PREVALENCE AND CHARACTERISTICS OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN OHIO DAIRY HERDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0227882
• Project Start Date: Sep 20, 2011
• Project End Date: Sep 19, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY - VET MED
1900 COFFEY ROAD, 127L VMAB
COLUMBUS,OH 43210

Performing Department
Preventive Medicine

Non Technical Summary
Staphylococcus aureus (SA) is the most common contagious mastitis pathogen in the US dairies and its control has for years been largely based on antimicrobial treatments. A highly resistant strain of SA (so called methicillin-resistant SA, MRSA) has become a great public health concern due to its zoonotic nature and resistance to most treatments. While SA is commonly found in milk of mastitic cows, data are missing on the occurrence and characteristics of MRSA in US dairies. The objective of this study is to estimate the prevalence of this resistant strain of SA in Ohio dairies and to characterize it to gain better understanding of its epidemiology transmission. This information is crucial for effective control of this organism to improve both animal and human health.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3450	1170	50%
311	3450	1100	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3450 - Milk;

Field Of Science
1100 - Bacteriology; 1170 - Epidemiology;

Keywords

staphylococcus aureus

mastitis

dairy herds

bulk milk tank

mrsa

Goals / Objectives
Methicillin resistant Staphylococcus aureus (MRSA) has emerged as a serious public health concern in the recent decade. While the resistant organism has commonly been isolated from dogs, cats and horses and transfer between animals and humans reported, food animals (mainly pigs and poultry) have only recently been reported as a potential source of MRSA. While the first animal MRSA was reported in a mastitic dairy cow in the 1970's, MRSA has not since been widely studied or reported as a problem in dairy production. S. aureus is the most common contagious mastitis pathogen in US dairy herds and beta-lactam antibiotics are commonly used in mastitis treatments and control. Studies on occurrence of MRSA in US dairies, however, are largely missing. The long-term goal of our research is to identify and develop cost-effective management practices in dairy herds that will promote both animal and public health and enhance sustainability of dairy production. The objective of the proposed study is to estimate the prevalence of and to characterize MRSA isolated from bulk tank milk in Ohio dairy herds. We hypothesize that MRSA can be found in bovine milk, but in low prevalence and will predominantly be of community-origin. This study is significant because without the knowledge on the presence and characteristics of this zoonotic pathogen of great public health concern, it is impossible to develop effective control strategies for its control. The information gained from this study will be important both to human and animal health. We plan to accomplish our overall objective and to test our central hypothesis by pursuing the following specific aims: 1. To assess the patterns of phenotypic antimicrobial resistance of S. aureus isolated from bulk tank milk samples in Ohio dairies. 2. To determine the prevalence of mecA in S. aureus isolates from bulk tank milk. 3. To genotypically characterize S. aureus strains form bulk tank milk to assess their epidemiologic origin. At completion of this study, we expect to have shown that MRSA can be found in US bulk tank milk only in low frequency (<5%). Additionally, we expect that MRSA strain detected in bulk tank milk in the US will be community acquired. This study is significant, because it will be on of the first in the US to evaluate the prevalence and characteristics of MRSA in US dairy herds. This is important, because without this knowledge it is impossible to effectively control this organism which causes most of the contagious and subclinical intramammary infections in US dairy herds and which additionally is a significant human pathogen and cause for great public concerns. The results from this study will help us further design intervention studies in dairies to evaluate their efficacy in controlling SA and particularly MRSA in them. We have included a graduate student on this study. It will serve as part of her doctoral research and she will be involved in all aspects of the study.

Project Methods
Aim 1: To assess the patterns of phenotypic antimicrobial resistance of S. aureus isolated from bulk tank milk samples in Ohio dairies. The working hypothesis is that beta-lactam resistance will be detected frequently in bovine S. aureus isolates. We will achieve this aim by performing antimicrobial susceptibility testing by applying disc diffusion method. Beta-lactamase production of the isolates will also be determined using cefinase discs. Aim 2: To determine the prevalence of mecA in S. aureus isolates from bulk tank milk. The working hypothesis for this aim is that mecA can be detected in S. aureus isolates from bulk tank milk, but in low prevalence. We will achieve this aim by applying a duplex PCR to detect mecA gen (which codes for methicillin resistance) and nuc gene (to confirm the identify of the isolates as S. aureus). Aim 3: To genotypically characterize S. aureus strains form bulk tank milk to assess their epidemiologic origin. The working hypothesis for this aim is that MRSA strains in bulk tank milk will predominantly represent community-acquired strains. We will achieve this aim by characterizing mecA and S. aureus isolates with PFGE (pulsed-field electrophoresis) and SCCmec typing and by performing PFGE also on phenotypically oxacillin-resistant and beta-lactamase positive isolates and on an equal number of a randomly selected oxacillin susceptible strains. Data analysis: Aims 1 and 2 is mostly descriptive. The proportion of isolates resistant intermediate and susceptible for each antimicrobial drug based on disc diffusion test and CLSI guidelines will be calculated. Similarly, the proportion of beta-lactamase producing isolates based on positive cefinase test will be calculated. For the statistical analysis, any isolate that is intermediate or resistant to ampicillin, cephalothin, ceftiofur, cefoxitin, oxacillin or penicillin in disc diffusion test will be considered positive for beta-lactamase production. Agreement between disc diffusion and cefinase tests regarding beta-lactamase production will be estimated using McNemar's chi-square test. Similarly, proportion of isolates carrying mecA gene will be calculated and compared to the proportion of isolates resistant to oxacillin phenotypically. Data analysis for aim 3 will be performed using Bionumerics software (Applied Maths, Kortrijik, Belgium) using "different brands" algorithm for clustering and the Unweighted Pair Group for Arithmetic Means (UPGMA) tree building approach with optimization of 1% and position tolerance of 1.5% (commonly used parameters that may be subject to modification). Visual inspection of the patterns will also be performed as a final step for analysis. Phenotypic and epidemiologic characteristics relevant to the analysis including antimicrobial resistance patterns will be included (information of the basic herd characteristics and their mastitis control practices are available for us through our mailed survey among the Ohio dairy producers). We will use known out-group strains (Staphylococci strains from dogs) in the fingerprint to assist in determining the threshold cut-off genetic similarity for clustering genotypic clonal groups.

Progress 09/20/11 to 09/19/12

Outputs
Target Audience:Other experts in the field, dairy veterinarians, dairy producers, extension and outreach. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?PI published one manuscript relevant to dairy producers and scientists and rpesented one paper at an international scientific meeting. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Methods and Results A mailed questionnaire was sent to 780 Ohio dairies to survey them regarding their mastitis control procedures, general herd characteristics and to estimate prevalence of S. aureus in BTM in OH. A total of 384 questionnaires were returned (49% response rate) and of these, 307 herds also allowed their bulk tank milk (BTM) to be cultured for detection of S. aureus. Prevalence of S. aureus in OH dairies was 69%. Phenotypic antimicrobial testing, using Kirby-Bauer disc diffusion method as well as screening for the presence of mecA gene in S. aureus isolates from different herds was performed. Majority (72%) of the isolates (one isolate per herd) were susceptible to all 11 tested antimicrobials. Most commonly resistance was found against ampicillin (13%) and penicillin (13%), followed by resistance to streptomycin (7%) and tetracycline (6%). MRSA isolates were found from BTM samples only in one herd, and despite the carriage of the mecA gene, this strain was phenotypically susceptible to oxacillin and genotypically had 86% similarity to USA200 strain which is often associated with healthcare related MRSA strains. Outcomes/Impact: The results of the study indicate that mecA gene can be found from S. aureus isolates in US bulk tank milk, but in a very low frequency and these isolates can be phenotypically susceptible to oxacillin. Even though the overall resistance levels in S. aureus isolates from bulk tank milk were relatively low, continuous monitoring of antimicrobial susceptibility of S. aureus in dairy farms is recommended. This will help in choosing the most effective antimicrobial drugs for controlling and treatment of this pathogen in dairy herds.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: da Costa, LC, Rajala-Schultz, PJ, Hoet, A, Van Balen, J, Schuenemann GM 2012: Screening Bulk Tank Milk for Methicillin Resistant Staphlylococcus aureus in Ohio Dairy Farms. 13th Conference of International Society of Veterinary Epidemiology and Economics (ISVEE XIII), Maastricht, The Netherlands, August 20-24, 2012.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: da Costa, LB, Rajala-Schultz, PJ, Schuenemann, GM 2015: Management practices associated with presence of Staphylococcus aureus in bulk tank milk from Ohio dairy herds. J. Dairy Sci. 99, 1364-73

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Staphylococcus aureus (SA) is the most common contagious mastitis pathogen in the US dairies and its control has for years at least partially been based on antimicrobial treatments. A highly resistant strain of SA (so called methicillin-resistant SA, MRSA) has become a great public health concern due to its zoonotic nature and resistance to most treatments. MRSA has been isolated from numerous animal species, including dogs, cats, horses, swine and poultry. While SA is commonly found in milk of mastitic cows, data are missing on the occurrence and characteristics of MRSA in US dairies. The objective of this study is to estimate the prevalence of this resistant strain of SA in OH dairies and to characterize it to gain better understanding of its epidemiology and transmission. This information is crucial for effective control of this organism to improve both animal and human health. The specific aims of this project are 1) to assess the patterns of phenotypic antimicrobial resistance of S. aureus isolated from bulk tank milk samples in OH dairies; 2) to determine the prevalence of mecA (a gene encoding for methicillin resistance) in S. aureus isolates from bulk tank milk; 3) to genotypically characterize S. aureus strains from bulk tank milk to assess their epidemiologic origin. Our central hypothesis is that MRSA can be found in bulk tank milk, but in very low prevalence. Progress: A mailed questionnaire was sent to almost 800 Ohio dairies to survey them regarding their mastitis control procedures and general herd characteristics. A total of 384 questionnaires were returned (49.2% response rate) and of these, 307 herds also allowed their bulk tank milk (BTM) to be cultured for detection of S. aureus. Prevalence of S. aureus in OH dairies was 67.7%. Phenotypic antimicrobial testing, using Kirby-Bauer disc diffusion method and test for beta-lactamase production as well as screening for the presence of mecA gene in S. aureus isolates from different herds and is currently on-going. Up to date, several tested isolates have been positive for beta-lactamase production, but only one isolate carrying mecA gene has been detected so far. In the light of a recent study from the UK reporting a finding of a novel mecA homologue (LGA251) from bulk tank milk in dairy herds and from humans, further research is warranted if and when phenotypic antimicrobial susceptibility testing in the current study suggests the presence of methicillin resistant S. aureus, despite the lack of mecA gene. PARTICIPANTS: Dr. Rajala-Schultz is a faculty member in the Department of Veterinary Preventive Medicine. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
At the completion of this study, we expect to have shown that MRSA can be found in US bulk tank milk only in low frequency. This study is significant, because it will be one of the first in the US to evaluate the prevalence and characteristics of MRSA in US dairy herds. This is important, because without this knowledge it is impossible to effectively control this organism which causes most of the contagious and subclinical intramammary infections in US dairy herds and which additionally is a significant human pathogen and a cause for great public health concerns. The results from this study will help us further design intervention studies in dairies to evaluate the efficacy in controlling SA and particularly MRSA in them.

Publications

• da Costa, LC, Rajala-Schultz, PJ, Hoet, A, Van Balen, J, Schuenemann GM 2012: Screening Bulk Tank Milk for Methicillin Resistant Staphlylococcus aureus in Ohio Dairy Farms. 13th Conference of International Society of Veterinary Epidemiology and Economics (ISVEE XIII), Maastricht, The Nether lands, August 20-24, 2012 (submitted).