Isolation and Identification of Adherent Gram-Negative Microorganisms from Four Meat-Processing Facilities

Abstract

Biofilms are described as a matrix of microorganisms which have adhered to and colonized a surface. Once formed, biofilms are difficult to remove and may be a source of contamination in food-processing environments. In this study, stainless-steel chips were fixed to surfaces adjacent to food-contact surfaces and cast-iron chips were suspended in the floor drains of four meat-processing plants. Biofilm formation was quantified by staining the attached cells and viewing them under epifluorescence microscopy. The stainless-steel and cast-iron chips removed from the plant environment showed some attached microorganisms. Floor drains appeared to provide an excellent environment for the formation of biofilms. Pseudomonas, Klebsiella, Aeromonas, and Hafnia species were identified as gram-negative microorganisms associated with the test surfaces.

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1135
Journal of Food Protection, Vol. 60, No.9, 1997, Pages 1135-1138
Copyright ©, International Association of Milk, Food and Environmental Sanitarians
Research Note
Isolation and Identification of Adherent Gram-Negative
Microorganisms from Four Meat-Processing Facilities
SCOTT K. HOODt and EDMUND A. ZOTTOLA*
Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, Minnesota 55108, USA
(MS# 96-212: Received 20 August 1996/Accepted 23 January 1997)
ABSTRACT
Biofilms are described as a matrix of microorganisms which
have adhered to and colonized a surface. Once formed, biofilms are
difficult to remove and may be a source of contamination in
food-processing environments. In this study, stainless-steel chips
were fixed to surfaces adjacent to food-contact surfaces and
cast-iron chips were suspended in the floor drains of four meat-
processing plants. Biofilm formation was quantified by staining the
attached cells and viewing them under epifluorescence microscopy.
The stainless-steel and cast-iron chips removed from the plant
environment showed some attached microorganisms. Floor drains
appeared to provide an excellent environment for the formation of
biofilms. Pseudomonas, Klebsiella, Aeromonas, and Hafnia spe-
cies were identified as gram-negative microorganisms associated
with the test surfaces.
Key words: Biofilm, meat processing, cell adherence, gram-
negative bacteria
Biofilms are described as bacteria that have attached to
and are growing on an inert surface. Studies of biofilms and
biofilm-forming bacteria have been conducted in various
environments including aquatic systems, oral surfaces and
medical implants (2, 9, 19). While biofilms can be useful, as
in the treatment of wastewater, biofilms are more often
studied because of the harm they can cause by serving as a
source of microbial contamination.
Over the last 15 years, researchers have suggested that
biofilms on food-contact surfaces may be a potential source
of concern in food-processing environments (10, 20). Micro-
organisms can be found throughout food-processing opera-
tions. Pathogens such as Listeria and Salmonella species
pose a risk to the safety of processed foods, and other
contaminants can reduce the keeping quality of foods. Data
compiled by Nelson (8) showed that Listeria monocytogenes
*Author for correspondence. Tel: 612-624-9274; Fax: 612-625-5272;
E-mail: ezotto1a@che2.che.umn.edu
tPresent address: Kohler Mix Specialities, Inc., White Bear Lake, MN.
could be isolated in 5% of over 8,800 environmental
samples in 62 dairy-processing facilities. Other researchers
have found similar levels of L. monocytogenes in processing
environments (17). Clearly, pathogenic microorganisms can
reside in processing environments, and it is possible that
biofilms harboring pathogens may also exist in the plant
environment.
If these environmental isolates have the opportunity to
reside on a surface, biofilms may be formed. Once attached
to a surface, microorganisms appear to be more difficult to
remove. Ronner and Wong (11) studied Salmonella typhimu-
rium and Listeria monocytogenes in biofilms on stainless
steel and Buna-N rubber. They found that biofilm cells were
more resistant than free-living cells to the sanitizers quater-
nary ammonium compounds, iodine, chlorine, and
anionic acids.
When microorganisms in a biofilm become dislodged
from a food-contact surface, they have the opportunity to
attach to the surface of a food, such as meat. Both
pathogenic and spoilage microorganisms can rapidly adhere
to the surface of beef tissue (1, 3). This adhered population
can then pose a threat to the safety and keeping quality of
meat products.
The objective of this study was to determine if biofilm
formation occurs in meat-processing environments, and if
so, to identify the types of microorganisms in such biofilms.
MATERIALS AND METHODS
Meat plants
The four plants included a slaughtering operation, hamburger
patty production, beef jerky packaging and sausage production.
Plant personnel were aware of the presence of testing materials.
Test surfaces
Stainless-steel chips (6 by 6 by 1.2 mm) were attached to
stainless-steel surfaces using double sided tape (3M, St. Paul, MN)
or RTV 108 silicone adhesive (GE Silicones, Waterford, NY). The
sites chosen were directly adjacent to surfaces where food would be
placed during the processing operation. Cast-iron chips (6 by 6 by
1.2 mm) were suspended in floor drains using nylon fishing line
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1136 HOOD AND ZOTTOLA
because neither adhesive method worked in the drains. Drains were
chosen to include areas with a high flow rate or a low flow rate of
water.
Isolation and enumeration of microorganisms
The chips were removed at predetermined time intervals and
rinsed with 0.1
%
peptone water. One chip from each location was
stained with acridine orange. Stained microorganisms were enumer-
ated by examining 10 microscopic fields at 1,000X (field area =
2.01 X 10-
4
cm
2)
using an Olympus BHT microscope with a
reflected UV light attachment.
The second chip was placed in tryptic soy both (TSB) (Difco
Laboratories, Detroit, MI). After mixing by vortex for 10 s, 1 ml of
broth was transferred to two additional tubes of broth each
containing 9 ml of TSB. The tube containing the chip was
incubated at 37°C for 24 h. The other two were incubated at 21°C
and 13°C for 48 hand 7 days, respectively.
Identification of microorganisms
The broth cultures were streaked for isolation of microorgan-
isms onto tryptic soy agar (TSA) (Difco) and incubated at 37°C or
21°C. Colonies of different morphology were transferred to TSB
using a sterile inoculating loop. Cultures were allowed to grow at
37°C or 21°C for 18 to 24 h and were then Gram stained. All
gram-negative cultures were identified using api 20e or the API
NFT systems (api, Plainview, NY).
In one processing facility, chips were removed and one placed
into lactose broth (LB) (Difco) and one in UMV broth (UVMB)
(Oxoid). The LB samples were then transferred to selenite cysteine
(SC) broth and streaked onto bismuth sulfite agar (BSA) and XLD
agar (Difco) to determine if Salmonella spp. were present. The
UMVB samples were transferred to Fraser broth and then streaked
onto modified Oxford agar (Oxoid) to determine if Listeria were
present.
RESULTS AND DISCUSSION
The four meat-processing plants included slaughter,
raw, and finished beef or pork operations. The locations of
the chips in each plant are shown in Table 1. Actual
food-contact surfaces were not used to make sure the
probability that the stainless-steel chip would become dis-
lodged and enter the food was minimized. Sites adjacent to
food-contact surfaces were located so that chips would
potentially be exposed to meat via the spraying of water or
other disloging processes.
The results of direct microscopic examination of stain-
less-steel and cast-iron chips placed at various points in the
plants are shown in Table 2. Stainless-steel chips in locations
adjacent to food contact surfaces did not appear to harbor
high levels
(>
10
4/cm2)of microorganisms. However, when
these chips were placed in broth and incubated, bacterial
growth occurred. This indicated that while true biofilms may
not have been present, viable microorganisms were adhering
to the stainless-steel surfaces.
Other researchers have observed various levels of
adherent microorganisms in other food-processing opera-
tions. Holah et al. (6) observed surface levels greater than
103cells/cm2on samples taken in processing plants produc-
ing baked beans, egg glaze, fish and buttermilk. The
traditional definition of the term biofilm suggests a surface
TABLE 1. Location of cast-iron and stainless-steel chips placed in
meat-processing plants. Cast-iron chips were used only in drains
Site
Plant no. Description
A
1Floor drain of a beef-slaughter operation
2Floor drain of a beef-slaughter operation
3 Under a sink
adjacenttoacuttingstationinaslaughter operation
4Next to a sink adjacent to a cutting station in a
slaughter operation
B
Drain in a refrigerated warehouse of a hamburger
operation (low flow volume
u)
2Drain in a room where hamburger patties were being
formed (high flow volume
U)
3The horizontal surface of a hamburger patty forming
maching
4The leg of a table where beef jerky and cheese were
packaged
C
Drain in a room where hamburger patties were being
formed (high flow volume
U)
2On a horizontal surface of a hamburger-patty-forming
machine
3On a horizontal surface of a steak-packaging machine
D
1Drain in a sausage-stuffing room (high flow volume
U)
2A vertical surface of a sausage-stuffing machine
3A vertical surface of a sausage-stuffing machine
4Drain in a cooked-ham-packaging room (low flow
volume
U)
5A vertical surface on a cooked-ham-cutting table
6A vertical surface on a cooked-ham-cutting table
U
The volume of water flowing down the drains was not deter-
mined. Low flow is used to describe areas where water was not
continuously flowing to the drain; high flow, where there was a
continuous supply of water to the drain.
which has been colonized by the microorganisms. A level of
103cells/cm2,where only individual cells are observed, does
not fit the traditional definition. However, these adherent
microorganisms still could contribute to the contamination
of food products. The term biotransfer potential may provide
a better definition to use where lower levels of cells are
present (7). Biotransfer potential can be used to describe
microenvironments that do not fit the classic description of a
biofilm, but do pose a risk of contamination to foods which
may contact that microenvironment. In cases such as the
meat plants in the present study, single adherent microorgan-
isms could be dislodged by high-pressure spraying during or
after a production run. If the dislodged bacteria became
airborne, they could contaminate the food.
The data in Table 2 suggests that floor drains with both
high-flow and low-flow volumes may contain biofilms at
significant levels. Spurlock and Zottola (14) showed that
L.
monocytogenes could survive in 0.1 % reconstituted nonfat
dry milk in a drain for 28 days. The significance of biofilms
in floor drains is an example of putting the concept of
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ADHERENT GRAM-NEGATIVE SPECIES FROM MEAT-PROCESSING PLANTS 1137
TABLE 3. Identification microorganisms isolated from stainless-
steel and cast-iron chips placed infour meat-processing plants
a
HP, hamburger patty.
b
TNTC, too numerous to count.
eDifficult to stain due to a layer of fat from the ground beef.
TABLE 2. Morphology and numbers of cells on stainless-steel and
cast-iron chips placed in four meat-processing plants
Time in Cell
Chip location place morphology Cells/cm2
Floor drain (low flow) 4h Rods, cocci 7.5 X 1()4
Floor drain (high flow) 4h Rods, cocci TNTCb
Adjacent to beef jerky line 4h <5.0 X 103
Adjacent to
Hpa
line 4h Pockets of rods
and cocci
Floor drain (low flow) 7 days <5.0 X 103
Floor drain (high flow) 7 days Rods, cocci TNTCb
Adjacent to beef jerky line 7 days <5.0 X 103
Adjacent to slaughter 8h <5.0 X 103
Floor drain (high flow) 8h <5.0 X 103
Floor drain (low flow) I h Rods, cocci 1.0 X 104
Floor drain (low flow) 4h Rods, cocci 1.0 X 104
Adjacent to
Hpa
machine I h Rods, cocci
e
Adjacent to
Hpa
machine 4h Rods, coci
e
biotransfer potential into practice. Biotransfer could occur
when an aerosol is formed, and the microorganisms can be
carried to other locations. Aerosols from floor drains have
been cited as a potential source of contamination in food-
processing facilities (4). An additional study by Spurlock
and Zottola (15) showed that L. monocytogenes could
survive in aerosols generated in 0.1 %reconstituted nonfat
dry milk for over 8 h.
The species of microorganisms isolated from the chips
are shown in Table 3. Only gram-negative isolates were
identified. Frequently isolated microorganisms were species
of Pseudomonas and Klebsiella. These are the microorgan-
isms which are commonly found in meat-processing environ-
ments (18).
Pseudomonas species have been studied extensively
and are known to adhere to food-contact surfaces (5, 13). In
addition, P.fragi can act as a primary colonizing microorgan-
ism, facilitating the adherence of L. monocytogenes to a
glass surface (12).
I. Chung, K., J. S. Dickson, and J. D. Crouse. 1989. Attachment and
proliferation of bacteria on meat. J. Food Prot. 52:173-177.
2. Dankert, J., A. H. Hogt, and J. Feijen. 1986. Biomedical polymers:
bacterial adhesion, colonization and identification. Crit. Rev. Biocom-
pat. 2:219-301.
3. Farber, J. M., and E. S. Idziak. 1984. Attachment of psychrotrophic
meat spoilage bacteria to muscle surfaces. J. Food Prot. 47:92-95.
4. Heldman, D. R., T. I. Hendrick, and C. W. Hal!. 1965. Sources of
air-bourne microorganisms in food processing areas-drains. J. Milk
Food Techno!.28:41-45.
5. Herald, P. J., and E. A. Zottola. 1989. Effect of various agents upon the
attachment of Pseudomonas fragi to stainless stee!. J. Food Sci.
54:461-464.
6. Holah, J. T., R. P. Betts, and R. H. Thorpe. 1989. The use of
epifluorescence microscopy to determine surface hygiene. Int. Biode-
ter.25:147-153.
7. Hood, S. K., and E. A. Zottola. 1995. Biofilms in food processing.
Food Contr. 6:9-18.
8. Nelson, J. H. 1990. Where are Listeria likely to be found in dairy
plants. Dairy Food Environ. Sanit. 10:344-345.
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10. Pontefract, R. D. 1991.Bacterial adherence: its consequences in food
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II. Ronner, A. B., and A. C. L. Wong. 1993. Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and Buna-n rubber. J. Food Prot.
56:750-758.
12. Sasahara, K.
c.,
and E. A. Zottola. 1993. Biofilm formation by
Listeria monocytogenes utilizes a primary colonizing microorganism
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Published as paper no. 22578 of the contribution series of the
Minnesota Agricultural Experiment Station conducted under project 18-56
supported by Hatch Funds and funds from the National Livestock and Meat
Board, Chicago, IL.
REFERENCES
ACKNOWLEDGMENTS
The mucus-like colony morphology of the Pseudomo-
nas and Klebsiella species isolated from the meat plants in
our study suggests that these microorganisms produce
extracellular material that could allow the entrapment of
other microorganisms. The existence of microorganisms that
may be primary colonizers in meat plants suggests that these
areas may have the potential to harbor pathogenic species.
Gram-positive microorganisms were also found but
were not identified. When selective enrichment was utilized,
no Salmonella or Listeria species were found. However, the
surface area tested in this study was small compared to the
total food-contact area in the processing environment. Other
studies have shown that Listeria spp. can be isolated from
equipment, floors, and drains in meat-processing plants by
using swabbing techniques (16).
It also must be noted that when several stainless-steel
chips removed from the processing environments were
incubated in TSB, growth did not occur. There are several
reasons that this may have occurred, including increased
awareness of cleaning procedures by personnel because a
study was being done. However it does indicate that utilizing
proper cleaning and sanitizing programs may adequately
eliminate problematic microorganisms.
Location
Adjacent to
HPa
machine
Floor drain (high flow)
Floor drain (low flow)
Floor drain (high flow)
Floor drain (high flow)
Floor drain (high flow)
Floor drain (low flow)
Fllar drain (low flow)
Floor drain (low flow)
Adjacent to
HPa
machine
Adjacent to
HPa
machine
Klebsiella oxytoca
Pseudomonas sp.
Pseudomonas sp.
C.freundii
Aeromonas sp.
Pseudomonas sp.
Klebsiella sp.
Klebsiella sp.
Hafnia alvei
p. fiuorescens
p. fiuorescens
Microorganism
a
HP, hamburger patty.
HHP-6-15-2
HGB-6-15-1
HW-72-15-1
HGB2-3.5-15-1
HGB2-19.5-15-1
HGB-2-19.5-15-3
HW2-25-15-1
HW2-19.5-37-2
RMHPI-24-37-1
RMHP4-64-15-1
RMHPI-72-15-2
Code
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1138 HOOD AND ZOTIOLA
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