Carriage of MRSA Among Dairy Farm Workers in the High Plains
Abstract No:
1698
Abstract Type:
Student Poster
Authors:
J Nunez1, S Reynolds2, J Schaeffer3
Institutions:
1Colorado State University, fort collins, CO, 2Colorado State University, Fort Collins, CO, 3N/A, Fort Collins, CO
Presenter:
Jessica Nunez, B.S
Colorado State University
Colorado State University
Faculty Advisor(s):
Dr Stephen Reynolds
Colorado State University
Colorado State University
Joshua Schaeffer
N/A
N/A
Description:
The nasal microbiome in dairy farmers contains two times the bacterial diversity (and abundance) as compared to non-farmers. It is believed that this diversity is protective against the colonization of methicillin- resistant Staphylococcus aureus (MRSA).
Situation/Problem:
Dairy farmers experience a heavy burden of bioaerosol-related respiratory ailments. Bioaerosols are known to contain inflammagens (specifically endotoxin), and a diverse bacterial community that is associated with upper respiratory inflammation and pulmonary decrement among workers. However, identifying casual agents (beyond endotoxin) is still an area that warrants further research. Industrialization and modernization of the dairy industry has led to dramatic changes to production, work organization and tasks. Consequently, exposure patterns have been altered. Recently, we demonstrated that the mass of dairy bioaerosols is predominantly present in particle size ranges that span 10-100 μm in aerodynamic diameter; these are known to deposit in the upper respiratory system (i.e., the nasopharyngeal region). The nose contains complex bacterial communities and this microbiome may play a role in the inflammatory response to bioaerosols. Recently, the nasal microbiome in dairy farmers was shown to contain over two times the bacterial diversity (and abundance) as compared to non-farmers. It is believed that this diversity is protective against the colonization of methicillin- resistant Staphylococcus aureus (MRSA). In contrast, persistent nasal carriage of MRSA, specifically livestock-associated strain, has been demonstrated in swine production workers. Recent evidence shows an increase in soft tissue infections caused by LA-MRSA among at risk populations. The objective of this research was to characterize the presence and carriage of Staphylococcus spp. with a focus on livestock associated MRSA in the nose of dairy workers.
Methods:
In the field, worker nasal lavages were collected by investigator by instilling five mls of sterile saline (control) or hypertonic saline (treatment) into each nostril over a timespan of ten seconds. Participant would immedialtey expell the saline liquid into a sterile sample collection cup. Samples were then quantified and preserved with protease cocktail inhibitor in the field to halt cellular metobolic processes. This research was done in conjunction with research into inflammatory properties related to the nasal microbiome of dairy workers.
• In the field, nasal lavage samples were kept between 2º - 8ºC until they could be placed in a laboratory refridgerator to await further processing (within 12 hours). Long-term preservation was achieved with a 40% Glycerol stock solution in a 1:1 ratio to create a 20% solution.
• Samples were then placed for long-term preservation in a -80ºC freezer.
• Preserved samples were thawed slowly prior to analyses.
• Tryptic Soy Agar (TSA), Chromagar MRSA, Chromagar Staph aureus, and Mannitol Salt Agar (MSA) were inoculated with each nasal lavage sample (pre and post).
• Inoculated media was incubated for 24 hours at 37.5ºC . A positive and negative control was also included for each media. Positive controls were :
Staphylococcus aureus ATCC 25923 (MSSA)
Staphylococcus aureus ATCC 700699 (MRSA)
• Post 24-48 hour incubation, samples were monitored for growth.
• Colonies from selective media were collected and propagated in Brain Heart Infusion Broth (BHI) for 24-48 hours at 37.5ºC.
• BHI samples were plated onto TSA plates to grow isolates of original media in preparation for Antibiotic Sensitivity Testing (AST) via the Kirby Disc Diffusion method. Plates were incubated at 37.5ºC for 24-48 hours.
• After appropriate incubation and growth, isolated colonies were selected to transfer into 2 mL of Saline, and vortexed to match a 0.5 McFarland standard.
• Within fifteen minutes of creating desired saline mixture, TSA plates were lawn streaked and allowed to dry for 5 minutes prior to placing antibiotic disks.
• Inoculated TSA plates were divided into four quadrants prior to antibiotic sensitivity testing (AST).
• Tetracycline, Vancomycin, Cefoxitin, and a blank disk were placed on each TSA plate. A blank disk was included as a control. Plates were incubated for 24 hours prior to interpretation.
• Organisms will be confirmed using Matrix Assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF).
• DNA extraction will be performed using Qiagen's DNeasy Blood and Tissue kit for use in multiplex PCR.
•Multiplex PCR will be performed targeting/amplifying mecA, scn (immune evasion cluster) and the PVL genes to determine LA-MRSA and CA-MRSA.
• In the field, nasal lavage samples were kept between 2º - 8ºC until they could be placed in a laboratory refridgerator to await further processing (within 12 hours). Long-term preservation was achieved with a 40% Glycerol stock solution in a 1:1 ratio to create a 20% solution.
• Samples were then placed for long-term preservation in a -80ºC freezer.
• Preserved samples were thawed slowly prior to analyses.
• Tryptic Soy Agar (TSA), Chromagar MRSA, Chromagar Staph aureus, and Mannitol Salt Agar (MSA) were inoculated with each nasal lavage sample (pre and post).
• Inoculated media was incubated for 24 hours at 37.5ºC . A positive and negative control was also included for each media. Positive controls were :
Staphylococcus aureus ATCC 25923 (MSSA)
Staphylococcus aureus ATCC 700699 (MRSA)
• Post 24-48 hour incubation, samples were monitored for growth.
• Colonies from selective media were collected and propagated in Brain Heart Infusion Broth (BHI) for 24-48 hours at 37.5ºC.
• BHI samples were plated onto TSA plates to grow isolates of original media in preparation for Antibiotic Sensitivity Testing (AST) via the Kirby Disc Diffusion method. Plates were incubated at 37.5ºC for 24-48 hours.
• After appropriate incubation and growth, isolated colonies were selected to transfer into 2 mL of Saline, and vortexed to match a 0.5 McFarland standard.
• Within fifteen minutes of creating desired saline mixture, TSA plates were lawn streaked and allowed to dry for 5 minutes prior to placing antibiotic disks.
• Inoculated TSA plates were divided into four quadrants prior to antibiotic sensitivity testing (AST).
• Tetracycline, Vancomycin, Cefoxitin, and a blank disk were placed on each TSA plate. A blank disk was included as a control. Plates were incubated for 24 hours prior to interpretation.
• Organisms will be confirmed using Matrix Assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF).
• DNA extraction will be performed using Qiagen's DNeasy Blood and Tissue kit for use in multiplex PCR.
•Multiplex PCR will be performed targeting/amplifying mecA, scn (immune evasion cluster) and the PVL genes to determine LA-MRSA and CA-MRSA.
Results / Conclusions:
Out of 238 samples (pre and post) collected from 31 volunteers over 5 consecutive days, 80 samples tested positive for MSSA (34%), 23 samples tested positive for MRSA (9.7%). Antibiotic sensitivity testing results were measured against standards set by the Clinical and Laboratory Standards Institute (CLSI). CLSI standards are as follows:
Tetracycline ("Resistant" = ≤ 14mm),
Vancomycin ("Susceptible"= ≥ 7mm) and
Cefoxitin (mecA+ = ≤ 21mm)
To date, all MRSA samples tested (only 13% have undergone AST) are susceptible to vancomycin, which we use to predict potential threat to public health. Additionally, resistance and susceptibility has been observed; one sample is resistant to tetracycline, and one sample is classified to have an intermediate resistance to tetracycline (resistance n=2). Of the samples tested, one sample is susceptible to tetracycline, and one sample is positive for mecA + gene against cefoxitin; two samples tested were susceptible to cefoxitin and therefore mecA -.
Preliminary results show a higher incidence of MRSA than anticipated. To date, AST results do not show any resistance to vancomycin, but results do show some gene variations that could indicate a positive predictor of LA-MRSA. Future analysis will confirm genetic presence with Multiplex PCR and MALDI-TOF.
Tetracycline ("Resistant" = ≤ 14mm),
Vancomycin ("Susceptible"= ≥ 7mm) and
Cefoxitin (mecA+ = ≤ 21mm)
To date, all MRSA samples tested (only 13% have undergone AST) are susceptible to vancomycin, which we use to predict potential threat to public health. Additionally, resistance and susceptibility has been observed; one sample is resistant to tetracycline, and one sample is classified to have an intermediate resistance to tetracycline (resistance n=2). Of the samples tested, one sample is susceptible to tetracycline, and one sample is positive for mecA + gene against cefoxitin; two samples tested were susceptible to cefoxitin and therefore mecA -.
Preliminary results show a higher incidence of MRSA than anticipated. To date, AST results do not show any resistance to vancomycin, but results do show some gene variations that could indicate a positive predictor of LA-MRSA. Future analysis will confirm genetic presence with Multiplex PCR and MALDI-TOF.
Primary Topic:
IH Profession
Secondary Topics:
Aerosols
Occupational and Environmental Epidemiology
Co-Authors
Please add your co-authors below. Co-authors are listed for professional courtesy and will not be communicated with regarding the decision notification or any on-site logistics, if accepted. Only the primary presenter listed is expected to attend and present the content on-site.
1. High Plains Intermountain Center for Agricultural Health and Safety,
2. Department of Environmental & Radiological Health Sciences, Colorado State University,
3. University of Wyoming, Department of Animal Science