
frozen chicken products, clinical samples, and from the the enrichment broths. Salmonella isolates were typed using
Onderstepoort Veterinary Institute, University of Pretoria, API 10S (bioMerieux, Durham, USA), and 3M Petrifilms for
South Africa. The source of the bacterial isolates is shown in coliforms and E. coli (3M Microbiology Products, USA) were
Table 1 and the antibiotics that each strain was resistant to are used to confirm the presence of E. coli. Standard microbiolog-
ical procedures were used to isolate E. coli and Salmonella
from the clinical samples and veterinary samples.
Table 1
The source of gastrointestinal pathogens examined in this study 2.4. Identification and susceptibility testing
Source Bacterial isolates Number
Poultry samples Salmonella #s 1–6 and E. coli #s 1–5 11 The tetracycline and ofloxacin resistance profiles of the
Humans with infectious Salmonella #s 7 and 8 and E. coli #s 8 4 bacteria were determined using disk diffusion susceptibility
intestinal disease and 9 testing on Mueller–Hinton agar plates with reference to the
Onderstepoort Veterinary E. coli #s 6 and 7 2 National Committee for Clinical Laboratory Standards
Institute
(NCCLS, 2001) criteria (disk content of each antibiotic shown
 C.A. Thorrold et al. / International Journal of Food Microbiology 113 (2007) 315–320
in Table 2). Only isolates with high level resistance were ana-
lyzed further. Susceptibility testing was repeated on all isolates
after serial passage (Suller and Russell, 2000) to confirm long
term retention of antibiotic resistance. Macro-restriction analysis
was performed as described (Botteldoorn et al., 2004; Vali et al.,
2004) to ensure that the bacteria used in this study were not
clonally related.
2.5. Ethidium bromide accumulation assays
Ethidium bromide accumulation was assayed as previously
described (Baranova and Neyfakh, 1997) with slight modifica-
tions. Briefly, bacteria were grown in brain heart infusion (BHI)
broth, pelleted by centrifugation, and resuspended to an optical
density of 0.2 at A600. Ethidium bromide was added to the cell
suspension at a final concentration of 2 μg/ml. Sample fluo-
rescence was used as a measure of the amount of ethidium
bromide incorporated by the cells and was recorded using a
standard visible range spectrofluorimeter at excitation and emis-
sion wavelengths of 530 and 600 nm, respectively. Five minutes
after ethidium bromide addition, carbonyl cyanide m-chloro-
phenylhydrazone (CCCP) (Sigma-Aldrich, MO, USA), an ef-
flux pump inhibitor, was added to the cell suspension at a final
concentration of 5 μg/ml. The natural fluorescence of the bac-
terial cells was subtracted by adjusting the zero point prior to
ethidium bromide addition.
2.6. Measurement of chemical accumulation
Chemical accumulation of the antimicrobial components in
several household detergents was assayed by the method of
Chapman and Georgopapadakou (1988) with slight modifica-
tions. Bacteria were grown overnight at 37 °C in Luria Bertani
broth, harvested by centrifugation, washed in phosphate buf-
fered saline (PBS), and adjusted to a calculated A600 of 10.0 in
PBS. The chemicals, sodium salicylate (Sigma Aldrich, MO,
USA), or triclosan (kind gift from Ciba Geigy, Manchester, UK),
or ortho-phenylphenol (Sigma Aldrich), were then added to
final concentrations of 10 mM, 4 mM and 6 mM, respectively
(the concentrations recommended by the household product
manufacturer). Samples (0.5 ml) were removed at timed inter-
vals as indicated in Fig. 3. Seven minutes after addition of the
chemical, CCCP was added to half of the reaction mixture to a
final concentration of 200 μM and the other half of the reaction
mixture was used as a control (no CCCP). Each of the aliquots
removed was immediately diluted in 1 ml of ice-cold PBS and
centrifuged at 5700 ×g for 5 min. The pellets were washed with
1 ml of ice-cold PBS, resuspended in 1 ml of PBS and boiled for
5 min to lyse the cells. The samples were centrifuged at 5700 ×g
for 10 min and the absorbance of the supernatant was measured
using a 3 series UV spectrophotometer (BioMate, NY, USA) at
wavelengths of 296 nm (for sodium salicylate), 382 nm (for
Triclosan) and 251 nm (ortho-phenylphenol), which were de-
termined to be suitable for the measurement of each aromatic
compound. The absorbance readings are indicative of the
amount of agent internalized by the bacterial cells and therefore
of accumulation. This procedure was done in duplicate on two
317
independent occasions for each isolate and the results plotted as
graphs of time vs. absorbance.
2.7. In-use disinfection procedure
Bacteria were grown in 10 ml BHI overnight and added to
50 ml of a 10% sterilized milk solution (Oxoid). One perforated
100% viscose fibre dishcloth (purchased from a supermarket and
autoclaved prior to use) was added to each isolate and squeezed
with gloved hands to distribute the soiling suspension throughout
the cloth. The cloth was transferred to a sterile plastic bag and left
to incubate at room temperature for 2 h.
The dishcloths were aseptically divided into 4 equal portions of
25 cm2. One portion was transferred to quarter-strength Ringer
solution for determination of the bacterial count without treat-
ment. The remaining cloth portions were immersed in the house-
hold disinfectant products (containing sodium salicylate, triclosan,
or 2-phenylphenol) at the recommended concentration, as well as
25% and 50% of this concentration. After 2 min, the cloth portions
were rinsed in running tap water for 30 s and a cloth portion from
each disinfectant treatment was transferred to flasks containing
10 ml of quarter-strength Ringer solution for determination of total
bacterial counts. Serial dilutions of the rinse fluid were plated onto
nutrient agar plates. The colony forming units (cfus) were deter-
mined and expressed as % reduction in bacterial numbers relative
to the cfus from the untreated aliquot. This procedure was repeated
in duplicate on two independent occasions for each isolate.
2.8. Statistical analysis
Multilevel linear models, also known as Hierarchical linear
models (HLM) were employed with strains at level-1 or lower
level and groups (Salmonella and E. coli) at level-2 or higher
level. STATA 8.2 (Stata Corporation, College Station, Texas,
USA) was used in the analysis of results. Results were considered
significant at the 5% level.
3. Results
3.1. Bacterial strains
Eight Salmonella and nine E. coli strains were examined in
this study. Using the National Committee for Clinical Labo-
ratory Standards (NCCLS, 2001) it was determined that 4 of the
Salmonella species were sensitive to tetracycline (disk content
30 μg) and ofloxacin (disk content 5 μg) and 4 were resistant to
tetracycline only. Four of the E. coli strains were sensitive and
five were found to be resistant to both the antibiotics. To test the
stability of the antibiotic resistance mechanisms, the isolates
were serially passaged for 20 days. Three of the 17 strains were
not viable after 2 years of storage and could not be tested. The
viable isolates were resistant to the original antibiotic(s) of
selection after this period (Table 2). This suggests that the genes
coding for resistance are chromosomal, but this was not inves-
tigated further. Dissimilar banding patterns were observed in the
macro-restriction analyses of all the bacteria examined, indicat-
ing that they were different strains.
318 C.A. Thorrold et al. / International Journal of Food Microbiology 113 (2007) 315–320

with a significant difference in the absorbance reading in the

presence and absence of CCCP (z = 10.76; p = 0.000), as a result
of a greater amount of cell-associated active component
(Fig. 2B). Similar results were observed with all three active
ingredients tested, demonstrating that the resistant strains had
efflux pumps that were extruding the active ingredients found in
commonly used household disinfectant products, and that these
efflux pumps were energy dependent.

3.4. In-use disinfection procedure

An in-use disinfectant test was used to assess the effect of

various concentrations of three commonly used household
Fig. 1. Representative figures of accumulation of ethidium bromide by (A) disinfectants, containing sodium salicylate (10 mM), triclosan
antibiotic sensitive E. coli # 2 and (B) resistant E. coli # 5. On addition of (4 mM), or 2-phenylphenol (6 mM), on the antibiotic sensitive
ethidium bromide, f luorescence intensity ( y-axis) increased, and then leveled and resistant bacteria.
off. (A) Addition of the eff lux pump inhibitor, CCCP, had no effect on After the disinfection procedure with all three household
f luorescence intensity indicating that no eff lux pumps were inhibited.(B)
products, there was a dramatic reduction in the number of
Addition of the eff lux pump inhibitor, CCCP, resulted in a sharp increase in
f luorescence indicating inactivation of eff lux pumps (n = 1). bacteria remaining on the cloths; as can be seen in Fig. 3A and B.
The greater the concentration of disinfectant used, the greater the
reduction in bacterial numbers; as seen in Fig. 3A and B.
3.2. Ethidium bromide accumulation With the antibiotic sensitive bacteria, the difference in the
number of surviving bacteria at different disinfectant concen-
Ethidium bromide accumulation was assayed to determine trations (Fig. 3A) was marginal. However, with the resistant
whether the bacteria had active efflux pumps. As shown in bacteria, there was a noticeable difference in the number of
Fig. 1A and B, the fluorescence intensity was seen to increase remaining bacteria at the different concentrations (Fig. 3B),
substantially on addition of ethidium bromide for each strain
examined. This was indicative of the cells taking up the dye, as
binding of ethidium bromide to intracellular components results
in increased fluorescence. By 5 min after the addition of ethid-
ium bromide a plateau in the fluorescence values was reached.
On addition of CCCP, the fluorescence intensity did not change
in the sensitive bacterial strains (Fig. 1A). These results indicate
that efflux pumps were not extruding the dye, since their in-
activation had no effect on the equilibrium. However, in each of
the resistant bacterial strains examined, addition of CCCP resulted
in a rapid and dramatic increase in fluorescence (Fig. 1B). This
was due to the inhibition of energy dependent efflux pumps,
resulting in a greater net accumulation of ethidium bromide.

3.3. Measurement of chemical accumulation

Cellular accumulation of the chemicals was measured in

order to establish whether efflux pumps could extrude the active
ingredients of three household disinfectants. In all the tetra-
cycline and ofloxacin sensitive bacterial strains, there was an
increase in the absorbance readings in both the presence and
absence of CCCP (Fig. 2A), with no significant difference be-
tween the presence and absence of CCCP (z = − 0.88; p = 0.381).
These results demonstrate the absence of efflux of the active
ingredients from the sensitive bacterial cells, and that the chemi-
cals were accumulating in the bacterial cells. This was observed
for all three of the active ingredients tested (4 mM triclosan,
10 mM sodium salicylate, and 6 mM ortho-phenylphenol). Fig. 2. Graph of accumulation of triclosan by (A) sensitive Salmonella # 2 and
(B) resistant Salmonella # 8. Absorbance on the y-axis is a measure of the levels
In the antibiotic resistant strains, the absorbance values in the of accumulated triclosan in the bacterial cells. Each data point represents the
absence of CCCP increased gradually with time. However, upon mean ± standard error of the mean of 2 independent experiments. Consistently
addition of CCCP, the absorbance values increased considerably, similar results were seen for the other strains and chemicals (n = 2).
 C.A. Thorrold et al. / International Journal of Food Microbiology 113 (2007) 315–320
Fig. 3. Representative bar chart graph showing the effect of different
concentrations of household disinfectant product (containing ortho-phenylphe-
nol) on (A) sensitive E. coli # 2 and (B) resistant E. coli # 7 in artificially
contaminated dishcloths. Each error bar represents the standard error of the
mean of 2 independent experiments (n = 2).
with a significant difference observed between the sensitive
and resistant bacteria at the 25% concentration (z = 30.20;
p = 0.000).
4. Discussion
The zoonotic organisms, Salmonella and E. coli, are known
to be associated with human gastrointestinal disease following
consumption of contaminated food. Similar to what has been
previously described, Salmonella and E. coli species resistant
to tetracycline and/or ofloxacin could be isolated from poultry
samples (Quednau et al., 1998). These organisms may find their
way into the home and be ingested if proper kitchen hygiene is
not practiced.
Tetracycline and ofloxacin were chosen as indicator anti-
biotics due to their well documented pathways of resistance due
to efflux pump activity (Heisig, 1996), which is the mechanism
most likely to be involved in cross resistance. The finding that
active efflux of ethidium bromide was solely associated with
antibiotic resistant organisms suggests that efflux mechanisms
could be responsible for the antibiotic resistance, but this was
not further investigated and other systems may also be involved.
Time-course sodium salicylate, triclosan and ortho-phenyl-
phenol accumulation experiments were performed, to determine
whether active efflux of these chemicals could be observed. The
use of the pump inhibitor CCCP allowed us to block energy
dependent efflux pumps, which resulted in an increase in the
cytoplasmic levels of the chemicals. A significant increase in
the amount of cell-associated components was seen with anti-
319
biotic resistant Salmonella and E. coli isolates, indicating that
the cells were unable to extrude the agents upon inhibition of the
pumps. The active components (sodium salicylate, triclosan and
ortho-phenylphenol) all contain aromatic groups, but are other-
wise not structurally related. The ability of the antibiotic resistant
strains to extrude all three chemicals indicates a promiscuity of
function of a single pump, or the activity of multiple pumps.
Cleaning cloths and sponges play a very important role in
domestic hygiene as they aid in the detachment of microbes and
soil present on the surface (Baranova and Neyfakh, 1997).
However, in many instances they remain moist for long periods
of time, contain residual amount of soil, and are not changed
frequently, providing ideal conditions for the survival and/or
growth of microorganisms (Bohn and Bouloc, 1998). Studies
have demonstrated that if cloths are not properly disinfected,
they can transfer bacteria to clean surfaces in the kitchen or
hands, resulting in disease (Bolhuis et al., 1995). Several brands
of household cleaning products are available that contain anti-
microbial agents, and these are marketed as being more effec-
tive at killing or inactivating bacteria than regular products. An
in-use disinfection procedure was used to test the efficacy of
several commonly used products containing salicylate, triclosan
and ortho-phenylphenol by simulating cleaning. The products
were used at the concentrations recommended by the manufac-
turer, as well as at half and quarter strengths. The rationale for
using the items at concentrations other than that recommended,
is that people may fail to adhere to the instructions and dilute the
agents to extend their use. This is particularly the case when a
100% (neat) application of a product is required for anti-
germicidal action, but it is most practically used with water for
cleaning purposes. As expected, the higher the concentration of
detergent product used, the greater the reduction in bacterial
numbers amongst both antibiotic sensitive and resistant bac-
teria. However, as the product was diluted to 25%, more anti-
biotic resistant bacteria survived than sensitive isolates. This
was seen with all three products tested and for both organisms.
The ability of tetracycline and ofloxacin resistant Salmonella
and E. coli to stably extrude the active ingredients of three
antimicrobial detergents used, suggests a mechanism of how
reduced susceptibility could occur. Efflux is thus postulated to
be involved in the reduction in efficacy of the ‘anti-germ’
household products observed at higher dilutions. While the use
of antimicrobial household detergents may assist in reducing
bacterial load in the kitchen and elsewhere, incorrect usage
could result in selection of bacteria with reduced susceptibility
to both antibiotics and antimicrobials. This could occur where
there is a sufficient concentration of the antimicrobial agent to
eliminate sensitive bacteria, but insufficient to reduce the num-
bers of the less susceptible, antibiotic resistant strains. This is
clearly undesirable in a setting such as that of South Africa,
where many individuals are immuno-compromised due to HIV/
AIDS and are receiving home-based medical care.
Acknowledgement
We thank Dr Jackie Picard, Onderstepoort Veterinary Insti-
tute, University of Pretoria, South Africa for E. coli isolates.
320 C.A. Thorrold et al. / International Journal of Food Microbiology 113 (2007) 315–320
References
Baranova, N.N., Neyfakh, A.A., 1997. Apparent involvement of a multidrug
transporter in the f luoroquinolone resistance of Streptococcus pneumoniae.
Antimicrobial Agents and Chemotherapy 41, 1396–1398.
Bohn, C., Bouloc, P., 1998. The Escherichia coli cmlA gene encodes the
multidrug eff lux pump Cmr/MdfA and is responsible for isopropyl-beta-D-
thiogalactopyranoside exclusion and spectinomycin sensitivity. Journal of
Bacteriology 180, 6072–6075.
Bolhuis, H., Poelarends, G., van Veen, H.W., Poolman, B., Driessen, A.J.,
Konings, W.N., 1995. The lactococcal lmrP gene encodes a proton motive
force-dependent drug transporter. Journal of Biological Chemistry 270,
26092–26098.
Botteldoor n, N., Herman, L., Rijpens, N., Heyndrickx, M., 2004. Phenotypic
and molecular typing of Salmonella strains reveals contamination sources in
two commercial pig slaughterhouses. Applied and Environmental Microbi-
ology 70, 5305–5314.
Butaye, P., Devriese, L.A., Haesebrouck, F., 2003. Antimicrobial growth
promoters used in animal feed: effects of less well known antibiotics on
gram-positive bacteria. Clinical Microbiology Reviews 16, 175–188.
Chapman, J.S., Georgopapadakou, N.H., 1988. Routes of quinolone permeation
in Escherichia coli. Antimicrobial Agents and Chemotherapy 32, 438–442.
Chopra, I., Roberts, M., 2001. Tetracycline antibiotics: mode of action,
applications, molecular biology, and epidemiology of bacterial resistance.
Microbiology and Molecular Biology Reviews 65, 232–260.
Cruchaga, S., Echeita, A., Aladuena, A., Garcia-Pena, J., Frias, N., Usera, M.A.,
2001. Antimicrobial resistance in salmonellae from humans, food and
animals in Spain in 1998. Journal of Antimicrobial Chemotherapy 47,
315–321.
Geornaras, I., Hastings, J.W., von Holy, A., 2001. Genotypic analysis of
Escherichia coli strains from poultry carcasses and their susceptibilities to
antimicrobial agents. Applied and Environmental Microbiology 67,
1940–1944.
Heisig, P., 1996. Genetic evidence for a role of parC mutations in development
of high-level f luoroquinolone resistance in Escherichia coli. Antimicrobial
Agents and Chemotherapy 40, 879–885.
Kusumaningrum, H.D., van Putten, M.M., Rombouts, F.M., Beumer, R.R.,
2002. Effects of antibacterial dishwashing liquid on foodborne pathogens
and competitive microorganisms in kitchen sponges. Journal of Food
Protection 65, 61–65.
Manie, T., Khan, S., Brozel, V.S., Veith, W.J., Gouws, P.A., 1998. Antimicrobial
resistance of bacteria isolated from slaughtered and retail chickens in South
Africa. Letters in Applied Microbiology 26, 253–258.
McDonnell, G., Russell, A.D., 1999. Antiseptics and disinfectants: activity,
action, and resistance. Clinical Microbiology Reviews 12, 147–179.
McEwen, S.A., Fedorka-Cray, P.J., 2002. Antimicrobial use and resistance in
animals. Clinical Infectious Diseases 34 (Suppl 3), S93–S106.
National Committee for Clinical Laboratory Standards, 2001. Performance
standards for antimicrobial susceptibility testing, 11th ed. pp. 42–43.
Parsonnet, K.C., Kass, E.H., 1987. Does prolonged exposure to antibiotic-
resistant bacteria increase the rate of antibiotic-resistant infection?
Antimicrobial Agents and Chemotherapy 31, 911–914.
Poole, K., 2000. Eff lux-mediated resistance to f luoroquinolones in gram-
negative bacteria. Antimicrobial Agents and Chemotherapy 44, 2233–2241.
Quednau, M., Ahrne, S., Petersson, A.C., Molin, G., 1998. Antibiotic-resistant
strains of Enterococcus isolated from Swedish and Danish retailed chicken
and pork. Journal of Applied Microbiology 84, 1163–1170.
Singer, R.S., Finch, R., Wegener, H.C., Bywater, R., Walters, J., Lipsitch, M.,
2003. Antibiotic resistance—the interplay between antibiotic use in animals
and human beings. Lancet. Infectious Diseases 3, 47–51.
Suller, M.T.E., Russell, A.D., 2000. Triclosan and antibiotic resistance in
Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 46, 11–18.
Vali, L., Wisely, K.A., Pearce, M.C., Turner, E.J., Knight, H.I., Smith, A.W.,
Amyes, S.G., 2004. High-level genotypic variation and antibiotic sensitivity
among Escherichia coli O157 strains isolated from two Scottish beef cattle
farms. Applied and Environmental Microbiology 70, 5947–5954.
Webber, M.A., Piddock, L.J., 2003. The importance of eff lux pumps in bacterial
antibiotic resistance. Journal of Antimicrobial Chemotherapy 51, 9–11.
White, D.G., Zhao, S., Sudler, R., Ayers, S., Friedman, S., Chen, S., McDermott,
P.F., McDermott, S., Wagner, D.D., Meng, J., 2001. The isolation of
antibiotic-resistant salmonella from retail ground meats. New England
Journal of Medicine 345, 1147–1154.