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.

Plot Box & Whisker:

28,0 RAX

27,6

27,2
R
A
X
26,8

26,4

±1.96*SE
±1.00*SE
26,0
97 98 99 Mean

YEAR

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 MIC breakpoint ∅ ∅ R ∅ 90 ∅ 50 MIC MIC
µg/ml), sensitive
(µ µg/ml), resistant
(µ mm mm 90 50
Rifaximin 1 4 19 10 25 27 0.386 0.291
 rifaximin
y=26,187-4,188*x+eps
32

28

24
Diameter

20

16

12

8
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
MIC

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.
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