Table of Contents

Meeting

Table of Contents

TABLE OF CONTENTS

Introduction....................................................................................................................

Goals and Objectives..................................................................................................

Methodology.................................................................................................................

Table of Antimicrobics.................................................................................................

Table 1. Top 15 serotypes..........................................................................................

Table 2. Distribution of isolates..................................................................................

Table 3. Total % sensitive, intermediate, resistant.................................................

Table 4. Total % resistance by species/sources ....................................................

Table 5. % resistance for non-clinical isolates.........................................................

Table 6. % resistance for HACCP samples..............................................................

Table 7. % resistance for clinical isolates.................................................................

Table 8. % resistance for top 15 serotypes...............................................................

Table 9. Multiple antimicrobial resistance.................................................................

Table 10. Most frequent resistance patterns.............................................................

Table 11. Most frequent resistance patterns for 5 or more antimicrobics.............

Table 12. Total number ACSSuT...............................................................................

INTRODUCTION

The emergence of resistance to antimicrobics has compromised control of many bacterial pathogens and is a global problem. Additionally, multiple resistance has emerged among many bacterial strains including Salmonella species. A penta-resistant strain of Salmonella typhimurium DT104 in which the resistance genes have been chromosomally integrated is proving to be particularly problematic resulting in increased morbidity and mortality in both animals and humans.

The development of resistant human pathogenic bacteria may result from direct use of antimicrobial agents in humans and animals and acquisition of resistant organisms or resistance factors from animal and environmental bacteria. The intestinal flora of animals that have been exposed to antimicrobial agents can serve as a reservoir of resistant bacteria.

Because of the public health concerns associated with the use of antimicrobics in food-producing animals, an antimicrobial resistance monitoring program was proposed by the Food and Drug Administration Center for Veterinary Medicine (FDA) as a post-marketing activity to help ensure the continued safety and efficacy of veterinary antimicrobics. In 1996, the CDC, the USDA, and the FDA established the National Antimicrobial Susceptibility Monitoring System to prospectively monitor changes in antimicrobial susceptibilities of zoonotic pathogens from human and animal clinical specimens, from healthy farm animals, and from carcasses of food-producing animals at slaughter. Non-typhoid Salmonella was selected as the sentinel organism.

Veterinary testing is conducted at USDA's Agricultural Research Service Russell Research Center in Athens, GA. Testing is done using a semi-automated system (Sensititre^TM Accumed, Westlake Ohio). This report summarizes the percentage of isolates collected during calendar year 1997 that were susceptible, intermediate, or resistant to 17 antimicrobics (n=2,391). The 17 antimicrobics were chosen to be representative of common antimicrobics (or classes of antimicrobics) used in animal and human medicine. A subsequent report will summarize the minimal inhibitory concentrations obtained for these isolates and will provide a discussion of the data. Questions regarding this report should be directed to any of the people listed below.

Paula J. Fedorka-Cray, PhD
USDA-ARS-RRC, Athens, GA
706-546-3305

Marissa Miller, DVM, MPH and Linda Tollefson, DVM, MPH
FDA-CVM, Rockville, MD
301-827-0186

David A. Dargatz, DVM, PhD and Nora E. Wineland, DVM, MS
USDA-APHIS-VS-CEAH, Fort Collins, CO
970-490-8000

GOALS AND OBJECTIVES

The goals and objectives of the monitoring program are to:

1) provide descriptive data on the extent and temporal trends of antimicrobial susceptibility in Salmonella and other enteric organisms from the human and animal populations;

2) facilitate the identification of resistance in humans and animals as it arises;

3) provide timely information to veterinarians and physicians;

4) prolong the life span of approved drugs by promoting the prudent and judicious use of antimicrobics; and

5) identify areas for more detailed investigation.

Information resulting from the monitoring program and follow-up outbreak investigations will be distributed to veterinarians, physicians, and food animal producer groups. Use of the information will be targeted to redirecting drug use so as to diminish the development and spread of resistance over the short term with directives involving long-term use developed in collaboration with the appropriate professional practitioner groups. Outbreak investigations and field studies will be initiated as a result of major shifts or changes in resistance patterns in either animal or human isolates.

METHODOLOGY

Isolation:

Salmonella isolates with known serotypes are struck onto 5% sheep blood agar (SBA) plates for isolation. Plates are incubates at 37^oC overnight. The following morning one well-isolated colony from each plate is picked and regrown on a second SBA plate which is incubated at 37^oC overnight.

Screening for resistance:

One sterile dd H₂0 tube and 1 Mueller- Hinton broth (MHB) tube is set in a rack for each isolate. One substrate strip is added to each MHB for a minimum of 15 minutes prior to inoculation (Note: Once substrate strips are added to MHB tubes, they must be used within 1 hour or discarded). Two to six colonies from the second SBA are collected with a sterile cotton tipped swab and used to inoculate the water tube. The tube is vortexed and the density is adjusted with the Nephlometer as per manufacturer's instructions (Note: the machine is calibrated with a McFarland standard prior to starting the procedure). A 10 ml disposable loop from Sensititre is used to transfer 10 ml from the inoculated water to a MHB tube containing the substrate strip. The MHB tube is vortexed and placed into the auto inoculator (typically one isolate per microtiter plate) as per manufacturer's instructions. The microtiter plate is incubated at 37^oC for 18 - 20 hours (Note: The time for reading plates is 18-20 h, ideally all plates are read as close to 18 hrs. as possible). Record the time the microtiter plate is inoculated and read on a sheet. Do NOT read plates or keep data from plates >20 h old). (Note: Ideally plates should not be stacked while in the incubator. If stacking is required, stack no more than 2 plates high.)

Microtiter plates are read as per manufacturer's instructions

Freezing clones:

Using a sterile disposable 1 ml inoculating loop 6 colonies from the second SBA plate are picked and inoculated (by vigorously shaking the loop to dislodge bacteria) into 1 ml LB broth plus 30% glycerol in cryo vials. The vials are stored frozen at -70^oC and labeled with the following information:

TABLE OF ANTIMICROBICS

* ranges were chosen to detect incremental changes in resistance based on previous 2 year data; ranges may be outside of the breakpoint value

RESULTS -
Veterinary Isolates

TABLE 1. Top 15 Salmonella serotypes identified for 1997 (N=2,391 total isolates) for all animal species

* typhimurium and typhimurium (copenhagen) isolates combined account for 328 (13.7%) of the total number of isolates

TABLE 2: Distribution of isolates by species and clinical status

CLINICAL (isolates collected from NVSL; N=763)

NONCLINICAL (N=1,628)

*includes 99 samples which are of unidentified clinical status **samples collected from carcasses at slaughter with the exception of eggs ***species unknown

TABLE 3: Total percent sensitive, intermediate or resistant

TABLE 4: Percent total resistance by species/sources (includes both clinical and nonclinical isolates)

TABLE 4: Percent total resistance by species/sources (includes both clinical and nonclinical isolates; continued)

Note: 2 isolates that were unidentified were not resistant to any antimicrobic

Table 5: Percent resistance for non-clinical isolates (excluding HACCP samples)

Note: Samples are included only if they were able to be identified as non-clinical. This does not include 99 samples of undetermined clinical status

Table 6: Percent resistance for HACCP samples

Table 7: Percent resistance for clinical isolates*

Note: Clinical isolates in Table 7 were all obtained from the National Veterinary Services Laboratories, Ames, IA

Table 7: Percent resistance for clinical isolates* (continued)

Note: Clinical isolates in Table 7 were all obtained from the National Veterinary Services Laboratories, Ames, IA

Table 8: Percent total resistance for the top 15 Salmonella serotypes from animal species/sources