Staphylococcus mastitis, biofilms, and antibiotic resistance: Barriers to milk quality and food safety on artisanal and farmstead cheese producing farms in Vermont

Staphylococcus mastitis, biofilms, and antibiotic resistance: Barriers to milk quality and food safety on artisanal and farmstead cheese producing farms in Vermont

Summary

The objective of this project is to support farmstead cheese producers with improved knowledge on mastitis, milk quality and food safety. The primary objective of this research is to quantify the diversity of Staphylococcus bacteria on farms that make artisan cheese and to describe the sources of potential pathogenic and beneficial members of this genus. Our goal is to identify potential practices that prevent persistence of pathogens while encouraging beneficial microbes. Work on this project was initiated in early November 2014. We finished molecular and phenotypic typing of Staphylococcus bacterial isolates obtained from four farms. We have accomplished genotyping of antimicrobial resistance markers (mecA and blaz), work on in vitro antibiotic resistance assays is still in progress. Biofilm assay analysis has already been done on 1/3 of the isolates. Biofilm genotyping is underway though we were delayed by some troubleshooting we had to do but we anticipate finishing this early 2016. We are on track to complete farm visits, sample collection and laboratory analysis in the final year of this two-year project.

Objectives/Performance Targets

Below are the objectives we intend to achieve in the remaining duration of the project. They have not changed as stipulated in the original proposal
1. To identify the sources of Staphylococcus species on farms that make farmstead cheese.
We will describe the detailed distribution of the CNS species on 5 dairy farms that make artisan or farmstead cheese. Under this objective we will use sequence-based molecular methods to describe the comparative distribution of bacteria isolated from various sources or niches on these farms.
The specific aims of this objective are to:
a. Identify the CNS species that are opportunistic mastitis pathogens and determine their sources on these farms.
b. Identify the CNS species that are normal flora of the udder and teat skin but are not commonly isolated from cases of mastitis, and describe these as a component of the beneficial teat skin commensal bacteria.
c. Identify the sources of CNS species that are known beneficial bacteria in cheese maturation.

Since funds became available; we have successfully completed speciation of 706 bacterial isolates from 4 artisan cheese farms (Table 1). Our target was to sample the fifth farm before the end of the year (2015). We were unable to meet this target because the farmer we had contacted took long to get back to us. We have scheduled to sample the remaining farm early February next year (2016) and we do believe, we will finish speciation of the isolates in the final farm early March 2016.

2. To quantify the association between antibiotic resistances or biofilm formation and the staphylococci species or strains isolated from various sources on dairy farms
The specific aims of this objective are to:
a. Quantify the prevalence of antimicrobial resistance among the various CNS species isolated from different sources on artisan cheese farms.
b. Quantify the prevalence of biofilm forming bacteria among the various CNS species isolated from different sources on artisan cheese farms

Work under this objective was started early this year. We successfully recruited 2 undergraduate students to work on the project in the summer of 2015. The students concentrated on objective 2 and accomplished the genetic PCR screening of antimicrobial resistance gene markers (blaz and mecA) for all the confirmed CNS at species level (706) from the four farms (Table 2). We have finished the in vitro antimicrobial assay analysis of approximately half of the 706 isolates. We anticipate finishing the remaining half plus the isolates from the fifth farm by the end of May 2016. For the in vitro biofilm assays, over 300 isolates have so far been done. Since, we run 30 isolates per 96 well plate, the remaining isolates should be finished by the end of May 2016. The summer undergraduate students also started on screening for biofilm gene markers. The bap primers did not work well on some of the CNS species and this slightly delayed our biofilm genotype analysis. We have redesigned new primer pairs and we hope to finish screening the remaining isolates by the end of April 2016. With biofilm assay, we are running isolates on two plate types (treated and untreated). This modification was not stipulated in the proposal. Our work on S. aureus biofilm analysis showed differential strain biofilm formation on the two plate surfaces with the most dominant strains forming much biofilms on untreated plates vs treated plates. This led us to explore this too in the CNS, we hypothesize that; this might be species or strain specific in the CNS.

• Figure 1A,B,C and D. CNS Species distribution by source.
• Table 1 and Table 2

Accomplishments/Milestones

We have demonstrated the proposed PCR-based species typing system is able to discriminate among the staphylococcus species isolated from these farms and we have established laboratory methods and protocols. This project is progressing as expected and we anticipate completing the sample collection of the remaining farm in February 2016. The two undergraduate students who worked on this project over the summer of 2015 contributed immensely to completion of some research objectives in year 1 of the project. In final year (2016), we anticipate completing laboratory, statistical analyses and outreach objectives. I had an opportunity for the invitation to the “across the fence TV show” where I briefly talked about this research and its impact to the farmstead cheese producers.

Impacts and Contributions/Outcomes

We anticipate improved understanding of Staphylococcus epidemiology and ecology on dairy farms will give more insight to the contribution of specific species and strains in this diverse group of bacteria. Our preliminary findings show some farms are dominated by unique species of bacteria that cause mastitis for example S. chromogenes, S. auricularis and S. haemolyticus (Table 1) and these are the most dominant causative agents of mastitis on these farms. Most species isolated from milk of infected cows are the same as those from cow skin suggesting that cow skin could be the primary source of these bacteria. However, what we haven’t elucidated yet, is whether the strains are the same. We rarely isolated environment-associated species in the cow’s milk. This suggests that most bacteria causing mastitis on these farms come from the cow skin surfaces. Cases of mastitis could be potentially reduced if farmers maintain cow teat end disinfection and hygiene. From the antimicrobial gene marker PCR screening, most isolates of S. chromogenes, S. auricularis and S. haemolyticus carry blaz gene (Table 2), which encodes for penicillin resistance. This suggests that penicillin is not a drug of choice for cow treatment in cases of mastitis because these are the same species predominantly causing mastitis on these farms. Some blaz positive isolates are susceptive to penicillin in our in vitro assays but we don’t know whether the resistance can be induced in vivo. Lincomycin is the second antibiotic most bacteria are resistant to from our in vitro assays. However, most isolates are susceptible to the  rest of the antibiotics tested. Most CNS species carrying mecA gene are susceptible to beta-lactam antibiotics. We intend to do more work to understand the genetic basis of this discordance in future. This data overall, will enable farmers and extension service providers to design better control strategies for the pathogenic species and better treatment options. Controlling staphylococcus mastitis on farms that make artisan cheese will improve profitability, product quality, and cattle health and welfare.

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