Food Microbiology 44 (2014) 168e172

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Short communication

Inactivation of avirulent pgmþ and Dpgm Yersinia pestis by ultraviolet

light (UV-C)
George C. Paoli a, *, Christopher H. Sommers b, O. Joseph Scullen b, Chandi Wijey a
a
Molecular Characterization of Foodborne Pathogens, US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center,
600 East Mermaid Lane, Wyndmoor, PA 19038, USA
b
Food Safety& Intervention Technologies Research Units, US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center,
600 East Mermaid Lane, Wyndmoor, PA 19038, USA

a r t i c l e i n f o a b s t r a c t

Article history: Yersinia pestis is the causative agent of bubonic plague. Though not considered a foodborne pathogen,
Received 8 April 2014 Y. pestis can survive, and even grow, in some foods, and the foodborne route of transmission is not
Received in revised form without precedent. As such, concerns exist over the possible intentional contamination of foods with this
27 May 2014
deadly pathogen. Here we report the inactivation of avirulent (pYV-minus) strains of Y. pestis by ultra-
Accepted 2 June 2014
Available online 17 June 2014
violet light (UV-C, 254 nm). Two strains of Y. pestis containing an intact pgm virulence locus (pgmþ) and
strains from which the pgm locus was spontaneously deleted (Dpgm) were tested using cells grown in
both logarithmic and stationary phase. The D10 values for inactivation (the UV-C dose required to
Keywords:
Yersinia pestis
inactivate one log of bacterial cells) of Y. pestis on the surface of agar plates ranged from 0.69 to 1.09 mJ/
Ultraviolet light cm2. A significant difference was observed between the inactivation of cells of Y. pestis strain Yokohama
Food grown in logarithmic and stationary phases, but no significant difference between growth phase
pgm locus sensitivity to UV-C was observed in Y. pestis strain Kuma. No difference in D10 values was observed
Irradiation between pgmþ and Dpgm strains of Yokohama grown to either logarithmic or stationary phase. A
measurable difference was observed between the D10 of Kuma pgmþ and Kuma Dpgm grown in loga-
rithmic phase, but this difference was diminished in the Kuma strains grown to stationary phase. Though
strain variations exist, the results showing that UV-C can inactivate Y. pestis cells on agar surfaces suggest
that UV-C would be effect in inactivating Y. pestis on food surfaces, particularly foods with a smooth
surface.
Published by Elsevier Ltd.

1. Introduction pharyngeal plague, gastrointestinal plague, or bubonic plague,

The genus Yersinia contains 11 species, 3 of which are known
human pathogens. Yersinia pestis is the causative agent of bubonic
plague and is typically transmitted to humans via the bite of a flea
from an infected rodent. Yersinia enterocolitica and Yersinia pseu-
dotuberculosis are foodborne pathogens causing nearly 100,000
illnesses in the US annually (Scallan et al., 2011).
While the foodborne transmission of Y. enterocolitica and
Y. pseudotuberculosis is well established, the risk associated with
consumption of foods contaminated with Y. pestis, either inciden-
tally or deliberately, is currently unknown. Although infrequent,
* Corresponding author. Molecular Characterization of Foodborne Pathogens, US
Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA. Tel.: þ1 215
233 6671; fax: þ1 215 836 3742.
E-mail address: George.Paoli@ars.usda.gov (G.C. Paoli).
sometimes resulting in death, can be acquired through the
handling or consumption of raw or undercooked animal products
(Arbaji et al., 2005; Bin Saeed et al., 2005; Christie et al., 1980; Leslie
et al., 2010). Furthermore, Y. pestis is capable of survival and growth
at refrigeration temperatures (Torosian et al., 2009) and in foods
such as ground beef (Bhaduri, 2010), frankfurters (Sommers and
Cooke, 2009), liquid egg product (Gurtler et al., 2010), and milk
(G. Paoli, unpublished).
Y. pestis is a deadly pathogen, but strains are rendered avirulent
or conditionally virulent upon spontaneous loss of either the 70 kb
pYV virulence plasmid or the 102 kb chromosomal pgm virulence
locus, respectively (Bearden and Perry, 2008). The pYV plasmid
(often referred to as pCD in Y. pestis), which is present in all three
human pathogenic species of Yersinia, encodes a type III secretion
system and a number of effector proteins required for infection
(Perry et al., 1998). The pgm locus, which is unique to Y. pestis,
contains the enteric pathogen high pathogenicity island encoding

http://dx.doi.org/10.1016/j.fm.2014.06.002
0740-0020/Published by Elsevier Ltd.
 G.C. Paoli et al. / Food Microbiology 44 (2014) 168e172
several proteins necessary for iron acquisition and a second region
which confers the pigmented phenotype (thus the name pgm) to
colonies grown on media containing the dye Congo Red (Buchrieser
et al., 1999). The deletion of the entire pgm locus is spontaneous and
frequent in vitro, involves a RecA-dependent recombination event,
and results in a loss of the pigment binding phenotype (Hare and
McDonough, 1999). In addition to the Y. pestis-specific pgm locus
and the 70 kb Yersinia virulence plasmid, pYV/pCD1, fully virulent
strains of Y. pestis contain 2 additional Y. pestis-specific plasmids
associated with pathogenicity, a large plasmid (100e110 kb) pMT1
and a small (~10 kb) plasmid pCP1 (Bearden and Perry, 2008).
Because of its deadly nature and potential for misuse, Y. pestis is
considered a select agent for food safety and defense (Anonymous,
2013; Takhistov and Bryant, 2006). As such, intervention technol-
ogies to inactivate Y. pestis should be investigated. Ultraviolet light
(UV-C, 254 nm) is a nonthermal intervention technology that can
be used for decontamination of water, food, and surfaces and is
used by many industries, including the medical industry, to reduce
the possibility of disease transmission (Koutchma, 2008; Koutchma
et al., 2009; Rutala et al., 2010; Yoosook et al., 2009). It has also been
proposed as a technology to inactivate foodborne pathogens of
relevance to biosecurity (Brickner et al., 2003; Rose and O'Connell,
2009).
Current trends in risk analysis often include not only potential
presence or absence of a pathogen, but, where possible, the path-
ogenicity and virulence of the microbial hazard (Anonymous,
2012). Yersinia spp. are a good model system to determine the ef-
fects of intervention technologies, in particular ultraviolet and
gamma radiation, on the reduction of both pathogen load and
pathogenicity. For example, studies on the Yersinia enterocolitica
pYV 70 kDa virulence plasmid (Butler et al., 1987; Sommers and
Bhaduri, 2001) and whether or not the presence of the plasmid
affects pathogen survival to gamma radiation (Sommers and Novak,
2002) were simplified by the pYV plasmid-associated crystal violet-
binding phenotype which can be used as a facile screen for plasmid
loss (Butler et al., 1987; Sommers and Bhaduri, 2001). Similarly,
Y. pestis represents a good model system to determine the effect of
chromosomal virulence genes on microorganism resistance to
genotoxic agents and interventions because of a simple pgm locus-
associated colorimetric screening assay (i.e., Congo Red binding).
The current study was undertaken to measure the inactivation
of Y. pestis by UV-C, with particular interest determining if there is
any difference in UV-C resistance between strains of Y. pestis with
or without the pgm locus, and if the frequency of loss of the pgm
locus is increased in survivors following exposure to UV-C.
2. Materials and methods
2.1. Yersinia pestis strains and growth conditions
Though fully virulent Y. pestis is a biosafety level 3 pathogen and
a select agent, strains that have lost either the pYV plasmid or the
pgm locus are attenuated for virulence and are considered biosafety
level 2, and are thus excluded from the select agent list
(Anonymous, 2013; Bearden and Perry, 2008). Y. pestis strains
Yokohama and Kuma, both of which are avirulent due to the loss of
the virulence plasmid pYV, were provided by Dr. Robert Brubaker
(Michigan State University, East Lansing, MI). Y. pestis was routinely
grown on Bacto™ Brain Heart Infusion (BHI) (Becton Dickinson,
Sparks, MD) broth or BHI plates solidified with 1.8% agar. Cultures
were inoculated from frozen (80  C) glycerol (15%) stocks, onto
BHI plates, incubated at 30  C for 72 h, and stored at 4  C until
needed.
Each Y. pestis strain was cultured independently in 25 mL of BHI
broth in a sterile 50-mL conical tube at 37  C with shaking
169
(150 rpm) for 18 h, to a density of approximately 107e108 CFU/mL,
using a New Brunswick Model G24 Incubator (Edison, NJ). To
generate logarithmic phase cultures, the overnight cultures were
diluted 1/10 in 25 ml of BHI and allowed to grow to an OD600 of
0.5e1.0. The cultures were then sedimented by centrifugation at
(1700  g) using a Fisher Scientific Marathon 21000R Centrifuge
(Needham Heights, MA). The cell pellets were then resuspended in
an equal volume of Butterfield's Phosphate Buffer (BPB) (Applied
Research Institute, Newtown, CT) for stationary phase cells and
reduced volume for logarithmic phase cells.
2.2. Phenotypic and genotypic characterization of the Y. pestis pgm
locus
The presence of the pgm-locus was determined phenotypically
by the ability of bacterial colonies to bind Congo Red (CR) on
Surgalla-CR medium. On this media pgmþ colonies bind CR and
appear red while Dpgm strains are non-pigmented. The Surgalla-CR
medium contained 1% Bacto™ Heart Infusion broth (Becton Dick-
inson, Sparks, Maryland) 0.2% Galactose, 0.01% CR and 2% agar
(Surgalla and Beesley, 1969). To prepare 1 L of media 10 g of heart
infusion broth was dissolved in 980 mL of water and 20 g of agar
was added prior to autoclaving. After autoclaving the media was
cooled to 55  C, 10 mL of a sterile (filtered) 20% galactose solution
and 10 mL of a sterile (autoclaved) 1% CR solution were added.
Inoculated plates were incubated at 26  C for 72 h.
The presence or absence of the pgm-locus was determined
genotypically by a multiplex PCR assay using primer pairs PP3þ/
PP14þ and DPGM1/DPGM2 (Bearden and Perry, 2008). Primers
PP3þ (GACGATTAACGAACCGGA) and PP14þ (ATTCAGGATGGCCTG
CTG) amplify a PCR product from the gene encoding the pesticin
receptor (psn) within the pgm locus yielding a 402 bp amplicon,
confirming the presence of the pgm locus. The deletion of the pgm-
region was confirmed using primers DPGM1 (GGCA-
GACGGACCATCCAG) and DPGM2 (CCCCGCCAGATCCTTACC) that are
complimentary to nucleotide sequences that lie outside of the
102 kb pgm-region and generate a 2020 bp amplicon that spans the
pgm-deletion site. The polymerase chain reaction was done using
GoTaq DNA polymerase as described by the manufacturer (Promega,
Madison, Wisconsin). Each 20 mL multiplex PCR assay contained 1X
Green GoTaq reaction buffer, 200 mM each dNTP, 0.8 mM each primer
(PP3þ, PP14þ, DPGM1, and DPGM2), 1.25 units of GoTaq DNA po-
lymerase, and 1 mL (~40 ng) of DNA template. PCR was done using an
iQ5 thermocycler (BioRad, Hercules, CA). Samples were denatured
for 90 s at 95  C, followed by 30 cycles of 95  C for 30 s, 55  C for 45 s,
72  C for 60 s, followed by a final extension at 72  C for 5 min. PCR
products were separated on 2% agarose gels, stained with ethidium
bromide and visualized with a UV transilluminator.
2.3. Selection of Y. pestis Dpgm strains
Individual CR-binding colonies of Y. pestis Kuma and Yokohama
were picked from Surgalla-CR plates and inoculated into 5 mL of
BHI broth. The cultures were grown overnight (~18 h), serially
diluted in phosphate buffered saline, and 100 mL of dilution ex-
pected to yield 100e200 cfu was spread onto 20 CR-Surgalla plates.
After incubating for 72 h at 26  C, 6 non-pigmented colonies of each
strain were checked for loss of the pgm locus by multiplex PCR. Each
of the isolates had lost the pgm locus (data not shown), and one
isolate of each strain was used for further experiments.
2.4. Exposure to ultraviolet light (UV-C)
For determination of D10 value, the UV-C dose needed to inac-
tivate 1 log10 of microorganism, the following protocol was used.
170 G.C. Paoli et al. / Food Microbiology 44 (2014) 168e172
Individual Y. pestis isolates were serially diluted in BPB and 0.1 mL
inoculated onto duplicate Surgalla-CR plates, which were then
allowed to dry for approximately 15 min. Exposures were carried
out using the UV-C source from a biological safety cabinet (NuAire,
Inc., Model NU-425-600, Plymouth, MN) with an intensity of
100 mW/cm2/s as determined by a calibrated UVX Radiometer (UVP,
Inc, Upland, CA). The logarithmic phase cells were then exposed for
ca. 0, 7.5, 15, 22.5, 30, and 37.5 s, to obtain total exposures of 0.75,
1.5, 2.25, 3.0, and 3.75 mJ/cm2, respectively. The UV-C dose was
extended to 4.5 mJ/cm2 for the stationary phase cells due to higher
cell density used in these experiments. For determination of fre-
quency of loss of the pigment binding phenotype, 0.1 mL of
appropriately diluted logarithmic or stationary phase culture was
inoculated onto 80 Surgalla-CR media plates (40 plates of 2
different dilutions). Half of the plates were not exposed to UV-C
(untreated controls) and the remaining plates of logarithmic and
stationary phase cultures were exposed to UV-C doses of 2.25 and
3.0 mJ/cm2, respectively (doses corresponding to approximately a 3
log reduction in cell number). Each treatment was conducted in
triplicate (n ¼ 3). For each replicate of each treatment, between
4000 and 20,000 colonies were screened for loss of pigment
binding by the bacterial colonies.
2.5. D10 values and log reduction
Data from the linear portion of the survival curves, 0e3.75 J/cm2
were used to determine D10. The mean plate counts of the treated
samples (N) were divided by the average control plate counts (N0)
to give a survivor ratio (N/N0). The log10 (N/N0) of the ratios were
then used for determination of D10 values and other statistical
analyses. D10 values were determined by the reciprocal of the slope
(Diehl, 1995). Each experiment was conducted independently 3
times (n ¼ 3).
2.6. Statistical analysis
Each experiment was conducted independently three times.
Statistical analysis functions of MS Excel (Microsoft Corp., Red-
mond, WA) were used for routine calculations, descriptive statis-
tics, and Analysis of Variance (ANOVA). ANOVA was used for
comparison of D10 values (Sommers and Novak, 2002).
3. Results and discussion
UV-C is used in a number of industries to inactivate bacteria
(Koutchma, 2008; Rutala et al., 2010; Yoosook et al., 2009) and has
been proposed as a technology for inactivation of microorganisms
of biosecurity concern (Brickner et al., 2003; Rose and O'Connell,
2009). The mechanism of killing by UV-C is primarily due to the
DNA damage through the formation of cyclobutane pyrimidine
dimmers and pyrimidine (6,4) pyrimidone photoproducts at
dipyrimidine sites causing error prone DNA replication resulting in
increased mutation frequency (Ikehata and Ono, 2011). UV-C irra-
diation also induces oxidative stress through the generation of
reactive oxygen species that can further damage DNA (Ikehata and
Ono, 2011) and proteins involved in several cellular process, in
particular molecules that contain iron or are involved in iron
acquisition and metabolism (Eisenstark, 1998). Due to the fact that
ultraviolet radiation affects DNA replication, faster growing cells
(i.e., cells in the logarithmic phase of growth) are usually more
sensitive to genotoxic agents such as ultraviolet and ionizing ra-
diation than are slower growing cells (i.e., stationary phase) (Child
et al., 2002; Dantur and Pizarro, 2004; Navarro Llorens et al., 2010).
Interestingly, certain aspects of UV-induced lethality may affect
or be affected by the Y. pestis pgm locus. First, several genes within
the pgm locus are involved in iron acquisition (Buchrieser et al.,
1999) and the intracellular iron pools may have an important
impact on the generation of reactive oxygen species resulting from
UV irradiation. Thus, the presence or absence of the pgm locus may
have an effect on the sensitivity of Y. pestis to UV-C. Secondly,
bacteria respond to UV assault by inducing the SOS response,
which, in repairing DNA damage, leads to replication errors and
mutagenesis (Schlacher and Goodman, 2007). Part of this SOS
response involves increased levels of the RecA-recombinase and
the high frequency loss of the pgm locus from Y. pestis occurs via a
RecA-dependent deletion (Hare and McDonough, 1999). Thus, it
would be interesting to determine if the Y. pestis that survives the
UV-C exposure shows any increase in pgm deletion frequency.
The D10 values obtained in this study are consistent with those
obtained in other studies of vegetative bacteria which commonly
report D10 values from 0.5 to 4 mJ/cm2 (Koutchma et al., 2009) and
differences in resistance to UV-C light within serovars and between
isolates of the same species (Koutchma et al., 2009). The D10 values
obtained in this study are similar to those obtained for Y. pestis
(Rose and O'Connell) and Francisella tularensis (Rose and O'Connell,
2009; Sommers et al., 2012), another Gram-negative bacterial
select agent that causes a plague-like ulceroglandular and pneu-
monic illness called tularemia. The results from UV-C inactivation
experiments obtained in this study are shown in Table 1 and Fig. 1.
Data from the linear portion of the survival curves, 0e3.75 mJ/cm2
(Fig. 1), were used to determine D10, which ranged from 0.69 mJ/
cm2 to 1.09 mJ/cm2. As such, Y. pestis seems to be on the more
sensitive side of the UV resistance scale for vegetative bacteria
(typical D10 values range from 0.5 to 4 mJ/cm2) as reported by
Koutchma et al. (2009). As expected, there were differences in the
D10 between isolates. There was no significant (ANOVA, p > 0.05)
difference between the D10 values for stationary phase cells versus
logarithmic phase cells for both pgmþ (D10 values of 0.82 and
0.83 mJ/cm2, respectively) and Dpgm (D10 values of 0.74 and
0.69 mJ/cm2, respectively) strains of Y. pestis Kuma. Unexpectedly,
logarithmic phase cells of both pgmþ and Dpgm strains of Y. pestis
Yokohama showed slightly higher UV-C resistance (D10  1.0 mJ/
cm2) than the same strains grown to stationary phase
(D10 < 0.80 mJ/cm2). While several reports have demonstrated an
increased UV-resistance in stationary phase cells (Child et al., 2002;
Dantur and Pizarro, 2004; Navarro Llorens et al., 2010) exceptions
to the correlation between specific growth rate and UV-sensitivity
have been reported (Bucheli-Witschel et al., 2010; Qiu et al., 2004).
A measurable difference was observed between the D10 of Kuma
pgmþ and Kuma Dpgm grown in logarithmic phase (p ¼ 0.043), but
this difference was reduced when cells were grown to stationary
phase (p ¼ 0.063) (Table 1). Y. pestis strain Yokohama demonstrated
Table 1
D10 values for inactivation of stationary and logarithmic phase cultures of pgmþ and
pgm (Dpgm) isolates of Yersinia pestis strains Yokohama and Kuma by ultraviolet
light.
Isolate Growth phase D10 (mJ/cm2) R2
Stationary
Kuma pgmþ 0.82 0.98
Kuma Dpgm 0.74 0.97
Yokohama pgmþ 0.77 0.89
Yokohama Dpgm 0.79 0.92
Logarithmic
Kuma pgmþ 0.83 0.97
Kuma Dpgm 0.69 0.97
Yokohama pgmþ 1.09 0.98
Yokohama Dpgm 1.00 0.97
D10 values are the average of 3 independent experiments. Analysis of variance was
used to determine significant differences in D10 values (P < 0.05) as indicated it the
text.
 G.C. Paoli et al. / Food Microbiology 44 (2014) 168e172
A Stationary Phase
0 with or without exposure to UV-C. Both stationary phase and logarithmic phase
-1
Log Reduction (N/No)
-2
-3
-4
-5
-6
-7
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
2 stationary phase. Furthermore, all 144 non-pigmented isolates and
UV-C (mJ/cm )
B Logarithmic Phase
0 associated plasmids (pMT1 and pCP1) present in avirulent (pCD1-
-1
Log Reduction (N/No)
-2
-3
-4
-5
-6
-7
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
UV-C (mJ/cm2)
Fig. 1. Inactivation of Yersinia pestis by ultraviolet light (UV-C). Strains Yokohama
(triangles) and Kuma (circles) with an intact pgm-locus (open symbols) or a deleted
pgm locus (filled symbols) were grown to (A) stationary or (B) logarithmic phase and
exposed to ultraviolet light (UV-C; 254 nm) as indicated. The slightly higher UV-C
exposure was used for the stationary phase cells as the result of higher cell densities
in these cultures. Each point is the average of 3 independent experiments. The error
bars indicate the standard deviation.
no difference in D10 values between pgmþ and Dpgm strains grown
to either logarithmic or stationary phases.
To address the question concerning possible differences in the
deletion frequency of the pgm locus in UV-C irradiated cells
compared to those that had not been exposed to UV-C, the pgmþ
cultures of strains Yokohama and Kuma were plated directly onto
Surgalla-CR media immediately prior to UV-C exposure. The ratio of
the non-pigmented colonies (Dpgm) to the total number of colonies
(most of which were CR-binding pgmþ) was used to determine the
frequency of the loss of the pgm locus. At least 24 non-pigmented
colonies and 3 CR-binding colonies from each experiment (a total
of 144 non-pigmented and 26 CR-binding colonies) were tested
using the pgm multiplex PCR. In every case the colony pigmentation
phenotype was consistent with the genotype as determined by the
pgm multiplex PCR. The frequencies of loss of the CR-binding
phenotype observed in this study (ranging from 1.6  102 to
7.7  103) are similar to those reported by Hare and McDonough
(1999). For strain Kuma, the frequency of the deletion of the pgm
locus in survivors of UV-C irradiation was 3.3e4.5 higher than that
of same culture that was not treated with UV-C (Table 2); however,
results of pair-wise analysis of variance using an F-test revealed no
significant difference between the two treatments (P < 0.05) for
171
Table 2
Frequency of the deletion of the pgm locus in Y. pestis strain Kuma and Yokohama
cultures were tested in three independent experiments.
Y. pestis Dpgm frequency (White colonies/Total colonies)
strain
Stationary phase Logarithmic phase
No treatment UV-C exposed No treatment UV-C exposed
Kuma 3.5  1003 1.6  1002 6.9  1003 2.3  1002
Yokohama 7.7  1003 7.7  1003 2.2  1003 6.7  1003
Frequencies of pgm loss are the average of 3 independent experiments. Pairwise
analysis of variance was used to determine significant differences in values
(P < 0.05) as indicated it the text.
either strain Kuma or Yokohama grown to either logarithmic or
26 pigmented isolates that were screened by multiplex PCR for the
presence of the pgm locus were also screened by a multiplex PCR
assay for the presence of the two Y. pestis-specific virulence-
minus) strains Yokohama and Kuma. No loss of either of these
plamsids was observed (data not shown); thus, Y. pestis survivors of
UV-C exposure do not appear to show any significant reduction in
virulence potential. Additional experiments to assess a larger
population of Y. pestis cells may be needed to more accurately
determine the frequency of CR-binding loss (loss of the pgm locus)
(Sommers and Bhaduri, 2001) and loss of virulence potential
resulting from exposure to ultraviolet light, as well as other geno-
toxic agents.
Acknowledgments
The authors would like to thank Dr. Robert Brubaker for
providing Y. pestis strains Yokohama and Kuma used in this study.
Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and does
not imply recommendation or endorsement by the U.S. Depart-
ment of Agriculture. USDA is an equal opportunity provider and
employer.
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