Detection of Methicillin Resistance in Staphylococci employing the UroQuick System' S' Roveta, S' Ca

1
Detection of Methicillin Resistance in
Staphylococci employing the Uro-Quick System.S.
Roveta, S. Cagnacci, F. Cavallini, D. Rocca, A.
Marchese, E.A.Debbia. Institute of Microbiology,
University of Genoa, Italy.
15th ECCMID, Copenhagen, April 2-5, 2005.
Paper 1386
Abstract DETECTION OF METHICILLIN RESISTANCE IN
STAPHYLOCOCCI EMPLOYING THE URO-QUICK SYSTEM. S.
Roveta, S. Cagnacci, F. Cavallini, D. Rocca, A.
Marchese, E.A.Debbia. Objectives Uro-Quick
system has been employed to detect
Methicillin-resistance (Met-R) in S. aureus and
coagulase-negative staphylococci (CoNS). In order
to achieve full agreement between the antibiotic
susceptibility results obtained by the reference
method (NCCLS) and the Uro-Quick system, the
optimal experimental conditions (inoculum size,
time of incubation, antibiotic and NaCl
concentration) were determined for S. aureus and
CoNS respectively. Methods 72 Met-R
staphylococci including different species (42 S.
aureus and 30 CoNS, in which 12 S. epidermidis)
heterogeneous in their expression of resistance
to b-lactam agents were screened with the
Uro-Quick System. S. aureus ATCC 29213 (mecA
negative) and S. aureus ATCC 43300 (mecA
positive) were used for quality control.
Oxacillin (in appropriate concentration following
the NCCLS breakpoints) was added in a vial
containing 2 ml of suspension of strain to tested
(a drug-free vial was used as control). After an
opportune time of incubation the instrument
printed the results no growth and a growth curve
like the control are representative of a
susceptible and resistant strain
respectively. Results The best results were
obtained using Mueller-Hinton broth and a
concentration of 106 cells/ml for S. aureus and
5x106 for CoNS. All the 42 S. aureus tested grown
within 5 hours of incubation and the met-R
phenotype was correctly detected within 6 hours
in 100 of strains (66.7 and 93.3 in 4 and 5
hours respectively). The 97 of CoNS strains
grown within 10 hours of incubation, an
insufficient growth within this period of time
was observed for 1 strain of S. haemolyticus
only. Met-R was correctly detected within 10
hours in 100 of strain grown at this time (55,
64 and 95 in 6 , 7 and 8 hours
respectively). Conclusion On the basis of the
present findings the Uro-Quick system appears to
be useful for the rapid detection of met-R
staphylococci. Excellent results were obtained on
S. aureus, concerning CoNS our result suggest
that there are differences in the growth rate
among the various members of this group and in
the incubation time necessary for the met-R
detection (more isolates of the respective
species must be analyzed to reach generalized
conclusion).
Methods The Uro-Quick System is an automated
rapid method, introduced by Alifax (Padua,
Italy), for bacteriuria screening which uses
laser-nephelometry (light scattering) to monitor
the growth of bacteria in vials containing broth
(Figure 2).The inoculated vials are incubated at
37C and magnetic stirred bars keep the samples
in suspension and well aerated to promote
bacterial growth. Antimicrobial susceptibility
test is carried out adding an aliquot (0.1 ml) of
the sample to a test tube containing Oxacillin in
appropriate concentration (following the NCCLS
breakpoints). A vial without drug is used as
control. After an opportune time of incubation
the instrument printed the results no growth and
a growth curve like the control are
representative of a susceptible and resistant
strain respectively (Figure 3). 100 Met-R
staphylococci including different species (48 S.
aureus and 52 CoNS 23 S. epidermidis, 15 S.
haemolyticus, 4 S. warneri, 4 S. saprophyticus, 4
S. capitis, 1 S. simulans and 1 S. hominis)
heterogeneous in their expression of resistance
to b-lactam agents were screened with the
Uro-Quick System. S. aureus ATCC 29213 (mecA
negative) and S. aureus ATCC 43300 (mecA
positive) were used for quality control.
Results The best results were obtained using
Mueller-Hinton broth and a concentration of 106
cells/ml for S. aureus and 5x106 for CoNS. The
97.9 of the S. aureus tested grown within 5
hours of incubation and the met-R phenotype was
correctly detected within 8 hours in all these
strains (76.6 and 93.6 in 5 and 6 hours
respectively). Only one strain (showing very
small colonies when cultured on to agar plate)
grown within 8 hours and was correctly detected
within 18 hours. The 98.1 of CoNS strains
grown within 8 hours of incubation, an
insufficient growth within this period of time
was observed for 1 strain of S. haemolyticus
only. Met-R was correctly detected within 8 hours
in all the 51 strains grown at this time (55,
90.2 and 98 in 6 , 7 and 8 hours
respectively). Met-R in the S. haemolyticus
growing within 10 hours was correcty detected
within 18 hours of incubation.
Introduction and Purpose The Uro-Quick (Figure
1), an automatic instrument used for the
screening and determination of bacteriuria in 3-5
hours, was recently employed to detect antibiotic
resistance in well characterized bacterial
strains and in uropathogens (Roveta et al., 2004)
. In this study Uro-Quick system has been
employed to detect Methicillin-resistance (Met-R)
in S. aureus and coagulase-negative staphylococci
(CoNS). In order to achieve full agreement
between the antibiotic susceptibility results
obtained by the reference method (NCCLS) and the
Uro-Quick system, the optimal experimental
conditions (inoculum size, time of incubation,
antibiotic and NaCl concentration) were
determined for S. aureus and CoNS respectively.
Figure 2 Light scattering
Conclusion On the basis of the present findings
the Uro-Quick system appears to be useful for the
rapid detection of met-R staphylococci. Our
results suggest that there are differences in
the growth rate among the various members of this
group and in the incubation time necessary for
the met-R detection (more isolates of the
respective species must be analyzed to reach
generalized conclusion).
Figure 1 Uro-Quick
Author for correspondence Eugenio A.
Debbia Institute of Microbiology, University of
Genoa. Largo R. Benzi 10 -16132 Genoa,
Italy. Tel [phone], Fax
39-010-3537698 eugenio.debbia_at_unige.it
Figure 3 Susceptible and resistant strains