Sensitivity to Rifaximin of strains of Staphylococcus aureus isolated from 1997 to 1999 - Comparison between years and calculation of the correlated MIC

Vicenzoni G. 1, Ponzoni A. 2, Madinelli R.1, Benini V. 1, Fiorini P. 1 & Farina R. 3 1 The Experimental Zooprophylactic Institute of Veneto - Verona Section 2 Study regarding consultancy and statistical analysis, PiQuadro Studio - Padova - Italy 3 FATRO - Ozzano Emilia - Bologna - Italy Summary The authors verified whether during 1997-1999 there had been a variation in sensitivity to Rifaximin of Staphylococcus aureus strains isolated from single quarter milk samples, through statistical analysis of frequency distributions of zones of inhibition. Furthermore, considering the breakpoints of Rifaximin, they extrapolated the MIC90 and MIC50 values, basing themselves on the diameter of the zones of inhibition within which 90% and 50% of tested strains are included. Calculation of the correlated MIC is based on the detection of a direct correlation between the MIC and the diameter of the zone of inhibition. The investigation detected no variation of pharmacosensitivity and established that the correlated MIC90 is 0.386 µg/ml Key words: Staph. aureus, Mastitis, Rifaximin, MIC Introduction Bovine mastitis is the principal pathology affecting dairy herds and Staphylococcus aureus is one of the pathogens most frequently isolated from both subclinical and chronic infections (1, 8). The use of specific control protocols (6) has decreased, in general, the incidence of mastitis, but in spite of this, Staphylococcus aureus remains one of the most important microorganisms associated with clinical and subclinical bovine mastitis. The one instrument available to the veterinarian for the control of this infection is the correct sanitary management of the herd. Therapy in the dry period and, when considered necessary, therapy in lactation, are an integral part of this management. Buiatrics base their therapeutic choices on clinical experience, comparing the results of the examinations performed in vitro with the results obtained in vivo. The laboratory, in addition to performing the antibiograms, should be able to highlight variations in pharmacological sensitivity of the isolated strains with regard to the various antimicrobial agents and to supply a constant report on the data recorded; to do this, it must have cheap, rapid and reliable instruments available. The objective of this research was to evaluate, through statistical methods, the frequency distributions of the zones of inhibition of Rifaximin and to also evaluate, over the period, any variations in pharmacological sensitivity to this antibiotic of Staphylococcus aureus, isolated from samples of single quarter milk of cows with subclinical mastitis; finally, to also determine the correlated MIC50 and the MIC90 for these strains. Materials and methods The research was performed at the Verona Laboratory of the Experimental Zooprophylactic Institute of Veneto and is based on data regarding measurements of the zones of inhibition of Rifaximin, for strains of Staphylococcus aureus isolated in the three-year period 1997-1999 from samples of single quarter milk of cows with subclinical mastitis. Over these years, 211, 305 and 422 antibiograms were performed, giving a total of 938 trials. These were performed using the Kirby Bauer method, following the internal procedures used at our Institute. The zones of inhibition for Rifaximin were measured with calipers, they were recorded on an Excel spreadsheet and elaborated with suitable statistical methods. The hypothesis of distributional normality was evaluated by means of the Kolmogorov-Smirnov test. For verification of the presence of significant differences in the distribution and in the mean of the diameter of the zones of inhibition between the three years considered, the non-parametric Kruskall-Wallis ANOVA test was used. As regards the correlated

MIC50 and MIC90 value, this was calculated on the basis of the breakpoint values indicated by the pharmaceutical company, corresponding to 1 µg/ml for a zone of inhibition ≤ 10 mm (resistance value) and 4 µg/ml for a zone of inhibition ≥ 19 mm (sensitivity value). Results The three graphs show the histograms of the frequencies of the zones of inhibition of Rifaximin, for the strains of Staphylococcus aureus found over the three years of observation.

In the Plot Box & Whisker graph are shown the mean values of the zone of inhibition of the three years and the respective standard errors.

For the purposes of verifying the existence of significant differences in the distribution of the frequencies of the zones of inhibition and in the mean of these over the three years, the hypothesis of normality in the distribution of the variable found was first verified. The hypothesis of normality of the values was evaluated by means of the Kolmogorov-Smirnov test. The results of the test rejected the hypothesis of normality of the distribution as a value of p < 0.01 was recorded throughout the three years. In consideration of the non-normal distribution of the values recorded, to verify the existence of significant differences in the distribution of the frequencies of the zones of inhibition and in the mean of these over the three years, it was decided to use the non-parametric Kruskall-Wallis ANOVA test. The result of this analysis showed that there are no significant differences in the distribution of the frequencies of the zones of inhibition and the mean over the three years examined. Based on the diameter of the zone of inhibition within which 90% and 50% of the strains examined, respectively, are included, the MIC90 and the MIC50 values were extrapolated. The calculation of the correlated MIC is based on evidence of a direct relationship between the MIC and the diameter of the zone of inhibition (2, 3, 4, 5, 7). 90% of the strains were included in a zone ≥ 25 mm, while 50% of the strains were included in a zone ≥ 27 mm. Considering the breakpoint values, corresponding to 1 µg/ml for a zone diameter of 19 mm and 4 µg/ml for a zone diameter of 10 mm, we determined the correlated MIC value corresponding to the value of the diameter including 90% and 50% of the strains examined. As no statistically significant differences were found between the various years, we decided to calculate the correlated MIC, considering the data recorded over the three years as a single sample. The MIC90 was 0.386 µg/ml while the MIC50 was 0.291µg/ml (Table 1). Table 1

MIC breakpoint (µµµµg/ml), sensitive

MIC breakpoint (µµµµg/ml), resistant

∅∅∅∅ mm

∅∅∅∅ R mm

∅∅∅∅ 90 ∅∅∅∅ 50 MIC 90

MIC 50

Rifaximin 1 4 19 10 25 27 0.386 0.291

±1.96*SE ±1.00*SE Mean

Plot Box & Whisker: RAX

YEAR

R A X

26,0

26,4

26,8

27,2

27,6

28,0

97 98 99

Discussion and conclusions One of the tasks of a public analysis laboratory should be that of constant observation of the course of antibiotic resistance, so as to supply periodic reports to the clinician on the real situation. The method most used to verify the appearance of antibiotic resistance by a microorganism to a given antibiotic is that of the determination of the Minimum Inhibitory Concentration (MIC). The classic method for determination of the MIC presents obvious difficulties and limits; for this reason, it is normally performed on a limited number of bacterial strains belonging to the same genus at multiyear intervals. A calculation based on a limited number of strains can lead to erroneous conclusions, as these may not be representative of the antimicrobial population which causes the pathology. To perform periodic reports, the laboratory needs methods which, in addition to being accurate, are also simple, rapid and cheap. For the purpose, we used the method of calculation of the correlated MIC. This method is based on the presupposition that there is a direct relationship between the diameter of the zone of inhibition and the Minimum Inhibitory Concentration. Knowing the breakpoints regarding the antibiotic being examined, which in the case of Rifaximin are 4 µg/ml for a zone ∅ of ≥10 mm (Resistance values) and 1 µg/ml for a zone ∅ of ≥19 mm (Sensitivity values), it is possible to construct a regression line. Using the values of the diameter of the inhibition measured and recorded in the various antibiograms performed by the laboratory routinely, it is possible to annually determine, mathematically, the diameter within which 90% and 50% of the strains examined are represented. Through a mathematical function, it is possible to determine the value in µg/ml corresponding to the diameter obtained. In this work, we have reported only the value regarding Rifaximin, but it is possible to perform this study on all the antibiotics tested in the space of these three years, on condition that the necessary breakpoint values with which to construct the regression line are known. As regards the antibiotic studied in this trial, in spite of its wide use in dry period therapy, it should be noted that it has not produced, over these 3 years, significant variations in the frequency of the diameter of the zones of inhibition and, consequently, in the level of the correlated MIC. As regards the correlated MIC90, it must be pointed out that the value determined by us, on 938 strains of Staphylococcus aureus examined, is lower than that reported by the manufacturing pharmaceutical company. This lower value, found in vitro, must be considered positive, as it indicates that lower concentrations are necessary to inhibit 90% of the strains examined. We believe that this procedure, in addition to supplying objective data, permits periodic reports to be supplied to the clinical veterinarian which, on the basis of these data, and knowing the microorganism involved, can direct therapy in a rearing establishment.

rifaximin y=26,187-4,188*x+eps

MIC

Dia

met

er

8

12

16

20

24

28

32

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5

References 1. Bramley AJ, Mastitis. In: Andrews AH, Blowey RW, Boyd H,Eddy RG (Eds), Bovine

Medicine: Diseases and Husbandry of Cattle, Blackwell Scientific Publication, (1992) Boston, pp.289-300;

2. Chapuis C, Delignette-Muller ML, Flandrois JP - An economic approach to the MIC, Zentralbl Bakteriol (1996) Jun;284(1):67-74;

3. Development of in vitro susceptibility criteria and quality control parameters for veterinary antimicrobial agents; approved guideline M37-A, vol. 19 No.3, NCCLS, February 1999;

4. Kronvall G, Analysis of a single reference strain for determination of gentamicin regression line constants and inhibition zone diameter breakpoints in quality control of disk diffusion antibiotic susceptibility testing, J. Clin. Microbiol. (1982) Nov; 16(5):784-793;

5. Kronvall G, Single-strain regression analysis for determination of interpretive breakpoints for cefoperazone disk diffusion susceptibility testing, J. Clin. Microbiol. (1983) Jun;17(6):975-980;

6. National Mastitis Council, 1996. Method of controlling mastitis in dairy cows. In: Current Concepts of Bovine Mastitis, 4th ed. pp. 39-46;

7. Performance standards for antimicrobial disk and dilution susceptibility tests for bactera isolated from animals; tentative standard M31-T, vol. 17 No. 11, NCCLS, August 1997;

8. Watts JL, Etiological agents of bovine mastitis, Vet. Microbiol. (1988), 16:41-66;