International Journal of Antimicrobial Agents 27 (2006) 538–540
Interpreting streptomycin susceptibility test results forSalmonella enterica serovar Typhimurium
Geraldine Doran a,∗, Martina NiChulain b, Niall DeLappe a, Colette O’Hare a,Geraldine Corbett-Feeney a,b, Martin Cormican a,b
a National Salmonella Reference Laboratory, University College Hospital, Galway, Irelandb Department of Bacteriology, National University of Ireland, Galway, Ireland
Received 12 January 2006; accepted 7 March 2006
Abstract
Resistance or susceptibility of Salmonella enterica to streptomycin is widely used as an epidemiological marker. However, there is no clearconsensus on the interpretation of streptomycin susceptibility test results. Comparison of results obtained with the Clinical and LaboratoryStandards Institute (CLSI) disk diffusion method, the minimum inhibitory concentration (MIC) determined by Etest and streptomycin resistanceg≤©
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enotype for 90 isolates of S. enterica serovar Typhimurium suggests that appropriate interpretive criteria for MIC results are susceptible at8 mg/L and resistant at ≥16 mg/L. For CLSI disk diffusion, we propose susceptible at a zone diameter ≥13 mm and resistant at ≤10 mm.2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
eywords: Streptomycin; Salmonella Typhimurium; Resistance; Disk diffusion; MIC; Resistance genotype
. Introduction
Although streptomycin has no role in the therapy ofalmonella infection, testing for susceptibility to strepto-ycin is widely used as an epidemiological marker. For
xample, resistance to streptomycin is part of the character-stic phenotype of resistance to ampicillin, chloramphenicol,treptomycin, sulphonamides and tetracyclines (ACSSuT)ssociated with Salmonella enterica serovar Typhimuriumefinite phage type DT104. External quality assessmentxercise involving laboratories across Europe reportingusceptibility test results for isolates of Salmonella foundhat agreement between laboratories, although overall quiteigh, was least satisfactory for streptomycin [1,2]. A reasonor the poor concordance identified in the report is a lack ofidely accepted criteria for interpretation of results of testingf susceptibility of streptomycin against Enterobacteriaceae.
The Clinical and Laboratory Standards Institute (CLSI)pecifies a disk content (10 �g), a method and interpretive
∗ Corresponding author. Tel.: +353 91 544 628.
criteria (susceptible ≥15 mm, intermediate 12–14 mm andresistant ≤11 mm) for disk diffusion susceptibility testingof streptomycin against Enterobacteriaceae [3]. Unlike mostantimicrobial agents, however, there are no correspondingCLSI interpretive criteria for dilution susceptibility testing[3]. The Neo-Sensitabs disk diffusion system of Rosco Diag-nostics, used in a number of published studies, specifies a100 �g disk content and criteria of susceptible at ≥26 mm,intermediate at 23–25 mm and resistant at ≤22 mm (Rosco,Taastrup, Denmark) [4,5]. As with the CLSI, correspondinginterpretive criteria for dilution susceptibility testing are notstated.
In their 2002 report, the US National AntimicrobialResistance Monitoring System (NARMS) performed brothmicrodilution susceptibility testing in Sensititre plates andapplied the criteria of susceptible at ≤32 �g/mL and resis-tant at ≥64 �g/mL [6]. Bywater et al. also considered thebreakpoint for resistance as ≥64 �g/mL [7]. The GlobalSalmonella Surveillance EQUAS exercise applies the crite-ria of susceptible at ≤8 �g/mL and resistant at ≥32 �g/mLbased on examination of population distribution (personal
E-mail address: [email protected] (G. Doran). communication).
924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.oi:10.1016/j.ijantimicag.2006.03.005
G. Doran et al. / International Journal of Antimicrobial Agents 27 (2006) 538–540 539
Streptomycin resistance in Enterobacteriaceae is mostoften attributable to aminoglycoside-modifying enzymes [8].The phosphotransferases encoded on genes strA (aph(3′′)-Ib) and strB (aph(6′′)-Id) are reported as being commonlypresent in Salmonella [5,9]. The aadA1 (ant(3′′)-Ia) gene wasreported as the most frequently identified aminoglycosideresistance determinant in Salmonella of multiple serovarsby Chen et al. [10]. The aadA2 (ant(3′′)-Ib) gene has alsobeen identified as an important mechanism of streptomycinresistance in Salmonella and is the basis for streptomycinresistance in the widely disseminated multidrug-resistant S.Typhimurium DT104 clonal group [11].
A review of our routine disk susceptibility test data for S.enterica indicated a consistent pattern of bimodal distributionover the years. The range of zone diameter values observedfor the apparent wild-type population of S. Typhimurium inour laboratory is 10–16 mm with a mode of 14 mm (cur-rently 12–14 mm is considered intermediate by the CLSI).For S. Enteritidis, the range is 13–20 mm with a mode of17 mm. Therefore, the modal value for the wild-type dis-tribution falls within the range categorised as intermediate(12–14 mm) for S. Typhimurium but not for S. Enteritidis.On seven occasions in 3 years of external quality assess-ment exercises, we reported as intermediate (six occasions)or resistant (one occasion) isolates of S. Typhimurium forwhich the intended result was susceptible. On each occasionoEafac
2
tpCl(r(a(D(Ddsdt2pa
were those previously described [12,13]. DNA of E. coli K12was included as a negative control in all amplification reac-tions.
3. Results and discussion
Table 1 shows the relationship between MIC, zone diam-eter and genotype. All isolates with MIC ≥16 mg/L werepositive for one or more of the three streptomycin resistancedeterminants. All isolates with MIC ≤8 mg/L were negativefor streptomycin resistance determinants. The diameter of thezone of inhibition showed an inverse relationship with MIC.There was no clear separation between the gene-positive andgene-negative populations by disk diffusion. However, all 42isolates with zone diameter ≥13 mm were resistance gene-negative and all 35 isolates with a zone diameter ≤10 mmwere resistance gene-positive. The 13 isolates with zonediameters in the range 11–12 mm included 4 resistance gene-positive and 9 resistance gene-negative isolates. Althoughthe numbers are small, there is a suggestion of a relationshipbetween the specific resistance genotype and the MIC. Forsix isolates with only aadA1, the median MIC was 16 mg/L,whereas for 20 isolates with only aadA2 the median MICwas 64 mg/L. Eleven isolates had aadA1 and aadA2 (medianMIC 32 mg/L) and two isolates had all three resistance deter-m
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ur result was reproduced in our laboratory, and results withscherichia coli ATCC 25922 were consistently within thecceptable range. This experience and the lack of any basisor a consensus in the literature prompted us to evaluate theppropriateness of current CLSI disk diffusion interpretiveriteria for streptomycin against S. Typhimurium.
. Materials and methods
Ninety isolates of S. Typhimurium comprising 40 resis-ant, 26 intermediate and 24 susceptible isolates based onrevious susceptibility results performed according to theLSI disk diffusion criteria [3] were selected. The iso-
ates were from human (n = 56), porcine (n = 14), bovinen = 3) and other animal (n = 10) sources as well as isolateseceived as part of external quality assessment programmesn = 7). The 83 routine isolates were collected during 2001nd 2003 and comprised the following phage types: DT104n = 10), DT104b (n = 30), DT12 (n = 2), DT120 (n = 4),T132 (n = 2), DT193 (n = 5), DT2 (n = 2), DT4 (n = 2), U302
n = 12), U310 (n = 4), U311 (n = 3), and one isolate each ofT18, DT21, DT40, DT41, DT85, DT99 and U302V. CLSIisk diffusion testing was repeated on all isolates and thetreptomycin minimum inhibitory concentration (MIC) wasetermined by Etest according to the manufacturer’s instruc-ions using Mueller–Hinton agar. Escherichia coli ATCC5922 was used as a control. All isolates were tested byolymerase chain reaction (PCR) for genes strA, aadA1 andadA2 [12,13]. PCR primers and amplification conditions
inants.These data show that the great majority (22 of 23 iso-
ates) of S. Typhimurium categorised as intermediate byurrent CLSI disk diffusion interpretive criteria lack strep-omycin resistance determinants and represent one end ofhe spectrum of zone diameter values observed within theild-type population. We suggest that the interpretive crite-
ia should be modified to resistant at ≤10 mm and susceptiblet ≥13 mm, with the range 11–12 mm considered intermedi-te/indeterminate. In our laboratory we now determine theIC for isolates with zone diameters of 11–12 mm. Our data
uggest that interpretive criteria for MIC results should beusceptible at ≤8 mg/L and resistant at ≥16 mg/L.
able 1inimum inhibitory concentration (MIC), zone diameter range and strep-
omycin resistance genotype of 90 isolates of Salmonella enterica serovaryphimurium
ICmg/L)
No. ofstrains
Zone diameterrange (mm)
No. genepositive
No. genenegative
4 14–17 0 413 12–17a 0 1334 11–17a 0 34
6 12 7–11a 12 02 7 7–12a 7 04 17 6–7 17 028 1 6 1 0256 2 6 2 0
otal 90 – 39 51a A total of five isolates gave a zone diameter of 11 mm (three gene positive,
wo gene negative) and eight isolates gave a zone diameter of 12 mm (oneene positive and seven gene negative).
540 G. Doran et al. / International Journal of Antimicrobial Agents 27 (2006) 538–540
Re-evaluation of interpretive criteria for susceptibil-ity testing of streptomycin against Enterobacteriaceae hasreceived a low priority because it is of little clinical interest.However, given the epidemiological applications of the strep-tomycin resistance phenotype, this subject merits a reassess-ment by the CLSI and other agencies that determine break-point interpretive criteria. Additional studies in independentlaboratories should be performed to provide additional evi-dence to support such a re-evaluation.
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