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Reta Duguma Abdi,1 Barbara Erin Gillespie,1 Jacqueline Vaughn,1 Caitlin Merrill,1 Susan Ivory Headrick,1
Desta Beyene Ensermu,1 Doris Helen D’Souza,2 Getahun Ejeta Agga,3 Raul Antonio Almeida,1
Stephen Paul Oliver,1 and Oudessa Kerro Dego1
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Abstract
Staphylococcus aureus is a frequent and major contagious mastitis bacterial pathogen. The antibiotic treatment
cure rates vary considerably from 4% to 92%. Staphylococcus aureus readily becomes resistant to antibiotics,
resulting in persistent noncurable intramammary infection that usually results in culling of infected animals.
Because of its notorious ability to acquire resistance to the commonly used as well as last resort antimicrobials
such as methicillin and vancomycin and the development of multidrug-resistant strains, antimicrobial resistance
(AMR) in S. aureus is of paramount importance in human medicine. The objective of this study was to evaluate
the prevalence of AMR and genetic diversity of S. aureus isolates from milk of dairy cattle. Staphylococcus aureus
isolates (n = 239) from 33 dairy farms in Tennessee were tested against 10 antimicrobials by broth microdilution
method using the Sensititer system. Genetic diversity of resistant isolates was evaluated by pulsed-field gel
electrophoresis (PFGE). Overall, AMR of the S. aureus isolates varied from as low as 1.3% for ceftiofur to as high
as 25% for sulfadimethoxine. Out of 239 S. aureus isolates, 82 (34.3%) of them were resistant to at least 1 of the
10 antimicrobials. The AMR isolates belonged to two major PFGE types, indicating the presence of dominant
clonal patterns among the resistant isolates. In general, there was a variation of prevalence of AMR within and
among farms over time, with an increasing trend in tetracycline resistance. Judicious use of antimicrobials in dairy
cattle farms can reduce the development of antimicrobial-resistant S. aureus.
Introduction Data from the 2007 USDA National Animal Health Mon-
Departments of 1Animal Science and 2Food Science, The University of Tennessee, Knoxville, Tennessee.
3
Food Animal Environmental Systems Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Bowling Green,
Kentucky.
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. Department of Agriculture. USDA is an equal opportunity provider and employer.
1
2 ABDI ET AL.
quarters are infected (Mellenberger and Keirk, 2001). Dairy of this study were (1) to evaluate the AMR of S. aureus
farmers rely on antimicrobials for the treatment and pre- isolates obtained from dairy cows and (2) to analyze genetic
vention of mastitis (Saini et al., 2012a, 2012b). diversity of resistant isolates to determine the scope of dis-
The prophylactic use of antibiotics as a blanket dry cow tribution of AMR traits among S. aureus isolates.
therapy exposes large number of healthy animals to antibi-
otics, thus increasing selective pressure on commensal bac-
Materials and Methods
teria. More than 90% of U.S. dairy farms use intramammary
antibiotics at dry off (USDA APHIS, 2008a) and 80% of Bacterial isolation, identification,
them treat all cows on the farm (Wagner and Erskine, 2013). and susceptibility testing
Antibiotics are administered to dairy cows through two pri-
A total of 239 S. aureus isolates were isolated from 187
mary routes: intramuscular and intramammary (USDA
dairy cows with mastitis from 33 dairy farms in Tennessee
APHIS, 2009), which can exert selective pressure both to the
during 2004–2016. The isolates were identified and stored by
mammary pathogenic bacteria and nonmammary commensal
Tennessee Quality Milk Laboratory at the University of
bacteria in the gastrointestinal tract. Nonprudent use of an-
Tennessee, Department of Animal Science, Knoxville, TN.
timicrobials in dairy farms increases selection pressure on
S. aureus isolates were tested for their sensitivity to a panel of
mastitis-causing and commensal bacteria (Barbosa and Levy,
10 commonly used antimicrobials for the treatment of mas-
2000; Barber et al., 2003). Once resistant bacteria emerge on
titis. All milk samples were collected and microbiologically
the farm, the resistant strains persistently circulate on the farm
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Table 1. Antibiotic Concentration Ranges on the Sensititer Mastitis Panel and Interpretive Criteria
Used for Testing Staphylococcus aureus Isolates Obtained from Dairy Cattle Farms
Concentrations Susceptible breakpoint
Antimicrobial class Antimicrobial agent used (mg/mL) MIC (mg/mL)
Beta-lactam Ampicillin 0.12–8 £0.25
3rd generation cephalosporin Ceftiofur 0.5–4 £2
1st generation cephalosporin Cephalothin 2–16 £8
Macrolide Erythromycin 0.25–4 £0.5
Beta-lactam Penicillin 0.12–8 £0.12
Beta-lactam Oxacillin 2–4 £2
Beta-lactam Penicillin/novobiocin 8/16–1/2 £1/2
Lincosamide Pirlimycin 0.5–4 £2
Sulfonamide Sulfadimethoxine 32–256 £256
Tetracycline Tetracycline 1–8 £4
The MICs were interpreted according to CLSI (2015) M31-A3.
MICs, minimum inhibitory concentrations.
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the control S. aureus strain NCTC 8325 was loaded into the Results
1st, 8th, and 15th combs and incubated at room temperature Prevalence of antimicrobial-resistant S. aureus
for 20 min. The comb was placed in the gel-casting platform
and the 1% SeaKem agarose was poured into it and kept at The prevalence of AMR among the 239 isolates tested is
room temperature for 20 min until solidified. The gel elec- shown in Figure 1. In total, 82 (34.3%) isolates were resistant
trophoresis was conducted using the CHEF Mapper at initial to one or more of the antimicrobials. Overall, AMR among S.
switch of 5 s, final switch of 40 s, and running time for 21 h at aureus isolates varied from as low as 1.3% for ceftiofur to as
200 V (6 V/cm) at temperature of 14C using ramp angle of high as 25% for sulfadimethoxine. The prevalence of AMR
120. The gel was stained by ethidium bromide (1.25 lg per was widely distributed throughout the farms at a varying
mL of water; Invitrogen, Carlsbad, CA) for 25 min and wa- proportion for different antimicrobials tested. About two-
shed twice for 30 min with fresh distilled water. Images were third of the farms had sulfadimethoxine-resistant isolates, one-
taken by ChemiDoc Touch Imaging System (Bio-Rad) and third had penicillin and erythromycin, a quarter had ampicillin,
exported to PulseNet by Image Lab 5.2.1 Software (Bio-Rad) penicillin–novobiocin, and one-fifth had tetracycline-resistant
and saved as a TIFF file. isolates (Fig. 2). Seven (21.2%) out of the 33 farms did not
have AMR S. aureus isolates, whereas 26 (78.8%) of the farms
Data analysis and interpretation had at least one S. aureus isolate resistant to one or more drugs.
Sulfadimethoxine-resistant S. aureus were the most widely
S. aureus isolates were classified as susceptible or resistant
according to the interpretative criteria (CLSI, 2015) of the
MIC values (Table 1). Intermediate values were considered
resistant. The prevalence of AMR isolates was compared in
two time intervals (2004–2011 and 2012–2016).
The TIFF images of PFGE were imported and analyzed
using GelCompar II software version 6.6 (Applied Maths,
Kortrijk, Belgium). The intra- and intergel PFGE runs were
normalized using control S. aureus strain NCTC8325. The
bands ranging between 10 and 674 kb were used for analysis.
The band patterns of the isolates were used for grouping as
follows: (1) same strains (no band difference), (2) closely
related strains (differed by two to three bands), (3) possibly
related strains (differed by four to six bands), and (4) unre-
lated strains (differed by seven or more bands). PFGE types
and subtypes were defined by groups formed at seven or
more band differences (to define PFGE types) and four to
six bands differences (to define PFGE subtypes) (Tenover FIG. 1. Prevalence of antimicrobial-resistant Staphylo-
et al., 1995). For band pattern comparisons within and be- coccus aureus isolates against commonly used antimicro-
tween different gels for isolates, the following settings were bials in dairy cattle. Out of 239 S. aureus isolates, the
prevalence of resistance to ampicillin (AMP) was 4.6%
used: optimization of 0.5% and position tolerance of 1.25%.
(n = 11), penicillin (PEN) 6.3% (n = 15), erythromycin
PFGE types and subtypes were defined by group differed (ERY) 5.4% (n = 13), oxacillin +2% NaCl (OXA+) 2.9%
by seven or more bands and four to six bands similarity (n = 7), pirlimycin (PIRL) 2.5% (n = 6), penicillin/novobio-
cutoffs, respectively, on a dendrogram constructed by the cin (P/N) 4.6% (n = 11), tetracycline (TET) 4.2% (n = 10),
unweighted-pair group matching algorithm (UPGMA) (Faria cephalothin (CEP) 2.1% (n = 5), ceftiofur (XNL) 1.3%
et al., 2008). (n = 3), and sulfadimethoxine (SDM) 25% (n = 60).
4 ABDI ET AL.
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of 4 (7.1%) of 56 isolates with one PFGE type were detected types, whereas erythromycin-resistant isolates grouped into
in three farms (Fig. 4 and Table 3). The abundance and dis- G and I types (Table 2). Changes of PFGE profiles over time
tribution patterns of the nine PFGE types in 11 farms showed indicated that some PFGE types were farm specific such as
hierarchy of genetic structure. Of the nine PFGE types, types PFGE type B that was persistently endemic over time in farm
G and I were abundant and widely distributed in 7 of 11 4, whereas some PFGE types such as G and I were distributed
farms. Both types G and I comprised dominant strains ac- overtime between different farms. Interestingly, some sub-
counting for 42 (75%) of the 56 isolates. Similarly, out of 11 types within PFGE type I such as I-iv and I-x were persis-
farms, 3 farms had type F and 2 farms had type D. A unique tently endemic over time in specific farms, particularly in
single PFGE type was observed in three separate farms, farms 3 and 5, respectively.
PFGE types A and B in farm 4, type C in farm 8, and types E
and H in farm 2. Four different PFGE types consisting of D,
Discussion
E, H, and I were detected in farm 2, whereas PFGE types
comprising C, F, G, and I were detected in farm 8. No farm Evaluation of S. aureus isolated from dairy cows for sen-
had more than four PFGE types. sitivity to antimicrobials showed that 34.3% of the isolates
Certain PFGE types were found to be associated with were resistant to at least one antimicrobial and the majority
AMR particularly to two PFGE types, that is, I followed by (65.7%) of the isolates was pansusceptible. Of the AMR
G. An isolate with single PFGE type was found resistant to isolates (n = 82), 74.4% showed resistance only to one anti-
multiple antimicrobials in this study. In this regard, those microbial as compared with 13.4% resistance to two anti-
from both G and I types were linked with seven AMR phe- microbials and 12.2% MDR (resistance to more than three
notypes. The sulfadimethoxine-resistant isolates showed se- antimicrobials). The resistant isolates showed 21 different
ven PFGE types, that is, B, D, E, F, G, H, and I, indicating resistance patterns. These findings indicated that about a third
multiple PFGE types with a single AMR pattern. of the S. aureus isolates were AMR and MDR was not ap-
Tetracycline-resistant isolates showed F, G, and I PFGE parently widespread. Even though it was not possible to
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FIG. 4. PFGE types of antimicrobial-resistant Staphylococcus aureus isolates from 33 dairy cattle farms in Tennessee. A
tree calculation (dendrogram) of genetic diversity was constructed using the UPGMA clustering method that is based on
average number of different bands of SmaI digested chromosomal DNA to evaluate PFGE patterns of 56 antimicrobial-
resistant S. aureus isolates. The S. aureus strain NTTC 8325 was used as control for the experimental variations between
duplicate experiments (intra-and intergel variation). Numbers under Staph PFGE indicate band numbers, numbers under
farm indicate dairy farm ID, letters under PFGE indicate PFGE types, and Roman numbers indicate PFGE subtypes. PFGE,
pulsed-field gel electrophoresis; UPGMA, unweighted-pair group matching algorithm.
6
AMR OF STAPHYLOCOCCUS AUREUS FROM DAIRY COWS 7
obtain accurate information on antibiotics use patterns in the (ampicillin, penicillin, ceftiofur, and cephaprin) and linco-
dairy farms included in this study, occurrence of high-level samide (pirlimycin), the two antimicrobial classes most
resistance to single antibiotics and low MDR may indicate commonly used for the treatment of mastitis in the United
variations in the antibiotic selection and use practices. States, was generally low. For the b-lactams, it ranged from
Similar observations were made from Wisconsin dairy cattle 1.3% (ceftiofur) and 2.5% (pirlimycin) to 6.3% (penicillin).
in which 25% (n = 116) of S. aureus isolates were AMR, and These findings are similar to reports from other parts of the
0.9% were MDR (Oliveira et al., 2012). However, in that United States (Pol and Ruegg, 2007; Oliveira et al., 2012;
study only five different resistance patterns were reported, Oliveira and Ruegg, 2014; Ruegg et al., 2015). Resistance
suggesting that AMR in the S. aureus tested in our study had to tetracycline (4.2%), erythromycin (5.4%), penicillin–
more diversity in their resistance profile. novobiocin (4.6%), and oxacillin +2% NaCl (2.9%) was
A relatively high prevalence (25%) of sulfadimethoxine generally comparable with reports from Wisconsin (Oliveira
resistance was observed among the isolates, which agree with et al., 2012), but lower than a more recent study in Wisconsin
a previous study (Pol and Ruegg, 2007). Higher prevalence (Ruegg et al., 2015). Oxacillin +2% NaCl is not prescribed
(49%) was also reported from Wisconsin (Sato et al., 2004). for veterinary use, particularly for mastitis in the United
Sulfadimethoxine is approved in the United States for the States. The 2.9% oxacillin resistance observed in this study is
treatment of septicemia caused by coliform mastitis, respi- comparable with the 2.1% reported for Minnesota dairy herds
ratory pathogens, and necrotic pododermatitis (Oliveira and (Haran et al., 2012). Some authors suggest that oxacillin re-
Ruegg, 2014). We did not evaluate antimicrobial usage pat- sistance is indicative of MRSA (Aarestrup and Schwarz,
terns in each farm, particularly sulfametoxide in treating 2006) with potential public health implication. In this study,
mastitis (Oliveira and Ruegg, 2014). The high sulfadi- all the oxacillin-resistant isolates were MDR (Table 2),
methoxine resistance prevalence requires further study to supporting the idea that methicillin/oxacillin resistance is
elucidate the level of sulfadimethoxine use in dairy cattle associated with multidrug-resistant S. aureus (Chambers and
production for treatments other than mastitis, and its associ- Deleo, 2009).
ation with sulfadimethoxine-resistant S. aureus. Interest- AMR in S. aureus isolates detected in this study was
ingly, resistance to antimicrobials that belong to b-lactams broadly distributed across the 33 farms. Anderson et al.
8 ABDI ET AL.
(2006) reported a 12.5% (3 of 24 farms) prevalence in North resistant isolates tested, two lineages coexisted in four farms,
Carolina and Virginia, which was lower than that in this three lineages coexisted in two farms, and four lineages co-
study. In general, specific antibiotic resistances were widely existed in another two farms, accounting for 39.3% (22/56),
distributed (Fig. 2). Sulfadimethoxine resistance was the 25% (14/56), and 28.6% (16/56) of the resistant isolates
most widespread (occurred in 63.6% of the farms), and cef- tested, respectively. This indicates that majority (92.9%) of
tiofur resistance was the least widespread (6% of the farms). the resistant isolates coexisted in multiple farms. Our findings
These results revealed that the percentage of farms that had and other reports from Pennsylvania and Ohio (Kapur et al.,
resistant isolates varied among the 10 antimicrobials tested. It 1995) showed that although few lineages dominate, the
is possible that different dairy farms use different antimi- S. aureus population in a particular farm/herd can be multi-
crobials and hence the percentage of resistant isolates was clonal. No farm was found to have more than four lineage
expected to vary with farm and type of commonly used an- types. Results of this study showed that the types of lineages
tibiotics (Saini et al., 2012b; Oliveira and Ruegg, 2014). involved in coexistence varied with farm. Coexistence of the
The prevalence of antimicrobial-resistant S. aureus heterogeneous lineages that accounted for AMR of 41.1%
showed some variations over the years. With the exception of within a specific farm has been reported from Canadian dairy
tetracycline resistance, which increased during the 2012– herds (Sabour et al., 2004). Existence of a single lineage was
2016 period, resistance to all other antibiotics declined from detected in three farms, accounting for AMR of 7.1% (4/56).
2004–2011 to 2012–2016 (Fig. 3). A decreasing trend for Single lineage existence per farm accounting for 58.6% of the
penicillin from 49% in 1994 to 30% in 2001 (Makovec and isolates in Canada (Sabour et al., 2004) and in Pacific regions
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Ruegg, 2003) to 20% in 2015 (Ruegg et al., 2015) was re- (Ritchie et al., 2014) was reported.
ported in Wisconsin. Over the 6-year period, from mid-1990 Interestingly, the phenotypic assay discriminated resistant
to early 2000, S. aureus isolates from bovine mastitis showed S. aureus isolates into 21 AMR patterns, whereas PFGE
either a decreasing trend or did not change their AMR level displayed 46 patterns. This indicated a better discriminatory
(Erskine et al., 2002). Despite the fact that in this study we power of PFGE than the phenotypic AMR assay method
did not evaluate the 33 farms over time, the AMR profile of (Zadoks et al., 2002; McDougal et al., 2003; Hallin et al.,
isolates in recent years seems lower than in previous years. 2007). Our results showed that the majority of the AMR
These could be due to several reasons, including (1) rising patterns were linked to a few PFGE types, particularly G and
awareness of farmers about prudent use of antimicrobials, (2) I. The G and I PFGE types were linked to multiple (seven)
aggressive culling of chronically infected cows, showing no AMR patterns, thus indicating that these are the major line-
response to antibiotic treatments, (3) changes in treatment ages responsible for AMR in the study area. Similarly, reports
protocols and intensity of drug use in dairy farms, (4) in- from different geographical areas indicated that frequently
creasing regulation and availability of guidelines on antimi- one or a few clones of S. aureus are responsible for resistance
crobial usage from FDA, and continuous monitoring of milk to antimicrobials (Anderson et al., 2006; Chambers and
quality standard from milk buyers and dairy farmers associ- Deleo, 2009; Sakwinska et al., 2011). In contrast, majority of
ation. Despite a general trend of reduction in AMR for most the S. aureus genotypes detected in this study seem to acquire
antibiotics over recent years, we noticed that resistance to resistance to sulfadimethoxine. Sulfadimethoxine resistance
tetracycline increased during 2012–2016 than during the was widely disseminated in seven PFGE types (B, D, E, F, G,
previous years. This might be due to increased usage of tet- H, and I) of the nine lineages detected, indicating multiple
racycline as the most preferred antibiotic to treat other health lineages had resistance to a single antimicrobial drug. Tet-
problems of dairy cows such as lameness, pneumonia, and racycline resistance was detected in F, G, and I PFGE types
reproductive infections (USDA APHIS, 2008a; Oliver et al., and erythromycin resistance was observed in G and I PFGE
2011). Therefore, variation in the prevalence of AMR among types.
farms and over years emphasizes the need for continuous
monitoring of AMR at the farm level overtime to determine Conclusion
effective mitigation measures.
The prevalence of antimicrobial-resistant S. aureus iso-
Among the 56 AMR S. aureus isolates tested, 46 PFGE
lates from dairy cows varied with farms, time, and types of
patterns were observed that were clustered into 9 major PFGE
antimicrobials tested. With the exception of sulfadimethox-
types, suggesting genetic diversity of the isolates. Studies
ine, resistance to most antimicrobials was low and there was
from different parts of the world indicate that S. aureus iso-
gradual decline in resistance to commonly used antimicro-
lates obtained from dairy cows tend to be genetically diverse
bials such as cephalothin, ceftiofur, and pirlimycin from
(Kapur et al., 1995; Fitzgerald et al., 1997; Zadoks et al.,
2004–2011 to 2012–2016. AMR S. aureus showed both
2002; Sabour et al., 2004; Anderson et al., 2006; Li et al.,
phenotypic and genotypic diversity, and most AMR patterns
2009). Lineage I that occurred in 55.4% of the resistant iso-
were associated with few PFGE types. Studies are required to
lates (n = 56) was the most prevalent PFGE type followed by
further understand the widespread occurrence of sulfadi-
lineage G that occurred in 19.6% of the resistant isolates.
methoxine and tetracycline resistance in S. aureus in dairy
PFGE types D and F together accounted for 14.2% (7.1%
cattle production systems despite the limited use of these
each) of the 56 isolates tested and the remaining PFGE types
antibiotics for the treatment of bovine mastitis.
(A, B, C, E, and H) were less abundant. The predominance of
PFGE type I may suggest clonal expansion of the isolates
Acknowledgment
with this PFGE type between and within the farms. Previous
reports support this observation (Anderson et al., 2006; This project was funded by the University of Tennessee,
Sakwinska et al., 2011). The PFGE typing showed that College of Veterinary Medicine, Center of Excellence in
multiple lineages coexisted in a particular farm. Of the 56 Livestock Diseases and Human Health (UT CVM-COE).
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