World's Poultry Science Journal
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Review of food grade disinfectants that are
permitted for use in egg packing centres
Andrew Wales, Emma Taylor & Robert Davies
To cite this article: Andrew Wales, Emma Taylor & Robert Davies (2022) Review of food grade
disinfectants that are permitted for use in egg packing centres, World's Poultry Science Journal,
78:1, 231-260, DOI: 10.1080/00439339.2022.1990741
To link to this article: https://doi.org/10.1080/00439339.2022.1990741
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Majesty’s Stationery Office and Animal and
Plant Health Agency. Published by Informa
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WORLD’S POULTRY SCIENCE JOURNAL
2022, VOL. 78, NO. 1, 231–260
https://doi.org/10.1080/00439339.2022.1990741
Review of food grade disinfectants that are permitted for use
in egg packing centres
Andrew Wales
a
, Emma Taylorb and Robert Daviesb
a
Department of Pathology and Infectious Diseases, School of Veterinary Medicine, University of Surrey,
Guildford, UK; bDepartment of Bacteriology, Animal and Plant Health Agency, Addlestone, Surrey, UK
SUMMARY
KEYWORDS
The handling and packing of eggs in commercial production creates
opportunities for the spread of those pathogenic micro-organisms that
can survive on the surfaces of eggs and equipment, and in organic
soiling such as egg contents and faeces. Salmonella Enteritidis is a key
zoonotic pathogen, and its spread between egg production premises
in recent years has implicated cross-contamination via egg handling
and packing equipment. Cleaning and disinfection to prevent such
spread have to be performed using food-grade agents, which limits
the options available to manufacturers of disinfectants and sanitisers.
The present review examines the active components of such products,
based upon the most frequently used disinfectants and sanitisers in
this part of the egg industry in the United Kingdom. Peer-reviewed
data are summarised for the main bactericidal elements, comprising
sodium hypochlorite and surfactants including quaternary ammonium
compounds. In addition, there is brief consideration of ancillary agents
with cleaning, pH-modulating, water-softening and additional bacter­
icidal effects. The amount of published surveillance and experimental
data for the effect of these agents on Salmonellaspp. and related
organisms is very variable, and in addition findings illustrate substan­
tial differences in biocidal effect between and within studies. Some of
these relate to test variables such as surface material, concentration,
exposure time, and the presence of organic soil or biofilm. Other
differences reflect inherent variability in disinfectant testing. It is sug­
gested that inadequate disinfection may occur under some foresee­
able conditions of application, even if concentration and exposure
time recommendations are followed. Further testing may be useful if
it more closely replicates the products and conditions of use.
Disinfectant; egg; Salmonella
Enteritidis; chlorine;
surfactant; quaternary
ammonium
Introduction
Salmonella enterica serovar Enteritidis (SE) is the dominant serovar for human salmonellosis
in Europe (EFSA and ECDC 2021), and remains very common among salmonellosis cases in
the UK (Public Health England n.d.). It has in recent decades been an intractable problem in
egg production in Europe and elsewhere, although concerted action has brought its pre­
valence down substantially in the UK and many European Union member states (APHA
CONTACT Andrew Wales
a.wales@surrey.ac.uk
Department of Pathology and Infectious Diseases, School of
Veterinary Medicine, University of Surrey, Vet School Main Building, Daphne Jackson Road, Guildford GU2 7AL, UK
© 2021 Crown Copyright. Reproduced with the permission of the Controller of Her Majesty’s Stationery Office and Animal and Plant Health
Agency. Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License
(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any med­
ium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
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DISINFECTANTS FOR EGG PACKING
2020; EFSA and ECDC 2021). There have been recent suspicions that SE can readily be
transferred between egg-producing flocks on different premises by the movement of reusable
egg trays and pallets, supported by investigations in Australia.
Australian egg production has historically been free of endemic SE flock infections.
A cluster of human salmonellosis cases caused by SE phage type 12 in 2018 and 2019 was
linked to consumption of eggs from producers in the states of New South Wales and Victoria,
21 of which had SE detected on their premises. Whole-genome sequencing established that
the SE strains on the farms and from the human cases were all closely related and that all the
farms involved were interconnected by movement of people, eggs or equipment linked to
certain egg packing centres (Australian Eggs n.d.; NSW Government 2019; NSW DPI 2019;
n.d.). In the absence of an identified common source via replacement pullets, it is likely that
spread of this SE strain occurred via fomite and/or human movement between these
producers’ sites, as well as trading in eggs between farms.
A UK study (Davies and Breslin 2003) established that egg packing facilities of SEinfected flocks were commonly contaminated with the organism, and indeed that eggs
could acquire SE contamination while passing through such affected facilities.
Equipment for handling and transporting eggs is subject to frequent soiling by faeces
and egg contents (Tachikawa et al. 2000) and in SE-affected flocks this soiling may
harbour the organism or be subject to surface contamination by it. Moisture, whether
present in the soiling or introduced by condensation, ineffective washing, etc. will allow
the multiplication of SE in situ. Inadequate cleaning and disinfection of such transfer­
rable equipment (particularly reusable egg trays, spacers and pallets) thus provides
a ready route for transfer of SE between packing facilities on different premises. When
egg trays are not shared between sites, there still remains the potential for their crosscontamination. This may happen at depots and also in transit, in vehicles that carry eggs
and trays in varying states of cleanliness and which may be cleaned and disinfected
relatively infrequently. A subtyping study in Thailand has provided supportive evidence
for such routes in the case of non-Enteritidis Salmonella serovars (Utrarachkij et al.
2012). The adoption of reusable plastic trays, in contrast to single-use fibre pulp trays,
may be an important factor in the spread of SE between premises. In addition to the
cyclical transport of washed and incompletely dried re-used trays, evidence also suggests
that under dry conditions there is more effective physical transfer of Salmonella organ­
isms from plastic compared with fibre surfaces (Regmi et al. 2021).
Once introduced via contaminated egg trays, etc. the ‘upstream’ transfer of SE from
egg handling areas into the environment of laying flocks may occur via the movements of
personnel, rodents or egg handling equipment. It has recently been documented that
hand and boot hygiene barriers are frequently absent or poorly implemented between
buildings on laying farms (Gosling et al. 2014; Sodagari et al. 2020). On some premises,
there may be a specific hazard of rodents accessing residual (Salmonella-contaminated)
condensation water in wrapped packs of egg trays.
Biocides and detergent chemicals used on egg handling and transfer equipment must be
food-grade, i.e. approved for use in close proximity to food. This heavily restricts the available
options, ruling out some highly effective bactericides that may cause flavour taint or are not
considered safe to ingest in any concentration. Examples include aldehydes, phenols and
cresols. In this context, the chemicals used for cleaning and sanitising products broadly fall
WORLD’S POULTRY SCIENCE JOURNAL
233
into four categories: surfactants, chlorine-based oxidising agents, pH-modifiers (principally
alkalinisers) and cation-sequestering agents that have antibacterial and water-softening
effects.
Beyond a simple microbicidal effect, this restricted set of cleaning and disinfecting agents
face challenges that include the killing and removal of bacteria in biofilms and in lipid- and
protein-rich adherent soiling resulting from broken eggs. Egg yolk appears to be highly
protective of embedded bacteria in the face of disinfectant application (Kuda et al. 2011), as
discussed below. Studies on cleaning of egg yolk from surfaces have shown alkaline condi­
tions, associated with lipoprotein and phospholipid breakdown, to be useful up to around pH
12 but further alkalinisation makes the protein component less soluble and more resistant to
dissolution (Helbig et al. 2015, 2019; Pérez-Mohedano, Letzelter, and Bakalis 2016). Yolk
deposits that have been denatured by heat treatment (80°C to 90°C) are harder to remove
with alkaline wash solution (Yang et al. 2019). Warm wash temperatures, in the range 40°C to
60°C, accelerate the removal process and reduce the significance of optimising pH.
The present review examines bactericidal efficacy data for the most common individual
components of UK-marketed products that are used in egg packing areas for sanitising
equipment. This includes data, where available, for performance against organisms protected
by biofilm and organic soil. Market authorisation data for sanitiser and disinfectant products,
which use combinations of these cleaning and microbicidal components, are consequently
outside the scope of this review of individual agents. As the principal issue is Salmonella
control, the main focus is on data relating to this genus plus other members of the family
Enterobacteriaceae.
Principal antimicrobial agents
Quaternary ammonium compound (QAC)
QAC are cationic surfactants, generally being organically substituted ammonium groups
bearing a polar (positively charged) ‘head’ plus one or two hydrophobic tails (Al-Adham,
Haddadin, and Collier 2013). They have weak cleaning (detergent) properties but exert
antibacterial activity at in-use concentrations by their destabilising interactions with
anionic phospholipids and associated proteins in cytoplasmic membranes and Gramnegative outer membranes (Lambert 2013; McDonnell and Russell 1999; Yoshimatsu and
Hiyama 2007). QAC are considerably more potent against Gram-positive than Gramnegative bacteria, such as Salmonella, they are regarded as susceptible to interference by
organic soil and also by anionic and certain non-ionic detergents, and are most active at
neutral to alkaline pH, being very poorly active below pH 3.5 (Al-Adham, Haddadin, and
Collier 2013). QAC have a low dilution coefficient and thus have a similar microbicidal
activity across a wide range of applied concentrations (Russell, Ahonkhai, and Rogers
1979) although other factors such as soiling may influence this in practical application.
Investigations in the 1950s using suspension tests indicated that natural or artificial
hard water (containing calcium and magnesium salts) could markedly reduce the micro­
bicidal effect of QAC against E. coli, and this could be ameliorated by water-softening
treatment (Butterfield, Wattie, and Chambers 1950; Chambers et al. 1955). The picture is
complicated by findings suggesting that culture conditions and prior exposure of the test
E. coli to the salts may be critical to the observed effect (Klimek and Bailey 1956). In
234
DISINFECTANTS FOR EGG PACKING
contrast with targets in suspension, surface disinfection tests appeared to be little impacted
by water hardness in studies examining Staphylococcus aureus (Kravitz and Stedman 1957)
and Salmonella spp. (Davison, Benson, and Eckroade 1996), although the exact composi­
tion of the QAC test products was not specified. A more recent suspension test study with
strains of Pseudomonas spp. supports the view that hard water diluent can reduce the
microbicidal effect of QAC (in this case benzalkonium chloride) against Gram-negative
bacteria (Langsrud and Sundheim 1997).
Benzalkonium chloride
Benzalkonium chloride (BAC, alternatively called alkyl dimethyl benzyl ammonium
chloride/ADBAC) is a mixture of closely related QAC, varying in the length of the
alkyl hydrophobic tail between eight and 18 carbon atoms (Al-Adham, Haddadin,
and Collier 2013). Its density (around 0.98) is close to water, meaning that in
aqueous solutions, percent concentrations expressed as weight or volume BAC per
weight or volume water (w/w, w/v or v/v) are nearly identical. Therefore, although
reports vary in their concentration units, equivalence may cautiously be assumed
between, say, ‘100 mg/L (or μg/mL)’ in one reference and ‘0.01%’ (units unspecified)
in another.
Surveys of minimum inhibitory concentration (MIC) of BAC have been reported for
various strain collections, using either broth dilution or agar medium techniques. MIC values
of BAC for 39 Salmonella strains from Spanish retail egg shells were in the range 50 to
100 mg/L (Fernández Márquez et al. 2016). Among 43 Salmonella isolates from Spanish
meats, 90% of MIC values were up to 50 mg/L, although there was one high MIC of 250 mg/L
(Garrido et al. 2015).
Salmonella field strain collections from non-European territories have yielded MIC
values similar to European isolates. Examples include 16 to 32 mg/L from turkey
processors in Texas, USA (Beier et al. 2011), 20 to 40 mg/L from multi-drug resistant
isolates from animals and retail meat in USA (Humayoun et al. 2018), 32 to 256 mg/L
from a broiler and an egg farm in China (Long et al. 2016), 32 to 64 mg/L from Brazilian
food environments (Haubert et al. 2019), and 32 to 256 mg/L from Thai poultry
(Chuanchuen et al. 2008).
Two studies have shown a pattern of lower MIC values among E. coli isolates, compared
with Salmonella spp. In a report by Morrissey et al. (2014), over 97% of 901 European
veterinary Salmonella isolates had MIC values up to 64 mg/L, with the highest being 128 mg/
L, whereas 98% of E. coli strains from Spanish hospitals showed MIC values up to 32 mg/L
and there was a maximum value of 64 mg/L. From Danish livestock strains (including many
broiler-derived isolates), the pattern is again of higher MIC values among Salmonella spp.
(128–256 mg/L) than E. coli (32–64 mg/L) (Aarestrup and Hasman 2004). In this latter study,
higher MICs overall compared with other studies may have been a consequence of the agar
dilution technique employed. A study of E. coli from Chinese retail chicken, using similar
methodology, reported 98% of 510 isolates to have an MIC up to 64 mg/L, although the
highest MIC was 256 mg/L (Sun et al. 2019). It is not clear for BAC whether the precise blend
of elements (i.e. relative proportions of eight- to 18-carbon chains) substantially alters its
effect against Salmonella and related organisms.
WORLD’S POULTRY SCIENCE JOURNAL
235
Field collections of Gram-positive bacteria reflect their greater sensitivity to QAC
compared with Salmonella and other Gram-negative genera. Typical BAC MICs for
Enterococcus spp. and Staphylococcus spp. were up to 8 mg/L (Morrissey et al. 2014;
Rizzotti, Rossi, and Torriani 2016), but occasionally as high as 16 mg/L with an agar
dilution technique (Aarestrup and Hasman 2004).
None of the survey studies cited have shown evidence of a bimodal distribution of
MIC values for Salmonella, E. coli or enterococci that, if present, would suggest the
presence of a separate, less-susceptible sub-population. Whilst studies on tolerance of
bacteria to BAC have tended to focus on elevated MIC as a measure of this, some
(discussed below) have considered the role of biofilm in reduced susceptibility. A large
survey of 740 E. coli and 500 Enterococcus isolates from Norwegian livestock around the
end of the 1990s showed all but one to have MIC values of below 31 mg/L, and no MIC
above 50 mg/L (Sidhu, Sørum, and Holck 2002).
Laboratory ‘training’ by serial culture in slowly increasing sub-MIC concentrations of
BAC has yielded isolates of Salmonella Typhimurium with MIC values elevated four to
16-fold, whose tolerance appeared stable when the selective pressure was removed and
was associated with upregulated efflux (Guo et al. 2014). Among E. coli exposed to subMIC concentrations of BAC, adaptations associated with improved tolerance included
increased membrane efflux pump activity, but other mechanisms (possibly leading to
increased cell membrane QAC resistance) also seemed likely to be involved (Moen et al.
2012). One study of biofilm-derived E. coli showed an enhanced biofilm phenotype
among strains trained to exhibit elevated MIC values (Pagedar, Singh, and Batish
2012), although a causal relationship between the two was not established.
In any event, the continuous exposure of planktonic bacterial monocultures to care­
fully calibrated low concentrations of BAC is unlike real-world conditions of intermittent
exposure in the context of mixed bacterial populations and other stressors. Furthermore,
neither MIC testing of isolate collections (as described above) nor a one-year longitudinal
monitoring study of E. coli isolates from pig and poultry accommodation exposed to
BAC-containing products (Maertens et al. 2020) have shown convincing evidence of
reduced or reducing susceptibilities to BAC in field strains of relevant bacteria. However,
it is plausible that working concentrations of BAC-based disinfectants and sanitisers may
sometimes be below the MIC of some strains of target species, and that use of BAC
against surface-adhered, biofilmed or organic soil-associated bacteria may prove poorlyeffective. Experience with persistent Salmonella contamination of hatchery environments
(UK Animal and Plant Health Agency, unpublished data) suggests that BAC preparations
used at less than UK General Orders concentration can be both ineffective and associated
with reduced susceptibility of the target Salmonella.
Surface disinfection tests using a small volume (0.1 ml) of disinfectant at room
temperature against organisms dried onto stainless steel showed greater than four log10
cycles (logs) reduction in E. coli and Staphylococcus aureus after 10 min’s exposure to
500 mg/L BAC in distilled water (Kuda, Yano, and Kuda 2008), although much more
modest reductions were seen even with 2000 mg/L BAC in the presence of heavy driedon organic soil (milk or gravy). On glass and using a similar methodology with
Salmonella Typhimurium, Kuda et al. (2011) showed greater than five logs reduction
by 2000 mg/L BAC in the presence of no soil or 25% egg albumen but markedly reduced
efficacy, amounting to less than 0.5 logs reduction, was seen with lipid-containing soil
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DISINFECTANTS FOR EGG PACKING
(25% egg yolk or 50% whole egg). From the same study, such high retention of viable
bacteria was shown to be susceptible to multi-stage washing and disinfection, albeit with
a focussed, experimental technique. Starting with ST dried on to glass with whole egg,
two 60-s phases of mechanical wiping in water followed by a 10-min soak and rinse with
water or anionic detergent reduced counts by nearly four or five logs, respectively. After
a final 1 mL application of BAC 2000 mg/L, no ST was detected, implying a further
reduction in excess of one log.
Using large volumes of detergent and BAC solution (immersion technique), E. coli
dried on to stainless steel with minimal organic soil demonstrated enhanced suscept­
ibility to 500 mL/L BAC after pre-soaking in detergent, but only with two of three
detergents (Walton et al. 2008). By contrast, all three detergents enhanced killing of
Listeria monocytogenes.
Geber et al. (2019) have provided insight into relationships between MIC and disin­
fection test performance for BAC, with a study using collected strains of S. aureus, E. coli,
Klebsiella pneumoniae and ST. Using hard water diluent, MIC values were below 10 mg/L
for the 12 staphylococci and were 50 mg/L for nine Gram-negative strains. In suspension
at room temperature, the same Gram-negative strains required two- to ten-times their
MIC of BAC to eliminate detectable bacteria within 5 min from aliquots initially
containing around 107 colony-forming units. The corresponding range with 30 min’s
exposure was one- to two-times MIC. However, when a light soil load (0.3 g/L bovine
albumin in the final mix) was introduced, the required BAC concentration for compar­
able performance at five or 30 minutes increased by up to tenfold (highest values being
1000 mg/L and 500 mg/L for 5- and 30-min exposures, respectively). This increase was
most marked for the longer exposure/lower concentration tests. On stainless steel at
room temperature with light soiling (1.5 g/L bovine albumin dried on with organism,
amounting to 0.75 grams per litre of subsequently-applied disinfectant), and with
a performance criterion of at least four logs reduction, between 5000 and 20,000 mg/L
BAC (100 to 400 times MIC) was required with 5 min’s exposure, or between 100 and
5000 mg/L (two to 100 times MIC) for 30 min. A direct strain-by-strain comparison of
5-min suspension and surface tests for Enterobacteriaceae in this study showed a five- to
40-times increase in required BAC concentration between suspension and surface tests.
There have been several studies of BAC efficacy against relevant organisms in biofilms and
of effects of BAC upon biofilming phenotype. However, in vitro methods for studying
biofilms are far from standardised, and are not representative of real-life situations. The
minimum biofilm eradication concentration (MBEC: elimination of viability following
incubation at 37°C overnight in presence of nutrient broth) for BAC for two SE strains in
three-day-old polystyrene surface biofilms was 800 to 1600 mg/L (Romeu, Rodrigues, and
Azeredo 2020). Tests examining disinfectant activity with conventional exposure times and
methodologies that vary in biofilm substrate and flow (shear) conditions (but which all
involve immersion of biofilm in excess disinfectant and at room temperatures) have reported
substantial variation in biocidal effect and are summarised in Table 1.
Among reports for five-minute exposures of Salmonella spp. biofilms, 200 mg/L BAC had
a modest (up to one log) effect onS. Agona, whilst a moderate (two to three logs) reduction
was seen for ST with 700 mg/kg BAC, and a high (four logs) microbicidal effect was seen for
SE with 500 mg/L with stirring (Ueda and Kuwabara 2007; Vestby et al. 2010; Wong et al.
2010a, 2010b). The lowest (200 mg/L) BAC concentration was that recommended by the
Oscillating
flow
— — As above — —
Stirred
bioreactor
— As above —
Unidirectional PVC tubing
flow
ST
As above
ST, SA (2 strains),
SE
As above
E. coli (French
disinfectant
testing strain)
Concrete
Polystyrene
pegs
7
5
2
3
3, 5 &
7
2
Age
(days)
10
Room
Room
Room
20 °C
5
20 °C
10 to Room
90
5
1
5
5
Strength (mg/L)
500 to 1000
Other
Immersed, stirred
— — — As above — — —
Variable
Substrate
immersed
Coupons immersed
Immersed
7,500 & 15,000 b
200
Immersed
Immersed
700 to 15,000 b
700
b
— — — As above — — —
200
Immersed
Time
(min) Temp.
5
Room?
Disinfectant
c
Wong et al. 2010
Vestby et al.
2010
Wong et al. 2010
c
, Wong et al.
2010 d
Reference
Ueda and
Kuwabara
2007
NtsamaEssomba et al.
1997
Concentration used was 2x Corcoran et al.
max. MIC for most
2014
tolerant strain
Little difference in biocidal
effect across a 20-fold
concentration range
Biofilms in low nutrient
culture.
Comments
≤ 0.3
≥5, at 20,000 mg/L For similar (5 log)
reduction, surface
(polyethylene plus 5%
skim milk) required
10,000 mg/L. Planktonic
cells required far less
(20 mg/L).
≤1
>6
1.5 to 3
2 to 3
> 4, at ≥ 500 mg/L
≤1
Effect (log cycle
reduction in
bacterial count)
4, at 500 mg/L> 4,
at 1000 mg/L
SE - Salmonella Enteritidis, SA - Salmonella Agona, ST - Salmonella Typhimurium. b mg/kg. c (Wong, Townsend, Fenwick, Trengove, et al. 2010). d (Wong, Townsend, Fenwick, Maker, et al. 2010)
a
Oscillating
flow
ST
Polystyrene
pegs
— — As above — —
Static culture Glass slide
E. coli O157
SA x2
Substrate
Stainless
steel
Technique
Static culture
Organism a
SE
Biofilm
Table 1. Summary of studies examining the effect of benzalkonium chloride on biofilms of Salmonella and certain other bacteria
WORLD’S POULTRY SCIENCE JOURNAL
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DISINFECTANTS FOR EGG PACKING
manufacturer in the study by Vestby et al. (2010). At very high concentrations of BAC
(≥7,500 mg/kg), an extreme microbicidal effect (at least six logs) was reported with fiveminute exposure, but interestingly a one-minute exposure did not show an increase in effect
beyond the moderate (up to three logs) kill achieved with a much lower concentration (Wong
et al. 2010b). On unsealed concrete test coupons, biofilms of SE, ST, and S. Agona appeared
resistant to the bactericidal effects of 200 mg/L BAC (at least twice MIC) for extended
exposures of up to 90 minutes (Corcoran et al. 2014).
For E. coli, Ntsama-Essomba et al. (1997) reported that minimum concentrations needed
for a five logs reduction performance standard were 500 to 1000 times higher for dried-on
(polyethylene substrate) or biofilmed (polyvinyl chloride substrate) organisms, respectively,
than for stationary-phase planktonic cells. However, the susceptibility of the suspended cells
seems high, in the context of other studies reporting MIC values and the suspension test
findings of Geber et al. (2019). Furthermore, the biofilmed cells in the report by NtsamaEssomba et al. (1997) showed an unusually low susceptibility to inactivation by comparison
with other studies in Table 1, for example Ueda and Kuwabara (2007).
There is some limited evidence of the effects of growing Salmonella in biofilms at sublethal
concentrations of BAC. When BAC was present at 1 mg/L (well below MIC) in media for
broth- and biofilm-grown SE, the biofilm cells showed a higher proportion to be subsequently
tolerant of 30 mg/L BAC in plate-count assays (Mangalappalli-Illathu, Vidovic, and Korber
2008). For the same serovar, the maintenance of biofilms in the presence of 25% to 50% of
MBEC did not alter the volume of biofilms subsequently formed by survivor cells (Romeu,
Rodrigues, and Azeredo 2020), and prior stepwise adaptation of S. Typhimurium for reduced
susceptibility to BAC was associated with slightly reduced biofilm volume in a laboratory
multi-well plate assay (Capita et al. 2017). By contrast, for E. coli field strains isolated from
dairy equipment biofilms in India, the strength of biofilm capability correlated positively with
MIC, both for isolates trained to have increased MIC and for those with a high MIC when first
isolated (Pagedar, Singh, and Batish 2012).
In summary, although there is a wide (in excess of tenfold) range of MIC values for BAC
reported from field strains of Salmonella spp., no resistant subpopulations have been identified
among livestock Enterobacteriaceae. Surface soiling substantially impedes the agent’s bacter­
icidal effect, and lipid-containing (yolk) soil appears to be particularly protective in this regard.
A study of a small number of Salmonella and E. coli strains suggests that conventional
performance thresholds are achieved in five-minute tests with up to 20 times MIC in
suspension (five logs reduction) and up to 100 times MIC on stainless steel (four logs
reduction). The tests were conducted using hard water and light protein soil. Similar BAC
concentrations achieve multiple log reductions in some biofilm assays using a large excess of
disinfectant, especially if agitated. Performance against concrete-grown biofilm is probably
substantially poorer, although only a modest BAC concentration (200 mg/L) was tested. There
is some indication of a ceiling effect, regardless of BAC concentration, with short (i.e. oneminute) exposures.
Didecyl dimethyl ammonium chloride (DDAC)
DDAC is a twin-chain (di-alkyl) quaternary ammonium compound, these reportedly
being more tolerant of hard water and organic soil than QAC(such as BAC) that bear
a single hydrophobic chain (Al-Adham, Haddadin, and Collier 2013). Findings by Walsh
et al. (2003), showing small effects upon performance of increasing the organic soil in
WORLD’S POULTRY SCIENCE JOURNAL
239
a suspension test (from 0.3 to 3 g/L bovine albumin), support this view. Reported MIC
values for E. coli include: 1.5 to 4 mg/L from French pig and pork sources (Soumet et al.
2016), 5 mg/L for an archived reference strain (Walsh et al. 2003) and 1.3 or 10 mg/L for
broth or agar MIC techniques, respectively, for another reference strain (Yoshimatsu and
Hiyama 2007). For Salmonellaspp., MIC ranges for field strain collections are on average
a little higher: 4 to 8 mg/L from French pig sources (Soumet et al. 2016) and 2 to 8 mg/L
from turkey processors in the USA (Beier et al. 2011). Attempts at stepwise training in
culture for reduced susceptibility were more frequently successful for E. coli than for
Salmonellaspp., with 50% and 3% of the researched strains, respectively, eventually yielding
subcultures showing MIC values elevated at least three-fold (Soumet et al. 2016).
When tested for the bactericidal effect in a five-minute 20°C suspension test, it is
remarkable how little difference a large change in concentration (1x MIC to 4000x MIC)
made , amounting to an additional approximately 0.5 logs from a base of 4.7 logs (low soil)
or 4.3 logs (high soil) for E. coli (Walsh et al. 2003). There was a larger concentration effect
for a Gram-positive test organism (Staph. aureus), amounting to between 1 and 1.5 logs.
Also in suspension, but using a low concentration of DDAC (0.3 to 0.6 ppm [≈ mg/L]) and
prolonged contact (3 h) with Staph. aureus, Gomi et al. (2012) demonstrated a synergy
between 37 non-ionic surfactants and DDAC for bactericidal effect. The degree of the
observed synergy varied with the DDAC-surfactant pairing.
Surface tests using a commercial product containing DDAC (‘D-128ʹ, of uncertain
overall composition as the study was conducted 20 years ago) showed that at recom­
mended dilution the product was apparently highly efficacious after 10 min’s exposure at
room temperature against Salmonella Typhimurium in suspension and on certain
surfaces (including stainless steel, plastic and painted cement), but viable organisms
remained after application to rubber and corroded steel surfaces (Ewart et al. 2001).
A study conducted with vegetable produce reported that 0.0125% (≈ 125 mg/L) DDAC
applied for 3 min did not significantly reduce viable, experimentally applied S.
Typhimurium on the surface of cucumber or parsley (Shirron et al. 2009).
Amphoteric surfactants
A few products incorporate amphoteric (zwitterionic) surfactant. Such molecules contain
a positively charged element (typically quaternary ammonium) plus a negatively charged
group, attached to a hydrophobic chain. Long-chain alkyl amine oxides are often used in
this role in food-grade products. Amphoteric surfactants combine the antimicrobial and
detergent properties of cationic and anionic surfactants, respectively. They are bacter­
icidal across a wide pH range, are similarly active against Gram-positive and Gramnegative organisms and are considered to be more tolerant than QAC of organic soil (AlAdham, Haddadin, and Collier 2013).
A study by Ntsama-Essomba et al. (1997) comparing five-minute, 20°C exposure tests
of E. coli in suspension (no soil), surface (5% skimmed milk soil) and biofilm (five-day)
included an amphoteric disinfectant (‘Tegol 2000ʹ). This class of agent showed increases
in minimum bactericidal concentration when moving sequentially from suspension to
surface then to biofilm tests, but these were less marked than for BAC. European (EN)
standard suspension tests reported by Taylor, Rogers, and Holah (1999) indicated that
amphoteric disinfectant products, used at their label concentrations, were generally
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DISINFECTANTS FOR EGG PACKING
effective against E. coli under both clean and dirty conditions, although one product
performed poorly at low (10°C) temperature. A study using suspension and immersedsurface tests with heavy poultry faecal soiling and disinfectants used around their UK
General Orders concentrations found that an amphoteric surfactant-based product
(Tego 2001) performed better against Salmonella in suspension than products based on
QAC, peroxygen or iodine (McLaren et al. 2011). With dried surface Salmonella plus soil,
performance compared with these other agents was generally similar or superior.
Non-ionic surfactants
Some surfactant molecules have a polar element that is not ionised. These non-ionic
detergents have a surfactant effect by virtue of hydrogen bonds; they are not bound or
precipitated by hard water ions, and are often compatible with QAC and other micro­
bicides. In consequence, such agents (typically ethoxylates) are very commonly found in
food-grade disinfectants and sanitisers. Although they are not considered as a group to
have significant intrinsic antimicrobial activity, they may act to sensitise Gram-negative
bacteria to the action of biocides via permeabilising effects on outer membranes (AlAdham, Haddadin, and Collier 2013).
N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine
N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine has several alternative names,
including laurylamine dipropylenediamine, bis-(3-aminopropyl)-dodecylamine,
dodecyl dipropylenetriamine and, commercially, ‘Triameen Y12D’ and ‘Lonzabac
12ʹ. It has a 12-carbon alkyl chain attached to polar amine groups, and is thus
a non-ionic surfactant that additionally demonstrates substantial antimicrobial activ­
ity. It has some structural and functional similarities to the (cationic) QAC , but
unlike them its activity against Gram-positive and Gram-negative bacteria is similar,
and it is more compatible with anionic surfactants (Anon n.d.; Borgmann-Strahsen.
2019). Company-published data on tests using 0.03% albumin soil load indicates
that Triameen, with a chelating agent, passes EN 1276 (five-minute quantitative
suspension test, five logs reduction standard) at 0.4% active ingredient. It also passes
EN 13697 (surface test on stainless steel, four logs reduction standard, exposure
time not specified, including Pseudomonas aeruginosa target) at 1.5% (BorgmannStrahsen. 2019).
One further study (Meade and Garvey 2018) compared the efficacy of concentra­
tions between 0.01% and 0.2% active agent (Triameen brand) on E. coli and Grampositive organisms (Staph. aureus, including meticillin-sensitive [MSSA] and meticil­
lin-resistant [MRSA] strains, and vancomycin-resistant Enterococcus spp.). In a fiveminute suspension test with light soil, 0.1% achieved a five-log threshold for E. coli
and MSSA. In a disk diffusion inhibition of growth assay, MSSA and E. coli appeared
similarly susceptible, but with a steel surface carrier test with soil, conducted over
30 minutes in an excess of 0.2% disinfectant, the Gram-positive test organisms were
substantially less inhibited (one to two logs reduction) than was E. coli (at least five
logs reduction).
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Sodium hypochlorite
Sodium hypochlorite (NaOCl) in aqueous solution forms various biocidal components:
hypochlorite anion (OCl−), hypochlorous acid (HOCl) and chlorine (Cl2) (ECHA 2017).
All of these are considered to represent ‘active’ or ‘available’ chlorine but their relative
proportions are heavily influenced by the prevailing pH and antimicrobial activity is higher
in the neutral to low pH range, dominated by undissociated hypochlorous acid and
elemental chlorine, both being more reactive than OCl−. Unmodified sodium hypochlorite
solutions are alkaline (as a consequence of the formation of HOCl from OCl− and H2O)
and there is usually excess hydroxide ion in concentrated stock solutions, further raising the
pH and improving chemical stability (OxyChem 2014). Thus, hypochlorite ions predomi­
nate in concentrated solutions, which consequently show lower biocidal activity compared
with more dilute solutions, of lower pH. The chlorine content of biocide solutions may be
expressed as percent (w/v) available chlorine or percent NaOCl (these being approximately
equivalent, as the molecular mass of NaOCl is similar to that of Cl2), or as parts per million
(ppm), where 10,000 ppm = 1% available chlorine (Anon 2020).
The mechanism(s) of chlorine’s antibacterial effect at conventional disinfectant concentra­
tions are not well understood, but disruption of bacterial nucleic acid and protein chemistries,
and of membrane function, have all been described (Al-Adham, Haddadin, and Collier 2013;
McDonnell and Russell 1999). As biocidal chlorine compounds in solution are highly reactive,
the disinfectant activity of NaOCl is susceptible to quenching by reactions with organic soil
(Al-Adham, Haddadin, and Collier 2013; Gélinas and Goulet 1983; Moats 1981).
Effects of hard water on the antibacterial effect of hypochlorite solutions have been
investigated, but only data for short-exposure and low chlorine concentration suspension
tests against E. coli O157:H7 have been found. At 4°C and in short (10 to 30 seconds)
exposure time tests, an inhibitory effect of CaCO3 concentrations between 50 and
500 mg/L was seen only with low hypochlorite concentration (≤0.0005%) (Swanson
and Tong-Jen 2017). At room temperature and with a hypochlorite concentration of
0.007%, 200 mg/L CaCO3 significantly inhibited killing, reducing the log reduction
values (compared with controls) by around one-third and one-fifth after 30 seconds
and 4 min, respectively (Pangloli and Hung 2013), although no such effect was seen
against a Gram-positive pathogen (Listeria monocytogenes) or at 100 mg/L CaCO3.
MIC values for NaOCl, using Mueller–Hinton broth dilution techniques, have been
reported for collections of field strains of several relevant bacteria. For 88 antibioticresistant Salmonella enterica isolates (including Typhimurium and Enteritidis serovars)
from animal and meat sources in the USA, MIC values using household bleach ranged
from 0.079% to 0.63% w/v NaOCl (Humayoun et al. 2018). The authors noted that freshly
opened bleach appeared more potent, with all MIC values in that circumstance being below
0.32%. This observation is supported by chemical industry data on hypochlorite solution
stability (OxyChem 2014) and by the findings of Rutala et al. (1998), who reported a decline
in active chlorine in diluted solutions (using either distilled or tap-water), hastened by the
influence of light, exposure to the atmosphere and lower pH values.
Using NaOCl from laboratory suppliers freshly-prepared in distilled water, MIC
values for Salmonella were in the range 0.026% to 0.41% w/v active chlorine (median
value 0.20%) for 901 European veterinary isolates (Morrissey et al. 2014). However, with
similar methodology, the MIC values of 10 multi-drug resistant Spanish poultry isolates
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DISINFECTANTS FOR EGG PACKING
were comparatively low, in the range 0.039% to 0.044% NaOCl (Molina-González et al.
2014). Morrissey et al. (2014) also examined E. coli (Spanish, 368 isolates, MIC range 0.1
to 0.82%) and Enterococcus spp. (worldwide, 109 isolates, MIC range 0.2% to 0.82%).
Other enterococci (21 isolates) from pigs in Italy showed MIC values from 0.22% to
0.52% NaOCl, in this case using iso-sensitest broth (Rizzotti, Rossi, and Torriani 2016).
As in the case of BAC (where a similar volume of peer-reviewed data exists) none of the
cited MIC studies identified a distinct subpopulation of less-susceptible strains. Laboratory
studies of tolerance have involved stepwise training with increasing sub-MIC concentrations
of NaOCl in broth culture. The 10 poultry Salmonella strains examined by Molina-González
et al. (2014) achieved modest (less than two-fold) increases in MIC by this method, which
varied by strain. A Salmonella Heidelberg isolate was similarly trained, yielding a derived
strain with a stable increase in MIC of around 25% (Obe et al. 2018). This less susceptible
strain also demonstrated enhanced volume and cell numbers in 48-h statically grown biofilms,
compared with the parent isolate. Similar findings, in respect of modest MIC increases with
training, associated with enhancement in biofilm mass and biofilm cells per unit area, have
been reported independently for S. Typhimurium (Capita et al. 2017). For NaOCl-adapted
E. coli, modest elevation of MIC and an increase in biofilm mass have also been observed also
(Capita et al. 2014).
A simple, short (1 min) suspension test at 25°C, using retail bleach and distilled water
diluent without organic soil achieved at least six logs reduction in mono-species cocktails
of S. Typhimurium or E. coli O157:H7 at NaOCl concentrations of 0.03% and above
(Yang et al. 2009). Also using household hypochlorite but with standard hard water as
diluent, 0.3% bovine albumin (‘low-level soiling’ in the EN 1656 veterinary suspension
test standard) and a 10-min exposure of SE at room temperature, reductions of two and
four logs were observed at NaOCl concentrations of 0.02% and 0.04%, respectively
(Kusumaningrum et al. 2003). An additional one log reduction was seen with a 30 minute
exposure.
S. Typhimurium declined by over four logs after 15 min’s exposure at 10°C to 0.05%
NaOCl, but by less than this four-log threshold when 1.2% protein soil was added to the
suspension (Kich et al. 2004). For a 5-min suspension test at room temperature with
a pass threshold of around six logs, the addition of 0.3% protein soil increased the
required NaOCl concentration between 10- and 50-fold for nine Salmonella, E. coli, or
Klebsiella strains (Geber et al. 2019). In this study, MIC values for these strains were
above NaOCl concentrations that passed no-soil suspension tests but below pass con­
centrations for suspension tests with soil.
In simulated washer water at 40°C with added NaOCl, the addition of 1% egg content
immediately reduced an initial 0.027% free chlorine by a half and by around 90% within
30 minutes (Moats 1981). No bactericidal effect was observed against S. Typhimurium
when the organism was added after a further 30 minutes.
Three studies have contrasted findings from suspension and stainless steel surface
tests, using the same within-study reagents and time/temperature conditions. Riazi and
Matthews (2011) reported that for both SE and E. coli O157:H7, 0.013% available
chlorine (in pH 7.2 buffered saline) at 22°C for 5 min yielded reductions of greater
than eight logs in suspension, but less than one log with a stainless steel surface test. A five
logs reduction of SE was seen at four times that chlorine concentration (0.05%) in the
surface test. Under similar time, temperature and concentration conditions (5 mins,
Oscillating flow
— — As above — —
Oscillating flow
— — As above — —
ST
As above
ST
As above
2
3
3, 5, 7
10
2&7
5
5
1
5
1
— As above —
10 & 15
10 to 90
20 °C
Room
Room
Room
Room
25 °C
Room
Time (min) Temp.
0.13 to
5.3%
0.05%
0.07-5.3%
0.13%
0.13 - 5.3%
0.02%
0.01%
0.05%
Strength
Disinfectant
Stainless
2
5 Room c. 0.06%
steel
Galvanised
4
1 to 3 Room 0.03 to
steel
0.1%
SE
Static culture
Stainless
10
5 Room? 0.0025% to
steel
0.02%
E. coli O157
— — As above — —
— — — As above — — —
E. coli (French disinfectant Unidirectional flow
PVC tubing
5
5 20 °C Variable
testing strain)
Glass
Polystyrene
pegs
Polystyrene
pegs
Stainless
steel
Concrete
Substrate
Age
(days)
2 days: 0.1 to 1.1 (ST), 0.1
to 0.5 (SA)7 days: 0.3
(ST), 0.2-1 (SA), 0.4-0.8
(SE)
> 4 (10 min) 5 (15 min)
Effect (log cycle reduction
in bacterial count)
Generally, > 5 at ≥ 0.03%
for ≥ 1 min
1 to 4
0.5 to 1
2 to 3
> 5 (0.13% to 0.53%)< 2
(5.3%)
>5
0.7 to > 5
Substrate
≥ 5 at ≥ 0.02%
immersed
Slides
immersed
Coupons
immersed
Coupons
immersed
Immersed,
stirred
As above
Immersed
Coupons
immersed
Coupons
4.4 (10 min) 5.3 (15 min)
immersed
Immersed
0.13%: > 45.3%: 3 (3 days),
> 4 (5 & 7 days)
As above
> 4 (3, 5 & 7d)
Coupons
immersed
Other
Comments
Reference
Corcoran et al.
2014
For similar (5 log) reduction,
surface (polyethylene plus 5%
skim milk) required 0.02%.
Planktonic cells required less
(0.004%)
Most consistent when ≥0.05% for
≥2 min
Progressive increase in effect
with increasing concentration
Ntsama-Essomba
et al. 1997
Møretrø et al.
2009 e
Ramesh et al.
2002
Ueda and
Kuwabara 2007
Polypropylene biofilms not
Iñiguez-Moreno et
consistently different from steel.
al. 2018
Bi-species films as susceptible as
monospecies Salmonella.b
Biofilms in low nutrient culture. Wong et al., 2010 c
Note relative insusceptibility of
3d biofilm and poorer effect of
higher hypochlorite
concentration.
Biofilms in low nutrient culture. Wong et al., 2010
d
Marked drop-off in effect above
~0.5% observed with 1 min
exposure but not with 5 min.
Vestby et al.
Concentration used was 2x max.
MIC for most tolerant strain
ST - Salmonella Typhimurium, SA - Salmonella Agona, SE - Salmonella Enteritidis, SS - Salmonella Senftenberg. b Staphylococcus aureus mono-species biofilm less susceptible than bi-species. c
(Wong, Townsend, Fenwick, Maker, et al. 2010). d (Wong, Townsend, Fenwick, Trengove, et al. 2010). e Comparative data for planktonic & surface tests in the same publication.
a
— — As above — —
Static culture, then
dried 1 hr
SA, SS
Oscillating flow,
then dried 1 hr
Salm. cocktail including ST Static culture
Static culture
Salm. spp, +/- Staph.
aureus
As above
SA
Stirred bioreactor
Technique
ST,SA (2 strains),SE
Organism a
Biofilm
Table 2. Summary of studies examining effect of hypochlorite on biofilms of Salmonella and certain other bacteria
WORLD’S POULTRY SCIENCE JOURNAL
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DISINFECTANTS FOR EGG PACKING
20°C, 0.057% NaOCl in distilled water) but with 0.3% added protein, four Salmonella
serovars from the Norwegian feed industry showed at least five logs reduction in
suspension but considerably less than a one log reduction on stainless steel (Møretrø
et al. 2009). Geber et al. (2019) performed the same comparisons for NaOCl as they did
for BAC (see BAC section), using thresholds of around six logs for suspension and four
logs for stainless steel surface tests with light soil. In contrast to BAC, results were broadly
the same between test types, with the required concentrations for five-minute surface
tests being between one-fifth and two times those for equivalent suspension tests.
The study by Riazi and Matthews (2011) reported reductions for E. coli and SE on
stainless steel of 2.3 and 3.8 logs, respectively, for 0.025% chlorine, but under similar
conditions (steel and plastic, 5 min, room temperature, 0.02% chlorine in hard water)
Baek, Kim, and Sang-Do (2011) reported reductions of around five logs for a standard
E. coli disinfectant testing strain. Performance on rubber (approximately 4.5 logs) and
wood (approximately three logs) was poorer. This apparently wide variation in
reported surface test performance of chlorine-based disinfectants is noted in a recent
systematic review of the subject (Gallandat et al. 2021), although this is likely to be
a result of variation in methodologies and test strain, and indeed similar variability has
been observed for other disinfectant agents (Bloomfield et al. 1994).
There are some full immersion (carrier) surface tests for Salmonella, using less
standardised methodologies. Choi et al. (2015) spiked chicken faeces with a mix of five
Salmonella serovars and dried it on to eggshells. Immersion in 0.02% NaOCl at room
temperature resulted in reductions of 1.5 and 2.5 logs in viable counts after one and
five minute’s exposure, respectively. A wet carrier test, involving coupons immersed first
in SE plus 0.1% serum albumin suspension then immediately transferred into 0.08%
NaOCl for 10 minutes, showed 4.5 and 2.7 logs microbicidal effects with steel and
polyethylene surfaces, respectively (Tondo et al. 2010).
Studies concerning disinfection of experimental Salmonella biofilms are summarised
in Table 2. Similar to those involving BAC, the reported methodologies vary substan­
tially, particularly in respect of substrate, age and flow (shear) stress during biofilm
development. However, the complete immersion of biofilm in excess disinfectant at
room temperatures is a set of features common to most tests. Several tests used 5 min’s
exposure and, at concentrations of 0.13% and above, reductions in ST in excess of four
logs were seen in this time (Wong et al. 2010b, 2010a). A lower NaOCl concentration
(0.05% to 0.06%) applied to two-day-old biofilms that had been dried before a fiveminute exposure, led to reductions of 0.5 to three log cycles among strains of serovars
Agona and Senftenberg (Møretrø et al. 2009; Vestby et al. 2010). Comparing these two
studies, a substrate of stainless steel (versus glass) and flow (versus static) conditions of
biofilm growth were associated with apparently lower susceptibility to the disinfectant
solution. With a stirred disinfectant solution during a five-minute exposure, a still-lower
hypochlorite concentration of 0.02% effected a substantial (four log cycle) reduction of
SE biofilm (Ueda and Kuwabara 2007).
With a one-minute exposure for ST biofilms on stainless steel, concentrations in the
range 0.07% to 0.5% effected reductions in excess of four logs (similar to five-minute
exposures), but interestingly a much higher concentration (5.3%) showed poorer efficacy,
up to three logs, with this short exposure time (Wong et al. 2010b, 2010a). This may
reflect loss of potency with elevated pH in stronger NaOCl solutions, as discussed earlier.
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A long exposure (10 to 15 minutes) showed a four- to five-log bactericidal effect for ST
with just 0.01% hypochlorite, albeit at a warm (25°C) temperature (Iñiguez-Moreno et al.
2018). By contrast, for Salmonella biofilms on concrete coupons, up to 90 min’s exposure
to 0.05% yielded little bactericidal effect, generally less than one log (Corcoran et al.
2014).
A field study of hypochlorite disinfection of poultry transport crates, constructed of
galvanised steel plus fibreglass, concluded that pressure-spray cleaning with
a disinfectant (sodium chlorite plus detergent) had no significant effect on coliform
bacteria surface counts (Ramesh et al. 2004). However, a subsequent two-minute immer­
sion in 0.1% NaOCl at 28°C resulted in a reduction of around four logs. Whether the
wetting, spray agitation and detergent application enhanced the subsequent hypochlorite
microbicidal effect was not examined, but in a situation such as this where organic soil is
present, pre-cleaning is likely to assist the subsequent action of disinfectant.
In summary, NaOCl MIC surveys using field isolates of Salmonella spp., E. coli and
Enterococcus spp., (including many from veterinary sources) show no evidence of
resistant subpopulations. By contrast to QAC, MIC values appear commonly to be
above concentrations that otherwise effect several log reductions in viable Salmonella
or E. coli in suspension tests, even with light soil added. This may reflect the conditions of
MIC tests that particularly inhibit the effects of NaOCl, for example, loss of chlorine over
hours to the atmosphere from high surface-area-to-volume ratio microwells and the
quenching effect on chlorine of organic matter in nutrient broth. Mueller-Hinton broth,
commonly used in MIC assays, contains a 2% mix of organic material in the form of
oligopeptides, beef infusion and starch. Inhibition by organic matter is particularly rapid
at the warmer temperatures used in MIC assays (Moats 1981).
The above data indicate that the performance of NaOCl against surface-dried
Salmonella and E. coli is often much poorer than in suspension tests with an equivalent
concentration of agent, but results vary substantially between studies and the effect of
added soil may complicate findings. Like BAC, performance against biofilmed organisms
appears to be similar to that against dried surface inocula, although biofilm tests have
involved full immersion in excess disinfectant (compared with small volumes used in
most surface tests), which may enhance apparent anti-biofilm performance. In one case,
stirring the disinfectant during exposure was associated with unexpectedly strong per­
formance against biofilmed SE at low NaOCl concentration.
Ancillary agents
These agents (EDTA, sodium hydroxide, sodium carbonate and anionic surfactants) are
included in disinfectant or sanitiser mixes to synergise with other biocides, to assist with
cleaning or to ameliorate constraints on performance caused by, for example, hard water
ions in diluting water.
Ethylene diamine tetra-acetic acid (EDTA)
EDTA has a structure with six potential binding sites that can surround and substantially
isolate a metal ion from its usual functional environment. The antibacterial properties of
EDTA are believed to derive from this chelating action on metal ions, including calcium,
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DISINFECTANTS FOR EGG PACKING
magnesium and iron. These are important ions in the cell wall of Gram-positive bacteria,
the outer membrane of Gram-negative bacteria, and the structure of bacterial biofilm.
Typically, EDTA is supplied as the disodium or tetrasodium salt, although some reports
do not specify which form was used experimentally. Metal ion bonding to EDTA
becomes increasingly stable at higher pH, which likely contributes to an observed
enhanced antibacterial activity at such pH values (Adler, DaMassa, and Scott 1979;
Boziaris and Adams 1999).
In Gram-positive organisms, calcium and magnesium form a large proportion of the
metal ions in the cell wall, and treatment of live cells with disodium EDTA 0.1%
(0.0034 mol/L) results in increased external leakage of nucleotides (Lee et al. 1994).
Chelation by EDTA of the same ions at the surface of the Gram-negative outer mem­
brane removes their stabilising effect on lipopolysaccharide, resulting in its loss and
increased permeability of the membrane to hydrophobic molecules (Vaara 1992).
Sequestration of cations in the extracellular polymeric substance of biofilms by EDTA
results in increased water solubility of the biofilm and increased permeability to other
antimicrobial substances (Finnegan and Percival 2015).
At high concentrations and with extended exposure, EDTA on its own can be
inhibitory or lethal to bacteria (Liu et al. 2018; Al-Bakri, Othman, and Bustanji 2009;
Chew, Tjoelker, and Tanaka 1985) and can disrupt or prevent formation of biofilms (AlBakri, Othman, and Bustanji 2009; Percival et al. 2005; Yamakawa, Tomita, and Sawai
2018; Liu et al. 2018). However, the various effects of EDTA also act to potentiate the
antimicrobial action of many other agents. Commonly, this is how it is used, at lower
concentration in a combination preparation. This mode may permeabilise bacteria or
biofilm or expose target sites and thus assist the co-applied microbicide such as BAC
(Adler and DaMassa 1975; Adler, DaMassa, and Scott 1979; Langsrud and Sundheim
1997), hydrogen peroxide (Du and Chen 2019) or bacteriocin (Yamakawa, Tomita, and
Sawai 2018). Its action with chlorine-based disinfectants appears to be more complex, as
EDTA is associated with a reduction in active chlorine in mixtures of the two agents
(Grawehr et al. 2003). Combinations created at the time of application showed undi­
minished activity against Gram positive (Enterococcus faecalis) organisms, but EDTA
interfered with chlorine-associated killing if premixed before application (Senna et al.
2018). Against Gram negative Salmonella Hadar (washed stationary-phase suspended in
distilled water), EDTA at 5 to 10 mM (0.17 to 0.34%) enhanced activity of a chlorinebased disinfectant at 37°C, but at lower concentration (0.017%) it inhibited killing of the
organism (Mullerat, Sheldon, and Arlene Klapes 1995).
Sodium hydroxide
Sodium hydroxide (NaOH) has well-established utility in cleaning (i.e. detergent) mixtures as
a consequence of various effects, principally alkaline hydrolysis of fats and oils (saponifica­
tion) which also aids surfactant activity by stabilising oil-in-water emulsions. Furthermore, by
raising the pH of solutions, NaOH can denature and aggregate proteins (Helbig et al. 2019)
and enhance the action of chelators of hard water ions (Ca2+ and Mg2+) such as EDTA
(Boziaris and Adams 1999). Such water-softening effects enhance the action of surfactants
and help stabilise detached surface soil in suspension.
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In addition to cleaning effects, NaOH (being a highly reactive compound) has micro­
bicidal activity although its potential as a general-purpose disinfectant is limited by its
hazardous and corrosive nature at practical biocidal concentrations. At a concentration
similar to QAC and hypochlorite disinfectants (0.012% NaOH w/v, pH 12), immersion of
Salmonella Infantis biofilms on stainless steel for up to 25 minutes was associated with
modest reductions (around two logs) in viability at 25°C (Speranza et al. 2017). In the
same study, much higher kill effects (at least six logs) were observed at higher tempera­
tures (55°C to 65°C), consistent with findings by Humphrey, Lanning, and Beresford
(1981) of substantially accelerated death of S. Typhimurium and coliforms in alkalinised
poultry scald-tank water at 52°C. The last study additionally posed a high suspended
organic soil challenge.
At a much higher concentration (4%), NaOH had a very substantial biocidal effect (greater
than seven logs reduction within 10 minutes) on two-day-old Salmonella biofilms on
concrete, but this was very much attenuated on one-week-old biofilms (Corcoran et al.
2014). It is possible that differing surface pH of the experimental concrete coupons was
a significant modulating factor in the effect on young versus old biofilms. A similarly
concentrated NaOH solution (3%) applied by spray to ox hides contaminated with
Salmonella- and E. coli-spiked faeces was associated with reductions of two to three logs in
viable organisms after rinsing (Carlson et al. 2008).
Sodium carbonate
Sodium carbonate (Na2CO3) dissolves to form an alkaline solution; the carbonate anion
is in equilibrium with bicarbonate (HCO3−) and hydroxide (OH−) anions, and the pH of
pure solutions of 0.01% to 1% w/v lies in the range 10.5 to 11.2 (Aquion n.d.). As such,
the cleaning properties of Na2CO3 share similarities with those of sodium hydroxide,
although the maximum pH (and corrosive hazard) is comparatively less.
Solutions of Na2CO3 also have bactericidal activity although, perhaps unexpectedly,
this is dependent on the action of carbonate ions (CO32-) and not on pH per se, although
the equilibrium with HCO3− shifts towards a higher concentration of CO32- under more
alkaline conditions (Diez-Gonzalez et al. 2000). The killing of S. Typhimurium and E. coli
was found to depend on carbonate anion concentration (Park and Diez-Gonzalez 2003),
an effect that may in part depend on carbonate binding of metal ion cofactors of bacterial
enzymes, especially Mg2+ (Jarvis et al. 2001). Lethal effects on E. coli O157:H7 at pH 8.5
were observed to intensify markedly between carbonate concentrations of 50 mmol/L
(approximately 0.5%) and 100 mmol/L, although conventional short-exposure disinfec­
tion tests were not done (Diez-Gonzalez et al. 2000). Using pH adjustment to obtain
a range of (calculated) free CO32- concentrations, Park and Diez-Gonzalez (2003) found
suppressive effects on E. coli and S. Typhimurium at concentrations above 2 mmol/L,
although conditions (37°C, 6 hours exposure) were not suitable for assessing disinfectant
effects.
Sodium carbonate has been shown to enhance the bactericidal effect of other biocides
or of elevated temperature, and both alkalinising and cation-binding effects of the
carbonate anion may be relevant in this respect. For planktonic E. coli and Salmonella
enterica subsp. arizonae, either benzalkonium chloride (0.0025% or 0.0013%, respec­
tively) or Na2CO3 (0.01%) achieved no measurable reduction in viable counts. However,
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DISINFECTANTS FOR EGG PACKING
when these two agents were combined, without altering their respective concentrations,
reductions in bacterial counts of three to five logs were observed after 30 minutes (Adler,
DaMassa, and Scott 1979). Use of Na2CO3 sufficient to adjust pH to 9 was reported by the
same authors to enhance the bactericidal activity of a BAC plus EDTA mix (Adler and
DaMassa 1975). Finally, as with sodium hydroxide, the use of Na2CO3 to alkalinise the
water in a poultry slaughterhouse scald tank was associated with greatly reduced survival
times for coliforms and S. Typhimurium at 52°C (Humphrey, Lanning, and Beresford
1981).
Anionic surfactants
Anionic surfactants are commonly incorporated into food-grade sanitising and disinfec­
tion products. For this application, such surfactants typically are sulphate or sulphonate
salts attached to hydrophobic carbon chains or rings. Although present principally as
detergents, they do have bactericidal activity in the low pH range (Al-Adham, Haddadin,
and Collier 2013) and may synergise with other antimicrobial components. Their
antagonism to the action of QAC precludes their use in products containing these agents.
Discussion
The principal bactericidal components of food-grade disinfectant products used in UK egg
packing centres are as follows: BAC, DDAC, sodium hypochlorite, N-[3-aminopropyl]N-dodecylpropane-1,3-diamine and amphoteric surfactants. There are substantial published
data on microbicidal effects for the first three of these. Five more agents or classes of agents
(sodium hydroxide, sodium carbonate, EDTA, anionic and non-ionic surfactants) are used to
synergise with the principal microbicidal agents. They also in some cases assist the removal
(and prevent re-deposition) of organic soil and/or manage the ionic composition of the
disinfectant solution (H+/OH−, Ca2+, Mg2+) so as to optimise the action of other components.
Much of the UK’s water supply is classified as hard or very hard (Aqua Cure 2020), so
counteracting this by the incorporation of suitable components in disinfectant products is
likely to be important. However, several studies have indicated that local water supplies used
as diluents can interfere with disinfectant effects in ways that do not relate to pH or hardness
(Butterfield, Wattie, and Chambers 1950; Davison, Benson, and Eckroade 1996; Wales et al.
2013), so that testing of disinfectants with local water may in some cases be useful.
Surveys of BAC and NaOCl MIC values for Salmonella and E. coli are not consistent
with there being widespread subpopulations harbouring reduced sensitivity to these
agents, although this does not preclude such phenomena happening in specific situations.
This is by contrast to patterns seen with (Gram-positive) Listeria spp., which are
recognised to more readily develop reduced susceptibility to biocides (Duze, Marimani,
and Patel 2021). For Listeria spp., bi-modal distributions of BAC MIC values have been
observed in field strain collections (Aarestrup, Knöchel, and Hasman 2007; Mereghetti
et al. 2000), and there is also limited evidence for reduced susceptibility to hypochlorite in
some strains recovered from intensive-use environments (Teixeira et al. 2020).
Experimentally, the potential for continual low-level exposure to substantially elevate
MIC among Salmonella and E. coli appears to be more marked for QAC than for
chlorine.
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For QAC, there is a well-recognised potential mechanism for resistance, in the
existence of the qac family of genes encoding multi-substrate membrane-located efflux
pumps. These are widespread amongst both Gram-positive and Gram-negative bacteria
(Jaglic and Cervinkova 2012), although their presence does not necessarily correlate with
reduced susceptibility to QAC, in Gram-negative bacteria at least (Kücken, Feucht, and
Kaulfers 2000). For the strains of Salmonella and E. coli trained to a state of reduced BAC
susceptibility discussed above, efflux via non-QAC multi-drug pumps (AcrAB-TolC and
AcrEF-TolC) was implicated, alongside changes in membrane permeability in some cases
(Moen et al. 2012; Guo et al. 2014).
The relationship between MIC values and effective disinfectant concentrations
appears to differ between disinfectants, at least in the cases of BAC (used typically at
many times MIC), DDAC (disinfection concentrations close to MIC) and NaOCl
(effective disinfection may occur below MIC). If comparison of MIC values is used as
a guide to relative susceptibilities between target species, it appears that salmonellae
appear in general to be a little less susceptible to QAC (BAC and DDAC) than do E. coli.
This may mean that efficacy against Salmonella is poorer than anticipated, if used at
a concentration determined by standard testing using an E. coli target. Although UK
Defra General Orders testing uses SE, few disinfectants that are used in food processing
environments are Defra-approved.
In contrast to QAC, susceptibilities to NaOCl (i.e. MIC values) are similar among
salmonellae, E. coli, and indeed Gram-positive organisms. However, one feature of
NaOCl solutions that may lead to unexpectedly poor performance in the field is the
loss of active chlorine, to the atmosphere and by chemical degradation, once containers
have been opened and solutions have been diluted.
For both BAC and NaOCl, there are data showing a substantial reduction of bacter­
icidal effect in the face of organic soil, and particularly by lipid-containing whole-egg
contamination, either on surfaces or in suspension in washer-type solutions. The more
recently developed agents (DDAC and N-[3-aminopropyl]-N-dodecylpropane-1,3-dia­
mine) are reputed to be less impacted by soiling. Whilst there are some data to support
this, severe challenges involving whole egg or yolk contamination are not reported, and
this is a notable gap in the published data. The effects of such soiling can be greatly
counteracted by effective cleaning before disinfection (see introduction), and this is an
area where matching of cleaning components with microbicides is critical. Having
unmatched products at the point of use can potentially result in reduced efficacy, as
(for example) the recognised antagonism of low pH and anionic surfactants to QAC
action (Al-Adham, Haddadin, and Collier 2013) shows. Such matching should be
integral to the development of cleaning and disinfection (C&D) systems or single C&D
products.
In egg handling and packing facilities, an important element of disinfection may be
performed by egg tray washers, where soiling may accumulate during recirculation of
wash water. One study showed how readily free chlorine can reduce to ineffective levels
in warm water with added egg content soiling. By contrast, acidification or alkalinisation
of water was shown in another study to greatly enhance kill rates in hot water, with the
added benefit of improved soil removal under alkaline conditions.
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DISINFECTANTS FOR EGG PACKING
The many differences between suspension and surface disinfection tests (Wales et al.
2021) can result in widely differing indications of efficacy from the two test types, even
with similar disinfectant exposure parameters of concentration, time and temperature.
For both NaOCl and BAC, the findings of Geber et al. (2019) are instructive, as they
concern the same bacterial strains (of Salmonella, E. coli and Klebsiella) and the same
diluent water, soil, disinfectant agent and neutraliser preparations, all at room tempera­
ture. For BAC, there was a modest (up to ten-fold) increase in required concentration
with the addition of suspended soil but a more marked (five to forty-fold) increase
required to pass surface versus suspension tests, both using light soil. By contrast, for
NaOCl, a marked increase in the required concentration was seen with the addition of
suspended soil (ten to fifty-fold) whereas there was little difference between concentra­
tions needed to pass the surface and suspension tests.
However, other investigators (Møretrø et al. 2009; Riazi and Matthews 2011) have
found more substantial differences between suspension and surface tests for NaOCl, and
it seems likely that surface disinfection tests are quite sensitive to the precise conditions
used, particularly when ‘borderline’ conditions of disinfectant efficacy are being exam­
ined. Such findings can be viewed in the context of generally quite wide variability in
disinfectant test results, attributable in part to variation in target organisms (even of
nominally the same strain) plus physical variables, and with such variability subject to
amplification under borderline conditions. One implication of this for field use is that
inadequate or inconsistent disinfectant efficacy may occur even when ‘label’ directions in
respect of exposure time and concentration have been followed, and even with exemplary
cleaning and application techniques.
One area of potential importance that has not yet been incorporated into standard
disinfectant testing is the resistance of organisms in surface biofilm communities to
disinfection. The existing data for the reviewed chemical agents suggests that the inter­
fering influence of biofilm is similar to that of organic soil. It should be noted, however,
that the published biofilm data arise from tests that are more varied in methodology than
surface tests and which involve immersion in excess disinfectant, which may over-state
disinfectant efficacy compared with standard (low disinfectant volume) surface tests.
In view of these findings, physical and chemical treatments that degrade and remove
both biofilm and soil are ideal. Investigations by Furukawa et al. (2010) have indicated
that many acidic and alkaline cleaners are effective against E. coli biofilm, but that
a Gram-positive (Staph. aureus) biofilm was removed effectively only by alkaline agents.
Whilst action against mixed-species biofilms (and indeed fatty soil) may in consequence
be better served by alkaline cleaners, this could conflict with a disinfectant agent more
active at lower pH such as hypochlorite and illustrates the care needed in selecting
compatible agents and/or adequate rinsing between cleaning and disinfection phases.
Less potentially hazardous cleaning agents tested by the same authors (including EDTA
and sodium dodecyl sulphate, both at 0.1%) were less effective, even against monospecies
E. coli biofilm.
In addition to biofilms, surface qualities (such as material, roughness, porosity and
damage or corrosion) evidently have substantial effects on the activity of applied disin­
fectants. Experimentally, most departures from a smooth, hard and nonporous surface
show a reduced microbicidal effect with QAC and NaOCl. It is not feasible to test and
licence products for every surface that they might be used on, but awareness of this
WORLD’S POULTRY SCIENCE JOURNAL
251
variation may help to foster an appreciation by operators that certain surface types will be
more resistant to effective disinfection, even when using products as recommended. It is
possible that future materials used for egg handling and transport will incorporate
antimicrobial surface qualities. Examples of such systems include heavy metals (elemen­
tal copper, complexed zinc, or silver nanoparticles), oxidisers (titanium dioxide) or
triclosan (Nichols 2004, 11; Depner et al. 2021). However, the possible benefits of such
materials in the field require further investigation.
It is notable that for both BAC and NaOCl there are data showing a limit to the
bactericidal effect with short exposure times (of around 1 min) regardless of the con­
centration of disinfectant applied. Thus, any disinfection protocols or practices that
employ such short exposure times may be vulnerable to failure, even if disinfectants
are used at higher than usual concentration. The mechanisms for such limitations are not
well explored and may differ between disinfectant classes. They might include barriers to
diffusion and (for hypochlorite, at least) an unfavourable pH of concentrated solutions.
In conclusion, the amount of peer-reviewed efficacy data for the principal active
components of food-grade disinfectants used in egg packing premises shows consider­
able variation according to the particular agent. Furthermore, interpretation of the
evidence is hampered by varied methodologies, in a field where it is recognised that
results vary substantially even with consistent experimental practice. Further investiga­
tions, using actual products plus standardised methodology reflecting field conditions (of
concentration, diluent, exposure time, soiling, temperature, surface materials and eggassociated SE targets), are likely to yield more insights into conditions where disinfection
of egg packing materials may prove inadequate. In addition, field studies can help to
uncover areas of disinfection vulnerability under ‘real-world’ conditions. These may
include limited access, suboptimal exposure times, lower temperatures, influence of
biofilms, inadequate cleaning, and idiosyncrasies of local water supply.
Disclosure statement
The authors declare no conflicts of interest.
Funding
This work was supported by the UK Department for Environment, Food and Rural Affairs (Defra),
project SV3998.
Notes on contributors
Andrew Wales is a veterinarian who qualified in 1993 and completed a PhD in Pathology and
Microbiology in 2003. Since then he has combined a career in general veterinary practice with
research interests in foodborne bacterial zoonoses. He has worked for many years in collaboration
with the UK Animal and Plant Health Agency and latterly has held a research fellowship at the
University of Surrey. Areas covered by research publications include: shigatoxigenic E. coli in
sheep, Salmonella in livestock (poultry, pigs, cattle and animal feed), Campylobacter in poultry,
antimicrobial resistance in livestock, disinfection and disinfectant testing, and microbiological
hazards for raw-fed companion animals.
252
DISINFECTANTS FOR EGG PACKING
Emma Taylor studied her Bachelor’s degree in Genetics with Microbiology at Queen Mary,
University of London and her Master’s degree in Medical Microbiology at the London School of
Hygiene and Tropical Medicine. She received her PhD from Imperial College London in 2020,
where she studied 16S rRNA methyltransferases. Currently, Emma is working for the Animal and
Plant Health Agency, where she conducts research on Shiga-toxin producing Escherichia coli
(STEC) and Salmonellain poultry.
Rob Davies is a veterinarian with 11 years’ experience of mixed farm practice and over 30 years’
experience of applied research on monitoring, detection and control of foodborne zoonoses,
including antimicrobial resistant organisms. Rob qualified from Bristol University in 1979 and
joined the Central Veterinary Laboratory (now part of the Animal and Plant Health Agency) at
Weybridge in 1990. He completed a PhD on Salmonella control in the poultry industry in 1997
and has worked on infection control and biosecurity across a wide range of animal and feed/food
production sectors, including field and laboratory studies of cleaning and disinfection. Rob also
leads the OIE Salmonella Reference Laboratory at Weybridge and is part of the team that delivers
the Defra disinfectant approvals scheme.
ORCID
Andrew Wales
http://orcid.org/0000-0002-8657-3007
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