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FDA Grant FD-U-001577-01 (September 30, 1998 – September 29, 2001)

Survey of Antimicrobial Resistant Enterococci in Animals

Marcus J. Zervos, MD

William Beaumont Hospital

Royal Oak, MI

Background:

During the past decade, enterococci have emerged as important pathogens associated with nosocomial infections.  Many human isolates of vancomycin resistant-enterococci (VRE) are now resistant to all clinically available antibiotics with the exception of quinupristin/dalfopristin (Synercid) and linezolid (Zyvox).  However, shortly after approval of these agents, resistance has been increasingly reported in human clinical isolates of Enterococci.  Resistance rates of E. faecium to quinupristin/dalfopristin from stool cultures as high as 7.4% have been reported in hospitalized patients in German hospitals.  Importantly these findings occurred before Synercid was approved for human use in Germany.  Although much has been learned over the last ten years about the epidemiology of nosocomial enterococci, there is little information on the epidemiology of drug resistant enterococci outside of the acute care hospital setting.  Sources and reservoirs other than hospitalized patients may play an important role in the spread of antimicrobial resistance.  Community acquisition of gentamicin resistant enterococci was documented in up to 7% of infections, which suggests a reservoir in the community.  In the United States acquisition and spread of VRE occurs in the hospital, while in Europe, community acquisition of VRE has occurred and has been associated with the use of avoparcin in food animals.  Non-human sources, such as farm animals receiving antibiotics in feed, have been increasingly suspected as a reservoir for some antibiotic-resistant bacteria.  It has been suggested that antibiotic use in food animals has resulted in "new" resistance genes and the emergence of multiresistant pathogens.  Based on these reports the prevalence of antimicrobial resistance needs to be further evaluated and risk factors for acquiring resistant isolates to be determined.

The three specific aims of the research in the proposal included

1.      To obtain information on State-wide (Michigan) prevalence rates of antibiotic-resistant enterococci in food animals.

Results: þNo vancomycin resistance was seen in any of the farms

þMean ciprofloxacin resistance rates in E. faecium were 24% in turkey, 4% in chicken, 42% in beef cattle, 38% in dairy cattle, and 45% in swine farms.  No ciprofloxacin resistance was seen in E. faecalis from any of the farms evaluated

þMean gentamicin resistance rates in E. faecium were 13% in turkey, 14% in chicken, 0% in beef cattle, 8% in dairy cattle, and 1.5% in swine farms.  Resistance rates in E. faecalis were 29% in turkey, 41% in chicken, 0% in beef cattle, 27% in dairy cattle, and 34% in swine farms

þMean quinupristin/dalfopristin resistance rates in E. faecium were 52% in turkey, 98% in chicken, 3% in beef cattle, 10% in dairy cattle, and 22% in swine farms

2.      To identify and characterize the resistance genes found in enterococci isolated from animal sources.  Characterization will include a) determining strain relatedness by pulsed-field gel electrophoresis (PFGE), b) evaluation of isolates for the presence, prevalence and location of known resistance genes and identification of novel resistance genes using PCR techniques, and c) comparison of isolates to already characterized human isolates from the same geographic areas in Michigan

Results: þSee table 1 for a summary of gentamicin resistance genes

þThree of the gentamicin resistance genes reported in humans were found in animal isolates from this study

þOne E. faecalis isolate from a chicken farm failed to generate a PCR product for any of the known gentamicin resistance genes suggesting the presence of a novel resistance gene in this isolate

þBy PCR all quinupristin/dalfopristin resistant isolates were negative for the vat(D) gene

þvat(E) gene was found in:

14/56 (25%) of chicken farm isolates

13/74 (18%) of the turkey isolates

The swine (n=59), dairy (n=51) and beef (n=3) isolates were negative for vat(E)

þOf the 31 Q-D resistant human isolates, one isolate from an outpatient was positive for the vat(E) gene.  All human isolates were negative for the vat(D) gene

þNone of the animal isolates had similar PFGE type to the human isolates evaluated

3.      To collect information on risk factors for antibiotic resistant enterococci in animal isolates including animal sources and age, size of farms, antimicrobial use and geographic and temporal clustering

þHigh ciprofloxacin resistance rates were found on farms with no reported enrofloxacin use: beef (40%), dairy (35%), turkey (21%), and swine (44%) farms.  However, ciprofloxacin resistance rates were high in the dairy farm (62%) and turkey farms (54%) using enrofloxacin

þDairy farms using aminoglycosides had a higher gentamicin resistance rate of 29% versus 4% in farms with no aminoglycoside use.  Swine farms using an aminoglycoside had a gentamicin resistance rate of 20% compared to 7% in farms with no aminoglycoside use.  No aminoglycosides were used in beef farms and gentamicin resistance rate was found to be 0%

þTurkey farms using virginamycin as a growth promoting agent had higher rates of Q/D resistance (50-100%, mean of 75%) compared to farms that did not use virginamycin: mean of 28% in turkeys, 4% in beef, 11% in dairy, and 20% in swine farms

þAll seven chicken farms reported use of virginamycin and the Q/D resistance rate ranged from 90-100% (mean 98%) among E. faecium isolated from these farms

Overall Conclusions

This study demonstrates that there is high rate of quinupristin/dalfopristin, gentamicin, and ciprofloxacin resistance rate among the various farms evaluated and use of antibiotics as growth promoting agents contributed to higher rates of resistance.  In chicken farms, which utilized virginamycin, the resistance rate ranged from 90-100% (mean of 98%). Although no virginamycin were used in beef, swine, and dairy farms, macrolides and lincosamides were commonly used, which may have selected for quinupristin/dalfopristin resistance among these farms. 

The vat(E) gene was the only previously described streptogramin-resistance gene found in farm animals and was found only in chicken (25%; 5/7 farms) and turkey(18%; 4/8 farms) isolates.  Only one human isolate contained the vat(E) gene and it was not the same PFGE strain type as any of the chicken or turkey isolates.  Quinupristin/dalfopristin resistant strains shared the same PFGE group within the same farm, but rarely were shared between farms.

The aac(6’)-Ie-aph(2”)-Ia was the most common gentamicin-resistance gene found in enterococcal isolates from farm animals, which is also the predominant gene found in human isolates.  In our study, the two gentamicin-resistance genes, aph(2”)-Ic and aph(2”)-Id, were more prevalent in animal isolates compared to humans (3%).

There was a large diversity of PFGE strain types among gentamicin resistant enterococci in farm animals.  Intra-farm dissemination of gentamicin resistant strains of enterococci was more common than inter-farm dissemination of the resistant strains.  Three PFGE strain types were shared between dairy, cattle and swine.

Manuscripts in Progress for Publication Based on this FDA Supported Research

• Prevalence of antimicrobial resistance in enterococci isolated from farm animals

• Molecular epidemiology of gentamicin resistance in enterococci isolated from farm animals

• Molecular epidemiology of quinupristin/dalfopristin resistance in Enterococcus faecium isolated from farm animals

Presentations at Scientific Meetings Based on this FDA supported Research

• Donabedian SM, Perri MB, Bozigar PS, Hershberger E, Angulo FJ, Rossiter S, Jones R, Debess E, Johnson S, Park M, Johnson J, Zervos MJ. Quinuprisitn/Dalfopristin Resistance in Enterococcus faecium Isolated from Humans, Farm Animals and Retail Food. 41^th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, December 16-19, 2001, Abstract #C2-1872.

• Donabedian SM, Bozigar PS, Perri MB, Hershberger E, Thal LA, Bartlett P, Chow JW, Jones R, Zervos MJ. Prevalence of Antimicrobial Resistance and Antimicrobial Use in Cows, Turkeys, Chicken and Swine. 41^th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, December 16-19, 2001, Abstract #C2-1873.

• Perri MB, Donabedian SM, Thal LA, Hershberger E, Angulo F, Jones R, Rossiter S, Debess E, Park M, Johnson S, Johnson J, Zervos MJ.  Common Molecular Mechanism of Gentamicin Resistance Among Enterococci Isolated from Animals, Food, and Humans . 41^th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, December 16-19, 2001, Abstract #C2-1874.

• Donabedian SM. Second FDA/FSI Extramural Research Annual Review. “Survey of Antimicrobial Resistant Enterococci in Animals” Cooperative Agreement FD-U-001 577-02.  College Park, MD, December 5-6, 2000.

• Donabedian SM, Thal LA, Bozigar PS, Perri MB, Hershberger E, Bartlett P, Chow JW, Jones R, Zervos MJ. Prevalence of Antimicrobial Resistance and Antimicrobial Use in Cows, Turkeys, and Swine. 40^th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, September 17-20, 2000.

• Zervos MJ.  NARMS. “Survey of Antimicrobial Resistant Enterococci in Animals” Cooperative Agreement FD-U-001 577-02.  Washington DC, April 2001.

• Thal LA, Hershberger E, Jones R, Joyce K, Hill B, Morano N, Rossiter S, Angulo F, Clark N, Tenover F, Gilbert L, Franko E, Steiner C, Johnson J, Debess E, Madden J, Zervos MJ.  Molecular Mechanism of Gentamicin Resistance Among Enterococci Isolated from Humans, Food Animals, Meat and Poultry. 40^th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, September 17-20, 2000 Abstract #167.

• Donabedian SM, Thal LA, Bozigar PS, Perri MB, Hershberger E, Bartlett P, Chow JW, Zervos MJ. Prevalence of Antimicrobial Resistance and Antimicrobial Use in Cows and Turkeys. 1^st International ASM Conference on Enterococci.  Banff, Canada February 28-March 2, 2000.

• Thal LA.  First FDA/FSI Extramural Research Annual Review. “Survey of Antimicrobial Resistant Enterococci in Animals” Cooperative Agreement FD-U-001 577-01.  College Park, MD, December, 1999.

TABLE 1.  Summary of Gentamicin Resistance Genes in Enterococci

*1 E. faecalis isolate from chicken with HLGR failed to generate a PCR product for any of the aminoglycoside resistance genes tested

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