Bacterial Epidemiology and Antimicrobial Resistance : Salmonella & Campylobacter-1998 Report

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 Resistance Monitoring System (NARMS) to prospectively monitor changes in antimicrobial susceptibilities of zoonotic pathogens from human and animal diagnostic 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 (Sensitre^TM, TREK^TM Diagnostics, Inc., Westlake Ohio). This report summarizes the percentage of Salmonella isolates collected during calendar year 1998 that were susceptible, intermediate, or resistant to 17 antimicrobics (n=3,318). A summary of the 17 antimicrobics were chosen to be representative of common antimicrobics (or classes of antimicrobics) used in animal and human medicine. A summary of the minimal inhibitory concentrations obtained for these isolates is also included. Additionally, Campylobacter isolates (n=194) were also tested using the E-test against 8 antimicrobics. A summary of the minimal inhibitory concentrations for these isolates is also included.

Questions regarding this report should be directed to any of the people listed below. Because of the amount of data and complexity of analyses involved, all permutations are not represented. Anyone requiring an analysis that is not represented in this report should direct their request to any of the people listed below.

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

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

Linda Tollefson, DVM, MPH
Marcia Headrick, DVM, MPH
FDA-CVM, Rockville, MD
301-827-0186 (Linda)
301-827-6523 (Marcia)

Kenneth E. Petersen, DVM, MPH
USDA-FSIS-EPZDD
Washington, DC
202-501-6695

Introduction

Antimicrobial resistance has become a concern for both human and animal health on a global scale. Expert scientific groups such as the Institute of Medicine, the American Society for Microbiology and the World Health Organization expressed apprehension about the national and global increase in antibiotic resistance and the complex issues surrounding the increase in the community and institutional setting (1-3).

In addition to concerns about treatment failures in veterinary medicine and human medicine related to resistant organisms, there is concern about the potential transfer of resistant organisms from animals to humans. The development of resistant human pathogenic bacteria may result from direct use of antimicrobial agents in humans and acquisition of resistant organisms or resistance factors from animal and environmental bacteria (4). Although antibiotics are typically not indicated for the treatment of uncomplicated salmonella infections in humans, their use in treating septic infections is critical.

Multiple resistance has also emerged among 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 (5-9). A decrease in susceptibility to fluoroquinolones (FQ) has been observed for some DT104 strains (10). One additional concern is the identification of FQ antibiotics in hospital wastewater (11) suggesting widespread exposure to both humans and animals which may confound treatment regimens.

History of Antimicrobial Susceptibility Monitoring in the US

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, especially FQs. In 1996, the CDC, the USDA, and the FDA established the National Antimicrobial Resistance Monitoring System (NARMS; referred to in previous publications as the National Antimicrobial Susceptibility Monitoring Program and subsequently changed to NARMS-Enteric Bacteria) to prospectively monitor changes in antimicrobial susceptibilities of zoonotic pathogens from human and animal diagnostic specimens, from healthy farm animals, and from carcasses of food-producing animals at slaughter (12,13). Non-typhoid Salmonella was selected as the sentinel organism.

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.

Materials and Methods

Salmonella:

Susceptibility testing is conducted at USDA’s Agricultural Research Service facility in Athens, GA. Isolates are selected from research studies conducted by the USDA or collaborators, from the National Animal Health Monitoring System studies, from diagnostic sources (such as the National Veterinary Services Laboratories and Sentinel Sites which are Veterinary Diagnostic Laboratories), and from raw product collected from federally inspected slaughter and processing establishments.

Testing is done using a semi-automated system (Sensitre^TM, TREK^TM Diagnostics, Inc., Westlake Ohio). All isolates are maintained at -70^oC to serve as a bank for future use. A description of the panel of antimicrobics and their concentrations is shown in Table 1a. National Committee for Clinical Laboratory Standards (NCCLS) guidelines (14) were followed throughout the testing procedure. Quality control strains E. coli ATCC 25922 and S. typhimurium ST129 (in-house strain) were used.

Isolation:

Salmonella isolates with known serotypes are struck onto 5% sheep blood agar (SBA) plates for isolation. Plates are incubated at 37^oC overnight. The following morning one well-isolated colony from each plate is picked and struck 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 :l disposable loop from Sensititre is used to transfer 10 :l 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). The time the microtiter plate is inoculated and read is recorded. Plates are not read if >20 h old.

(Note: Ideally plates should not be stacked while in the incubator. If stacking is required, they are stacked no more than 2 plates high.)

Microtiter plates are read as per manufacturer’s instructions. Isolates are classified as susceptible, intermediate, or resistant based on (NCCLS) established breakpoints used in human medicine with the exception of Cephalothin, Kanamycin, Streptomycin, Sulfamethoxazole, and Trimethoprim/Sulfamethoxazole (15). Currently, comparable veterinary breakpoints are not available.

Freezing clones:

Using a sterile disposable 1 :l 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.

Interlab Comparison:

Twelve samples from each laboratory (CDC and ARS) were tested in both laboratories for MICs to all 17 antimicrobics for interlab comparison and quality control.

Campylobacter:

Susceptibility testing is conducted at USDA’s Agricultural Research Service facility in Athens, GA. Isolates were obtained from chicken carcass rinses at Federally inspected slaughter plants during a study to refine methods and protocols for Campylobacter isolation. Initial characterization of Campylobacter included use of dark field motility and hippurate hydrolysis testing. PCR using primers to ceuC and HipO were used to speciate C. coli and C. jejuni, respectively. Isolates not reacting with either primer were classified as C. other and are under further study.

Antimicrobial testing was done using the E-test (AB BIODISK, Solna, Sweden) as per manufacturer’s instructions. All isolates are maintained at –70^oC to serve as a bank for future use. A description of the panel of antimicrobials and their concentrations is shown in Table 1b. Breakpoints are determined using, when available, NCCLS standards. Isolates are maintained at –70^oC to serve as a bank for future use.

Isolation:

Campylobacter isolates are struck onto 5% sheep blood agar (SBA) plates for isolation. Plates are incubated at 37^oC for 48 h under a reduced atmoshpere (5% O₂, 10% CO₂, balance N₂). At 48 h one well-isolated colony from each plate is picked and struck on a second SBA plate and incubated as described.

Screening for Resistance:

One sterile dd H₂O tube is inoculated with 10-15 colonies, vortex and the density is adjusted with a Nephlometer which is calibrated with a McFarland standard prior to starting the procedure. A sterile cotton swab is used to make a lawn on two 150mm MH agar plate containing 5% horse blood. E-test strips (4/plate) are placed as per manufacturer’s instructions and the plates are incubated 48h at 42^oC under a reduced atmosphere as described above. MICs are determined as per manufacturer’s description.

Sentinel Sites:

Sentinel sites were chosen to augment the numbers of diagnostic isolates and to provide a geographic representation that complemented states submitting human isolates to CDC. Three veterinary diagnostic laboratories (affiliated with schools) one each from California, New York and Washington State agreed to participate in 1999. Because of the small number of Salmonella isolates received in any year, the laboratories agreed to send all isolates up to a maximum of 500/site/year. Receipt of isolates did not begin until August and is reflected in the small number of submissions. Because of the small number of isolates that were received, data from all three sites were combined in 1998 (Table 8). Additional states will be added in subsequent years and as numbers of isolates and sites increase, data will be reported by geographic region.

Results and Discussion

Overall there were 3,318 Salmonella isolates of veterinary origin collected in late1997 and 1998 that were tested. These isolates represented a broad range of species and came from diagnostic cases, healthy animals on farm and federally inspected establishments (Table 2). Diagnostic isolates were randomly collected from isolates submitted from throughout the country to the National Veterinary Services Laboratories, Ames, IA. Care was taken to exclude isolates from California, New York and Washington so as not to duplicate testing of sentinel site isolates. The majority of diagnostic isolates from all species were submitted as a result of a primary or secondary salmonella associated clinical illness in the host, although salmonellosis might not have been the primary etiologic agent in all cases. For the non-diagnostic isolates, 78 cattle isolates were obtained as part of a national study in beef cattle on-farm (NAHMS). The salmonella slaughter samples were typically collected as part of the HACCP implementation testing. Samples were most often, but not exclusively, obtained from chilled carcasses at slaughter. For this report, descriptions of isolates are confined by major species. A more detailed description of isolates, particularly slaughter isolates will be presented in forthcoming peer reviewed manuscripts.

Overall most of the isolates were considered sensitive to most antimicrobics. Resistance was most common to Tetracycline (38.1% of isolates), Streptomycin (34.7%), Sulfamethoxazole (31.9%), and Ampicillin (17.9%) (Table 3).

Percent resistance by species/sources is shown in Table 4. The largest number of isolates was from swine (n=1056) and all isolates were susceptible to Amikacin and Ciprofloxacin. The highest resistance among swine isolates was observed for Tetracycline (53.9%) which may reflect the higher use of tetracycline compounds in the industry compared to other species. It is comforting to note that close encounters with exotic species (even being bitten by bears or snakes) will not result in the acquisition of highly resistant salmonellae (although iguanas, lizards, snakes and other reptiles are often contaminated with Salmonella).

Very little resistance for beef isolates obtained from a national study is observed (Table 5). This suggests that use of antimicrobics among operations submitting samples for this study did not result in a high prevalence of resistance.

Percent resistance of slaughter samples is shown in Table 6. Compared to 1997 results, percent resistance remained the same or decreased for a majority of the antimicrobials. The exception is observed for the cephalosporins (Ceftiofur, Ceftriaxone, and Cephalothin). Resistance to Ceftiofur, Ceftriaxone and Cephalothin increased in cattle and chicken isolates. Resistance to Ceftiofur decreased in swine and turkey isolates while resistance to Ceftriaxone decreased for turkey isolates and resistance to Cephalothin decreased in swine and turkey isolates. The significance of these results is unknown and may be attributed to differences in total numbers of isolates between years, seasonal variations (1997 slaughter data reflects a fall/winter collection time), geographic origin, serotypes among the species, or changes in drug use patterns. Further research in this area is encouraged.

Differences in percent resistance of veterinary isolates by species and diagnostic v. non-diagnostic or slaughter status are noted in Table 5 vs Table 7. Percent resistance for diagnostic isolates is greater than observed for non-diagnostic or slaughter isolates. This should be expected since diagnostic isolates generally represent the problematic isolates in medicine. Many of these isolates would be submitted for serotyping because of a clinical episode that has lead to a diagnostic effort. Since these diagnostic efforts are expensive, it is likely that something about these isolates has stimulated the producer and the veterinarian to go to greater lengths than usual to determine the etiologic agent in a particular case. In addition, since these are from clinical cases, it is likely that many of these organisms have been exposed to antimicrobial therapy. Therefore it should not be surprising that there is more resistance among diagnostic isolates than is seen for non-diagnostic isolates. The final outcome of these isolates with respect to entry into the food chain is unknown.

More of the diagnostic isolates from dogs (n=57) and cats (n=29) were resistant to the antimicrobials tested than those from the exotics (Table 7). Isolates recovered from exotic animals (n=75) were sensitive to many of the antimicrobials tested. Resistance among exotic isolates was only observed for Ampicillin (2.7%), Kanamycin (2.7%), Streptomycin (6.7%), Sulfamethoxazole (2.7%), Tetracycline (4.0%), Ticarcillin (2.7%), and Trimethoprim/Sulfamethoxazole (1.3%). Resistance to Nalidixic Acid (2.7%) was observed for the first time compared to other years.

Resistance among isolates submitted from the sentinel sites (Table 8) mirror resistance among diagnostic isolates (Table 7). Differences between Tables 7 and 8 may be accounted by numbers of isolates among species and geographic origin.

One hundred-twenty different serotypes of Salmonella were identified in 1998, an increase of 22 serotypes from the 98 reported in 1997. The top fifteen serotypes for 1998 are identified in Table 9. Although S. heidelberg is listed as number one, S. typhimurium and S. typhimurium var. Copenhagen are often combined and would account for 557 isolates. Interestingly, difference in percent resistance can be observed between the S. typhimurium and S. typhimurium var. Copenhagen isolates (Table 10). Resistance among serotypes varies widely as does recovery of serotypes by animal species (see MIC Figures). Interpretation of these data require careful analysis of both serotype and source of isolates before accurate changes in patterns can be determined.

Resistance to multiple antimicrobials is a concern. As organisms become resistant to more antimicrobials, treatment may be compromised. In 1998, 51.9% of all isolates were pan-sensitive and 8.1% were resistant to only one antimicrobial (Table 11), which was most commonly Tetracycline (Table 12). Although this represents a decrease in total numbers of isolates susceptible to all antimicrobials compared to 1997 in which 65.7% of all isolates were pan-sensitive, comparison of slaughter versus diagnostic isolates indicates that more slaughter isolates were pan-sensitive in 1998 (54.5%) versus 1997 (46.1%) and fewer diagnostic isolates were pan-sensitive in 1998 (48.6%) than 1997 (71.2%) (data not shown). Differences in numbers of isolates between years, geographic, species and serotype distribution, and drug use practices may account for these results and require further investigation.

The most frequent resistance patterns for five or more antimicrobials are listed in Table 13. Resistance to Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline is most often observed for S. typhimurium DT104. With the Sensititre System there is the possibility of one dilution variance between tests. Serendipitously, we observed that a quadra-resistant pattern consisting of resistance to Ampicillin, Chloramphenicol, Sulfamethoxazole, and Tetracycline is also observed for S. typhimurium DT104. The dilution obtained for Streptomycin in these cases is most often 32, which is one dilution below the breakpoint. Tables 14 and 15 provide the percent resistance for S. typhimurium and S. typhimurium var. Copenhagen with the penta- or quadra-resistant patterns and their subsequent characterization as to phage type. Total numbers of S. typhimurium and S. typhimurium var. Copenhagen increased from 328 in 1997 to 557 in 1998 as did the total number combined of those isolates with the penta-resistant pattern 58 (17.7%) in 1997 versus 163 (29.3%) in 1998. The total numbers of isolates subsequently identified as DT104 also increased between 1997 and 1998 from 37 in 1997 (1.5%; 37/2391) to 75 (2.3%; 75/3318) in 1998.

Although the penta- or quadra-resistant pattern for these isolates is most often associated with the DT104 or DT104 complex phage types, other phage types are also observed (Table 16). The most common phage type for S. typhimurium other than DT104 appears to be DT193 while the most common phage type for S. typhimurium var. Copenhagen is U302, which is similar to DT104.

MIC Figures

Data are presented in the following Figures as minimal inhibitory concentrations (MICs); NOTE: The scales of the Y-axis of the graphs are different dependent on the range of the response values. Please check Y-axis for correct interpretation. MIC histograms provide a truer representation of the data in that the distribution of values for the data set is depicted over the entire range of concentrations tested. One dilution variance can be obtained for any isolate. Additionally, numbers used for S. typhiumrium in these graphs are a combination of BOTH S. typhimurium and S. typhimurium var. Copenhagen.

Figures 1 through 26 provide the actual MIC distributions, which compliment Tables 3 through 10 and provide additional information regarding major serotypes (top three) by species. In Figure 1, the MICs are given for all Salmonella isolates regardless of species. Arrows have been placed at the breakpoint. An absence of an arrow (for Amikacin) indicates that the ranges tested were outside of the breakpoint and the actual breakpoint is listed in the lower left area of the graph (IE, 64 for Amikacin).

MICs by antimicrobial agent for Salmonella isolates from slaughter by species are shown in Figure 2. With the exception of Ampicillin and Tetracycline for turkey isolates (and those noted above for the Cephalosporins, there appears to be a decrease in resistance among slaughter isolates for 1998 as compared to 1997. MIC values also reflect this trend.

A comparison of MICs by antimicrobial agent for Salmonella isolates from cattle, swine and turkey by clinical status of isolates (non-clinical on-farm, slaughter and/or diagnostic) are shown Figures 3 through 7. For all species, and for most antimicrobials, there is a tendency for more diagnostic isolates to have higher MICs than slaughter isolates, which has also been observed for 1997. This is not unexpected for reasons outlined above. Figures 8 through 11 provide MIC distributions for isolates collected from all other species. Cat (Figure 8) and dog (Figure 9) isolates have higher MIC values for most antimicrobials as compared to all other species, possibly reflecting the treatment frequency related to domestic pets. This was also observed in 1997. Interesting, cat, dog and horse (Figure 11) isolates have more resistance to Ceftriaxone than all other species.

MICs for isolates from all species by serotype are shown in Figures 12 through 17. Serotypes chosen were based on total number of isolates as described in Table 10, except that S. typhimurium and S. typhimurium var. Copenhagen were combined (n=557). Although these graphs are useful in providing information about a specific serotype, use patterns, management, and other production considerations during production mandate that resistance among serotypes be critically analyzed by species. Figures 18 through 21 provide total percent resistance for the top 10 serotypes by major species regardless of clinical status. Differences between percent resistance become obvious from these graphs. Some serotypes (S. typhimurium, S. heidelberg, S. bredney, etc.) more than others (S. cerro, S. enteritidis, S. johannesburg, etc.) are often associated with resistance. However, this cannot be generalized among species as very little resistance is observed for S. montevideo from cattle isolates (Figure 18), while more resistance is observed for S. montevideo from chicken isolates (Figure 19). Similar observations are also noted for S. anatum.

TO further define the differences between serotypes within species, MICs for the top three serotypes within the major species as defined by clinical status are shown in Figures 22 through 30. Analysis should be conducted between serotypes within species. Interestingly, serotypes for cattle overlap more often between diagnostic (Figure 18) and slaughter (Figure 19) sources than from on-farm sources (Figure 20). MICs also tend to be higher for diagnostic isolates. For chicken isolates, the opposite appears to be indicated (Figures 21 and 22) in that there is more resistance among S. heidelberg and S. typhimurium isolates from slaughter than diagnostic sources. This may be a reflection of the low numbers of diagnostic isolates and requires further research.

MICs for Campylobacter isolates are shown in Figures 31 (C. jejuni) and 32 (C. others which includes 63 isolates identified as C. coli and 3 isolates which cannot be speciated with our primers). Higher MICs are shown for C. jejuni as compared to C. other.

Interlab Comparison:

Overall, 355/408 (86%) of MIC results obtained in CDC and ARS laboratories were identical. Of the 53 (14%) results that differed, 52/53 (98%) differed by one dilution. The one instance with >1 dilution involved Ticarcillin (3 dilutions higher). Overall, there was a 99% agreement rate with respect to interpretation of the results obtained by both laboratories.

References

1. The American Society for Microbiology Public and Scientific Affairs Board: Report of the ASM Task Force on Antibiotic Resistance. Washington, DC, March 16, 1995

2. Institute of Medicine Committee on Emerging Microbial Threats to Health in Emerging Infections: Microbial Threats to Health in the United States (ed. Lederberg, J., Shope, R.E., Oaks, S.C.) 159-160 (Washington, DC, National Academy Press, 1992).

3. US Congress, Office of Technology Assessment: Impacts of Antibiotic-Resistance Bacteria, OTA-H-629, Washington, DC, US Government Printing Office, September, 1995, 72.

4. Holmberg, S.D., Osterholm, M.T., Senger, K.A., et al.other authors. Drug-resistant Salmonella from animals fed antimicrobial agents. N Engl J Med 311, 617-622 (1984).

5. Threlfall, E.J., Frost, J.A., Ward, L.R., & Rowe, B. Epidemic in cattle and humans of Salmonella typhimurium DT104 with chromosomally integrated multiple drug resistance. Vet. Rec. 134, 577 (1994).

6. Wall, P.G., Morgan, D., Lamden, K., Ryan, M., Griffin, M., Threlfall, E.J., Ward, L.R., & Rowe, B. A case control study of infection with an epidemic strain of multi-resistant Salmonella typhimurium DT104 in England and Wales. Commun. Dis. Rep. 4, R130-R135 (1994).

7. Wall, P.G., Threlfall, E.J., Ward, L.R., & Rowe, B. Multiresistant Salmonellatyphimurium DT104 in cats: a public health risk. Lancet 348, 471 (1996).

8. Anonymous. Salmonella in animal and poultry production 1992. Ministry of Agriculture. Fisheries and Food. Welsh Office, Agricultural Department, Scottish Office, Agriculture and Fisheries Department. (1993).

9. Wall, P.G., Morgan, D., Lamden, K., Griffin, M., Threlfall, E.J., Ward, L.R., & Rowe, B. Transmission of multi-resistant strains of Salmonella typhimurium from cattle to man. Vet. Rec. 136, 591-592 (1995).

10. Threlfall, E.J., Ward, L.R., & Rowe, B. Increasing incidence of resistance to trimethoprim and ciprofloxacin in epidemic Salmonella typhimurium DT104 in England and Wales. Eurosurveillance 2, 81-84 (1997).

11. Hartmann, A., Alder, A.C., Koller, T., & Widmer, R.M.. Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater. Environmental Toxicol Chem 17, 377-382 (1998).

12. Centers for Disease Control and Prevention: Notice to Readers: Establishment of a national surveillance proven for antimicrobial resistance in Salmonella. Morbidity and Mortality Weekly Reports 45, 110-111 (1996).

13. Tollefson, L. FDA reveals plans for antimicrobial susceptibility monitoring. JAVMA 208, 459-460 (1996).

14. NCCLS. Methods for Dilution Antimicobial Susceptibility Test for Bacteria that Grow Aerobically – Fourth Edition; Approved Standards. NCCLS Document M7-A4. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1997

15. NCCLS. Performance Standards for Antimicrobial Susceptibility Testing: Eighth Informational Supplement. NCCLS Document M100 – S8. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1998

TABLE 1a. ANTIMICROBIALS - Salmonella

Antimicrobic	Antimicrobic Concentrations (ug/ml)*	Breakpoint
		(S)	(I)	(R)
Amikacin	4 - 32	<16	32	>64
Amoxicillin/Clavulanic Acid	0.5/0.25 - 32/16	<8	16	>32
Ampicillin	2 – 64	<8	16	>32
Apramycin**	2 – 16	<8	16	>32
Ceftiofur**	0.5 – 16	<2	4	>8
Ceftriaxone	0.25 – 16	<8	32	>64
Cephalothin	1 – 32	<8	16	>32
Chloramphenicol	4 – 32	<8	16	>32
Ciprofloxacin	0.015 – 2	<1	2	>4
Gentamicin	0.25 – 16	<4	8	>16
Kanamycin	16 – 64	<16	32	>64
Nalidixic Acid	4 – 64	<16		>32
Streptomycin**	32 – 256	<32		>64
Sulfamethoxazole	128 ? 512	<256		>512
Tetracycline	4 ? 64	<4	8	>16
Ticarcillin	2 ? 128	<16	32	>128
Trimethoprim/ Sulfamethoxazole	0.12/2.4 – 4/76	<2/38		>4/76

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

**breakpoints based on those used for human isolate testing

TABLE 1b. ANTIMICROBIALS - Campylobacter

Antimicrobic	Antimicrobic Concentrations (ug/ml)*	Breakpoint
		(S)	(I)	(R)
Azithromycin	0.016 – 256	<0.025	0.5 -1	>2
Chloramphenicol	0.125 – 256	<8	16	>32
Ciprofloxacin	0.016 – 32	<1	2	>4
Clidamycin	0.032 – 256	<0.5	1 – 2	>4
Gentamicin	0.025 – 16	<4	8	>16
Erythromycin	0.047 – 256	<0.5	1 – 4	>8
Nalidixic Acid	0.047 – 256	<16		>32
Streptomycin**	32 – 256	<32		>64
Tetracycline	0.023 - 32	<4	8	>16

TABLE 2: Distribution of Salmonella isolates by species and clinical status

DIAGNOSTIC (isolates collected from NVSL; n=1101)*

Species	Total Number
Cattle	181
Swine	256
Chicken**	203
Exotic	75
Turkey**	226
Dog	57
Horse	74
Cat	29

* isolates were obtained from the National Veterinary Services Laboratories, Ames, IA; the majority of isolates were obtained from a primary or secondary infection.**chicken and turkey isolates are primarily monitor samples

NON - DIAGNOSTIC (n=1,964)

Species	Number
Cattle	78
Slaughter***	1,886
Chicken	562
Turkey	240
Swine	798
Cattle	284
Egg	2

***samples collected from raw product

TABLE 2(cont.): Distribution of Salmonella isolates by species and clinical status

DIAGNOSTIC - SENTINEL (n=253)

State	Species	Number
California	Cattle Slaughter*** Chicken	15
	Horse	21
	Dog	1
	Seal	1
	Sea Turtle	1
New York	Cattle Slaughter*** Chicken	104
	Reptile	38
	Horse	16
	Cat	3
	Monkey	3
	Swine	2
	Bear	1
	Beetle	1
	Goat	1
	Sheep	1
	Walrus	1
Washington	Cattle	21
	Horse	6
	Dog	1
	Elk	1
	Iguana	1
	Mink	1

TABLE 3: Total percent veterinary Salmonella isolates sensitive, intermediate, or resistant

	Sensitive		Intermediate		Resistant
Antimicrobic	n	%	n	%	n	%
Amikacin	3318	100.0	0	0.0	0	0.0
Amoxicillin/Clavulanic Acid	2982	89.9	233	7.0	103	3.1
Ampicillin	2721	82.0	3	0.1	594	17.9
Apramycin	3271	98.6	2	0.1	44	1.3
Ceftiofur	3225	97.2	0	0.0	93	2.8
Ceftriaxone	3242	97.7	35	1.1	41	1.2
Cephalothin	3078	92.8	82	2.5	158	4.8
Chloramphenicol	3063	92.3	8	0.2	247	7.4
Ciprofloxacin	3318	100.0	0	0.0	0	0.0
Gentamicin	2902	87.5	51	1.5	365	11.0
Kanamycin	2803	84.5	27	0.8	488	14.7
Nalidixic Acid	3286	99.0	0	0.0	32	1.0
Streptomycin	2167	65.3	0	0.0	1151	34.7
Sulfamethoxazole	2258	68.1	0	0.0	1060	31.9
Tetracycline	2030	61.2	23	0.7	1265	38.1
Ticarcillin	2718	81.9	35	1.1	565	17.0
Trimethoprim/ Sulfamethoxazole	3214	96.9	0	0.0	104	3.1

TABLE 4: Percent total resistance by species/sources (includes both diagnostic and non-diagnostic Salmonella isolates)

Antimicrobic	SPECIES/SOURCE
	Cattle n=683	Swine n=1056	Chicken n=765	Turkey n=466	Horse n=117
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	4.8	1.1	1.8	4.7	12.8
Ampicillin	23.7	17.2	12.8	16.7	35.9
Apramycin	0.4	3.0	0.3	1.3	0.0
Ceftiofur	4.7	0.9	1.6	4.1	12.8
Ceftriaxone	1.5	0.4	0.5	3.2	4.3
Cephalothin	5.1	1.0	5.0	9.7	15.4
Chloramphenicol	9.1	10.6	2.2	1.5	24.8
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	3.2	2.8	16.2	33.9	19.7
Kanamycin	21.2	11.4	4.4	30.5	25.6
Nalidixic Acid	0.1	0.0	0.1	5.6	1.7
Streptomycin	28.6	36.7	28.8	55.4	37.6
Sulfamethoxazole	28.4	36.6	24.2	44.4	41.9
Tetracycline	30.6	53.9	20.9	53.4	32.5
Ticarcillin	22.4	17.0	11.8	16.5	31.6
Trimethoprim/ Sulfamethoxazole	3.8	1.7	1.2	3.6	24.8

TABLE 4 (cont.): Percent total resistance by species/sources (includes both diagnostic and non-diagnostic Salmonella isolates)

Antimicrobic	SPECIES
	Exotic* n=75	Dog n=61	Cat n=32	Egg n=2	Other n=61**
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	0.0	6.6	6.3	0.0	1.6
Ampicillin	2.7	24.6	46.9	0.0	0.0
Apramycin	0.0	1.6	0.0	0.0	0.0
Ceftiofur	0.0	6.6	6.3	0.0	0.0
Ceftriaxone	0.0	3.3	3.1	0.0	3.3
Cephalothin	0.0	8.2	12.5	0.0	0.0
Chloramphenicol	0.0	21.3	21.9	0.0	0.0
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	0.0	9.8	6.3	0.0	0.0
Kanamycin	2.7	11.5	25.0	0.0	0.0
Nalidixic Acid	2.7	0.0	0.0	0.0	4.9
Streptomycin	6.7	34.4	50.0	50.0	1.6
Sulfamethoxazole	2.7	32.8	46.9	0.0	3.3
Tetracycline	4.0	32.8	43.8	50.0	0.0
Ticarcillin	2.7	21.3	43.8	0.0	0.0
Trimethoprim/ Sulfamethoxazole	1.3	4.9	3.1	0.0	0.0

*from NVSL: iguana n=13, lizard n=8, reptile n=24, snake n=30

**from Sentinel sites; avian n=10, bear n=1, beetle n=1, elk n=1, goat n=1, iguana n=1, mink n=1, monkey n=3, reptile n=38, seal n=1, sea turtle n=1, walrus n=1

Table 5: Percent resistance for non-diagnostic Salmonellaisolates (excluding slaughter samples)

Antimicrobic

Beef Cattle

N=78

Amikacin	0.0
Amoxicillin/Clavulanic Acid	0.0
Ampicillin	1.3
Apramycin	0.0
Ceftiofur	0.0
Ceftriaxone	0.0
Cephalothin	0.0
Chloramphenicol	0.0
Ciprofloxacin	0.0
Gentamicin	2.6
Kanamycin	0.0
Nalidixic Acid	0.0
Streptomycin	11.5
Sulfamethoxazole	11.5
Tetracycline	2.6
Ticarcillin	1.3
Trimethoprim/Sulfamethoxazole	0.0

Table 6: Percent resistance for Salmonella isolated from slaughter samples

Antimicrobic	SPECIES
	Cattle n=284	Swine n=798	Chicken n=562	Turkey n=240	Egg n=2
Amikacin	0.0	0.0	0.0	0.0	0
Amoxicillin/Clavulanic Acid	2.5	0.4	2.0	.04	0
Ampicillin	9.2	13.3	13.0	10.4	0
Apramycin	0.0	1.4	0.2	0.8	0
Ceftiofur	2.1	0.1	2.0	0.4	0
Ceftriaxone	0.7	0.0	0.5	0.0	0
Cephalothin	2.1	0.1	4.4	5.0	0
Chloramphenicol	5.6	8.8	2.8	0.8	0
Ciprofloxacin	0.0	0.0	0.0	0.0	0
Gentamicin	1.8	1.0	15.5	18.3	0
Kanamycin	9.5	7.3	3.2	17.1	0
Nalidixic Acid	0.4	0.0	0.2	2.1	0
Streptomycin	16.2	29.7	27.8	40.8	50.0
Sulfamethoxazole	15.5	29.3	23.8	32.1	0
Tetracycline	24.3	47.9	20.5	45.8	50.0
Ticarcillin	8.5	13.3	11.7	10.8	0
Trimethoprim/ Sulfamethoxazole	2.5	0.5	1.4	2.5	0

Table 7: Percent resistance for diagnostic Salmonella isolates*

Antimicrobic	SPECIES
	Cattle n=181	Swine n=256	Chicken n=203**	Turkey n=226**	Horse n=74
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	7.2	3.1	1.5	9.3	10.8
Ampicillin	35.9	29.3	12.3	23.5	33.8
Apramycin	0.6	8.3	0.5	1.8	0.0
Ceftiofur	7.2	2.7	0.5	8.0	10.8
Ceftriaxone	3.9	1.6	0.5	6.6	5.4
Cephalothin	8.3	3.5	6.4	14.6	13.5
Chloramphenicol	13.3	16.0	0.5	2.2	23.0
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	3.9	8.6	18.2	50.4	23.0
Kanamycin	30.9	23.8	7.9	44.7	23.0
Nalidixic Acid	0.0	0.0	0.0	9.3	2.7
Streptomycin	39.2	58.6	31.5	70.8	41.9
Sulfamethoxazole	38.7	59.4	25.1	57.5	43.2
Tetracycline	40.3	72.3	22.2	61.5	33.8
Ticarcillin	34.3	28.5	11.8	22.6	31.1
Trimethoprim/ Sulfamethoxazole	6.1	5.1	0.5	4.9	25.7

*diagnostic isolates in Table 7 were all obtained from the National Veterinary Services Laboratories, Ames, IA; a majority of the isolates were obtained from a primary or secondary infection **although the chicken and turkey isolates were collected from the National Veterinary Service Laboratories some may be monitor samples.

Table 7 (cont.): Percent resistance for diagnostic isolates

Antimicrobic	SPECIES
	Exotic n=75	Dog n=57	Cat n=29
Amikacin	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	0.0	7.0	6.9
Ampicillin	2.7	24.6	48.3
Apramycin	0.0	1.8	0.0
Ceftiofur	0.0	7.0	6.9
Ceftriaxone	0.0	3.5	3.4
Cephalothin	0.0	8.8	13.8
Chloramphenicol	0.0	21.1	20.7
Ciprofloxacin	0.0	0.0	0.0
Gentamicin	0.0	10.5	6.9
Kanamycin	2.7	12.3	27.6
Nalidixic Acid	2.7	0.0	0.0
Streptomycin	6.7	33.3	51.7
Sulfamethoxazole	2.7	31.6	48.3
Tetracycline	4.0	31.6	44.8
Ticarcillin	2.7	21.1	44.8
Trimethoprim/ Sulfamethoxazole	1.3	5.3	3.4

Table 8 Percent resistance for all sentinel site Salmonella isolates

Antimicrobic	SPECIES
	Cattle n=140	Horse n=21	Reptile n=38	Other* n=25
Amikacin	0.00	0.00	0.00	0.00
Amoxicillin/Clavulanic Acid	9.29	16.28	2.63	3.13
Ampicillin	50.00	39.53	0.00	9.38
Apramycin	1.43	0.00	0.00	0.00
Ceftiofur	9.29	16.28	0.00	3.13
Ceftriaxone	0.71	2.33	0.00	0.00
Cephalothin	10.00	18.60	2.63	6.25
Chloramphenicol	15.71	27.91	0.00	9.38
Ciprofloxacin	0.00	0.00	0.00	0.00
Gentamicin	5.71	13.95	0.00	0.00
Kanamycin	44.29	30.23	0.00	3.13
Nalidixic Acid	0.00	0.00	0.00	0.00
Streptomycin	49.29	30.23	2.63	18.75
Sulfamethoxazole	50.71	39.53	2.63	12.50
Tetracycline	46.43	30.23	2.63	18.75
Ticarcillin	47.14	32.56	0.00	6.25
Trimethoprim/ Sulfamethoxazole	5.71	23.26	0.00	3.13

*avian=10, bear n=1, beetle n=1, cat n=3, dog n=4, elk n=1, goat n=1, iguana n=1, mink n=1, monkey n=3,
sea turtle n=1, seal n=1, sheep=1, swine, n=2, walrus n=1

TABLE 9. Top 15 Salmonella serotypes tested for 1998 (n=3,318 total isolates) for all animal species and status.

Serotype	Serogroup	Frequency (n)	Percent of Total
Heidelberg	B	324	9.8
Typhimurium (copenhagen)*	B	306	9.2
Typhimurium*	B	251	7.6
Derby	B	205	6.2
Kentucky	C3	202	6.1
Anatum	E4	140	4.2
Hadar	C2	115	3.5
Agona	B	96	2.9
Infantis	C2	96	2.9
Johannesburg	R	93	2.8
Senftenburg	E4	90	2.7
Muenster	E1	81	2.4
Montevideo	C1	79	2.4
Bredeney	B	74	2.2
Muenchen	C2	67	2.0

Typhimurium and Typhimurium var. Copenhagen isolates combined account for 557 (16.8%) of the total number of isolates

Table 10: Percent total resistance for the top 15 Salmonella serotypes tested from all animal species/sources

Antimicrobic	Heidelberg n=324	Typh(cop) n=306	Typhim. n=251	Derby n=205	Kentucky n=202
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	3.1	7.2	6.4	0.0	0.0
Ampicillin	21.6	77.8	47.8	2.0	1.5
Apramycin	1.5	1.6	0.4	3.9	0.5
Ceftiofur	2.8	6.9	4.4	0.0	0.0
Ceftriaxone	0.6	2.9	0.8	0.0	0.0
Cephalothin	9.3	9.2	10.8	0.0	0.0
Chloramphenicol	3.7	35.3	25.1	1.0	0.5
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	20.1	4.9	15.1	4.4	5.0
Kanamycin	20.4	43.1	31.5	2.0	3.0
Nalidixic Acid	0.0	0.0	0.0	0.0	0.0
Streptomycin	44.1	82.7	53.8	56.6	19.3
Sulfamethoxazole	31.5	82.4	55.8	54.6	10.9
Tetracycline	28.1	79.7	50.2	59.5	20.3
Ticarcillin	21.3	74.8	47.0	2.0	1.5
Trimethoprim/ Sulfamethoxazole	3.4	3.3	7.2	0.0	1.0

Table 10 (cont.): Percent total resistance for the top 15 Salmonella serotypes tested from all animal species/sources

Antimicrobic	Anatum n=140	Hadar n=115	SEROTYPE Agona n=96	Infantis n=96	Johannes. n=93
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	0.0	0.9	7.3	5.2	0.0
Ampicillin	5.7	10.4	15.6	5.2	1.1
Apramycin	1.4	0.0	2.1	4.2	1.1
Ceftiofur	0.0	0.9	7.3	5.2	0.0
Ceftriaxone	0.0	0.0	2.1	4.2	0.0
Cephalothin	1.4	3.5	8.3	5.2	0.0
Chloramphenicol	0.7	0.9	13.5	5.2	2.2
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	6.4	18.3	13.5	4.2	1.1
Kanamycin	5.7	13.9	17.7	3.1	2.2
Nalidixic Acid	0.0	0.9	0.0	1.0	0.0
Streptomycin	12.9	80.0	32.3	14.6	4.3
Sulfamethoxazole	12.9	27.0	57.3	16.7	4.3
Tetracycline	41.4	87.0	57.3	16.7	32.3
Ticarcillin	5.7	10.4	11.5	5.2	1.1
Trimethoprim/ Sulfamethoxazole	5.0	3.5	12.5	3.1	0.0

Table 10 (cont.): Percent total resistance for the top 15 Salmonella serotypes tested from all animal species/sources

Antimicrobic	Senften. N=90	Muenster N=81	SEROTYPE Monte. n=79	Bredeney N=74	Muechen n=67
Amikacin	0.0	0.0	0.0	0.0	0.0
Amoxicillin/Clavulanic Acid	3.3	0.0	0.0	23.0	0.0
Ampicillin	8.9	8.6	2.5	24.3	6.0
Apramycin	1.1	2.5	1.3	1.4	1.5
Ceftiofur	1.1	0.0	0.0	23.0	0.0
Ceftriaxone	1.1	0.0	0.0	20.3	0.0
Cephalothin	4.4	4.9	0.0	24.3	0.0
Chloramphenicol	1.1	4.9	1.3	1.4	4.5
Ciprofloxacin	0.0	0.0	0.0	0.0	0.0
Gentamicin	36.7	22.2	10.1	56.8	4.5
Kanamycin	18.9	22.2	7.6	55.4	10.4
Nalidixic Acid	0.0	4.9	0.0	25.7	1.5
Streptomycin	43.3	29.6	12.7	66.2	22.4
Sulfamethoxazole	23.3	24.7	11.4	60.8	28.4
Tetracycline	21.1	32.1	12.7	58.1	32.8
Ticarcillin	8.9	8.6	1.3	21.6	6.0
Trimethoprim/ Sulfamethoxazole	2.2	2.5	1.3	0.0	1.5

Table 11: Multiple antimicrobial resistance for Salmonella isolates

Number of Antimicrobics Resistant to	No. Isolates	Percent of Isolates
0	1723	51.9
1	270	8.1
2	265	8.0
3	349	10.5
4	120	3.6
5	119	3.6
6	291	8.8
7	46	1.4
8	38	1.1
9	32	1.0
10	15	0.5
11	30	0.9
12	14	0.4
13	4	0.1
14	2	0.1
Total	3318	100

Table 12: Most frequent resistance patterns for tested Salmonella isolates

AntimicrobicsAntimicrobics	No. Isolates	Percent
Tet	217	6.5
Strep/Sulfa/Tet	155	4.7
Amp/Kan/Strep/Sulfa/Tet/Tic	133	4.0
Amp/Chlor/Strep/Sulfa/Tet/Tic	123	3.7
Strep/Tet	98	3.0
Gen/Strep/Sulfa	67	2.0
Sulfa/Tet	63	1.9
Gen/Strep/Sulfa/Tet	55	1.7
Kan/Strep/Tet	52	1.6

Table13: Most frequent resistance patterns with 5 or more antimicrobics for tested Salmonella isolates

Antimicrobics	No. Isolates	Percent
Amp/Kan/Strep/Sulfa/Tet/Tic	133	4.0
Amp/Chlor/Strep/Sulfa/Tet/Tic	123	3.7
Gen/Kan/Strep/Sulfa/Tet	40	1.2
Amp/Kan/Strep/Sulfa/Tic	20	0.6
Amp/Strep/Sulfa/Tet/Tic	18	0.5
Amp/Ceph/Gen/Kan/Strep/Sulfa/Tet/Tic	18	0.5
Amp/Chlor/Kan/Strep/Sulfa/Tet/Tic	16	0.5
Gen/Kan/Nal/Strep/Sulfa/Tet	14	0.4
Amo/Amp/Cefti/Ceftr/Gen/Kan/Strep/Sulf/Tet/Tic	13	0.4
Amp/Gen/Strep/Sulfa/Tic	13	0.4
Amo/Amp/Ceft/Ceph/Chlor/Gent/Kan/Strep/Sulfa/Tet/Tic/Trisul	9	0.3
Amp/Chlor/Gen/Kan/Strep/Sulfa/Tet/Tic/Trisul	8	0.2
Amp/Gen/Strep/Sulfa/Tet/Tic	6	0.2

Table 14: Total S. typhimurium percent resistance with ACSSuT or ACSuT pattern

	Typhim.	Percent of Typh Total n=251 n=3318		Typh(cop)	Percent of T(cop) Total n=306 n=3318		Total	Percent of T+cop Total n=557 n=3318
No. of Isolates	251	100	7.6	306	100	9.2	557	100	16.8
ACSSuT (penta-resistant)	56	22.3	1.7	107	35.0	3.2	163	29.3	4.9
ACSuT (quad-resistant)	3	1.2	0.1	1	0.3	0.03	4	0.7	0.1
Total	59	23.5	1.8	108	35.3	3.3	167	30.0	5.0

Table 15: Number of DT104 and DT104 complex isolates with ACSSuT and ACSuT patterns

	Typhim.	Percent of Typh Total n=251 n=3318		Typh(cop)	Percent of Typh Total n=306 n=3318		Total	Percent of T+cop Total n=557 n=3318
ACSSuT (penta-resistant)
DT104	33	1301	1.0	42	13.7	1.3	75	13.5	2.3
DT104A	1	0.4	0.03	2	0.7	0.06	3	0.5	0.01
DT104B	2	0.8	0.06	32	10.5	1.0	34	6.1	1.0
DT104C	0	-	-	0	-	-	-	-	-
ACSSuT (quad-resistant)
DT104	1	0.4	0.03	0	-	-	1	0.2	0.03
DT104A	0	-	-	0	-	-	0	-	-
DT104B	0	-	-	1	0.3	0.03	1	0.2	0.03
DT104C	0	-	-	0	-	-	-	-	-
Total	37	14.7	1.1	77	25.2	2.3	114	20.5	3.4

Table 16: Phage types other than DT104 within ACSSuT and ACSut resistance patterns

	Typhim.	Percent of Typh Total n=251 n=3318		Typh(cop)	Percent of Typh Total n=306 n=3318		Total	Percent of Typh Total n=557 n=3318
ACSSuT (penta-resistant)
DT12	1	0.4	0.03	4	1.3	0.12	5	0.9	0.15
DT120	1	0.4	0.03	0	-	-	1	0.18	0.03
DT189	1	0.4	0.03	0	-	-	1	0.18	0.03
DT192	1	0.4	0.03	0	-	-	1	0.18	0.03
DT193	7	2.8	0.2	1	0.3	0.03	8	1.4	0.24
DT208	0	-	-	1	0.3	0.03	1	0.18	0.03
RNDC	4	1.6	0.12	0	-	-	4	0.72	0.12
U302	2	0.8	0.06	12	3.9	0.36	14	2.5	0.42
Untypable	4	1.6	0.12	12	3.9	0.36	16	2.9	0.48
Total	21	8.4	0.63	30	9.8	0.9	51	9.2	1.5
ACSSuT (quad-resistant)
DT12	-	-	-	-	-	-	-	-	-
DT120	-	-	-	-	-	-	-	-	-
DT189	-	-	-	-	-	-	-	-	-
DT192	-	-	-	-	-	-	-	-	-
DT193	2	0.8	0.06	-	-	-	2	0.36	0.06
DT208	-	-	-	-	-	-	-	-	-
RNDC	-	-	-	-	-	-	-	-	-
U302	-	-	-	-	-	-	-	-	-
Untypable	-	-	-	-	-	-	-	-	-
Total	23	9.2	0.69	30	9.8	0.9	53	9.5	1.6