a Department of Clinical and Biological Sciences, University of Torino; b Laboratory, Agnelli Hospital, Pinerolo (Torino); c Laboratory, Paediatric Hospital, Torino, Italy
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Abstract |
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Introduction |
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Group A streptococci are still susceptible to penicillin and for patients allergic to this antibiotic, erythromycin or other macrolides are the drugs of choice.3 Lately, however, an increase in macrolide resistance has been observed in different countries.48 Resistance to most macrolides, lincosamides and streptogramin B (MLS resistance) can be constitutive or inducible. It is linked to a target modification occurring at the level of the ribosomes via the ermAM gene which encodes a 23S rRNA methylase.910 Recently, Seppala et al.11 found that inducible resistance can also be due to a novel gene, named ermTR, which does not belong to the ermAM class of genes. Another mechanism of erythromycin resistance, mediated by an efflux pump and encoded by the mefA gene, which is specific for 14- and 15-membered macrolides and type B streptogramin molecules but not for lincosamide antibiotics, has been described.12,13
In this work we have analysed 221 S. pyogenes isolates from throat swabs of untreated children with uncomplicated pharyngotonsillitis. These isolates, from Torino and a nearby city situated in the north-west of Italy, were assessed for their macrolide resistance phenotype. Since a relationship of T protein agglutination pattern and opacity factor (OF) production with M protein in group A streptococci has been found14 and since protease production is related to invasive isolates,15 the isolates were evaluated for these phenotypic characteristics.
All drug-resistant and some susceptible strains were then genetically analysed by pulsed-field gel electrophoresis (PFGE). This technique is interesting as a potential alternative to serotyping because it has been reported that individual M types give distinct PFGE patterns,16 and may be useful for study of the diffusion of particular clones.
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Materials and methods |
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In 1997 we collected 221 S. pyogenes isolates (129 in Torino, 92 in Pinerolo) from throat swabs of untreated children with uncomplicated pharyngotonsillitis.
T typing
T typing was performed by slide agglutination technique with trypsin-treated bacteria and T antisera from Chemapol (Prague, Czech Republic).17
Opacity factor (OF)
Overnight cultures were centrifuged at 300g for 15 min and 10 µL aliquots of supernatant were added to each well containing 100 µL of horse serum (Sigma-Aldrich, Milano, Italy) in a 96-well microtitre plate. After overnight incubation at 37°C and the addition of 100 µL of physiological solution (0.9% w/v NaCl in H2O), the production of the OF protein by bacteria was revealed by opacity of wells, while negative wells appeared clear on visual examination.18
Resistance phenotype
All strains were screened for MLS resistance by the agar disc diffusion assay according to Sutcliffe et al.12 A standardized bacterial suspension was used as inoculum for streaking the surface of a Columbia blood agar plate (bio-Mérieux, Roma, Italy). Erythromycin (15 µg) and clindamycin (2 µg) discs (Oxoid, Milano, Italy) were placed on the plates approximately 10 mm apart. Plates were incubated overnight at 37°C.
Constitutive resistance (CR) was indicated by the growth of colonies around clindamycin and erythromycin discs; blunting of the inhibition zone around the clindamycin disc indicated inducible resistance (IR); resistant to erythromycin and susceptibile to clindamycin characterized resistance mediated by an efflux pump, named new resistance (NR) phenotype.
Protease assay
The protease activity of each isolate was determined by the casein plate assay.15 It was considered positive when hydrolysis was present around the inoculum zone.
Pulsed-field gel electrophoresis
Cultures in 6 mL of Todd Hewitt broth (Difco, Detroit, MI USA) were incubated for 8 h, then centrifuged at 200g for 15 min. According to the technique reported by Cocuzza et al.,6 the pellet was washed in 1 mL of PIV buffer (10 mM Tris pH 8.0 and 1 M NaCl), resuspended in 200 µL of the same buffer and diluted to an OD of 5 at a wavelength of 620 nm. This bacterial suspension was incorporated in 100 µL of 1.5% low melting point agarose (Bio-Rad, Milano, Italy) in PIV buffer and agarose plugs were prepared and maintained at 4°C. Bacteria were lysed by incubating the plugs in EC lysis solution (6 mM Tris pH 8.0; 1 M NaCl; 100 mM EDTA pH 8.0; 0.2% sodium deoxycholate; 0.5% sodium laurylsarcosine; 0.5% Brij 58) with 50 mg/L RNAse A for 16 h at 37°C. Plugs were then incubated at 50°C for 17 h in ES solution (0.5 M EDTA pH 9) containing 1 g/L proteinase K (Sigma-Aldrich). After four washings with TE buffer (10 mM Tris pH 7.5 and 1 mM EDTA pH 8) and gentle agitation for 1 h, plugs were stored in TE at 4°C. Plugs were incubated in 1 mL of pre-SmaI buffer (6 mM Tris pH 8; 20 mM KCl; 6 mM MgCl2 and 6 mM 2-mercaptoethanol) for 30 min at 37°C and then treated overnight at 30°C with 10 U SmaI (Sigma-Aldrich). The gel was prepared with 1% pulsed-field-certified agarose (Bio-Rad) in 0.5 x TBE buffer (45 mM Tris-borate and 1 mM EDTA) and sealed with 0.75% low melting point agarose. A 501000 kb (Sigma-Aldrich) molecular-weight marker was used. PFGE was performed with a CHEF Mapper XA System (Bio-Rad). The run was performed at 200 V for 23 h at 11.3°C with an initial pulse time of 1 s and final pulse time of 30 s. Following ethidium bromide staining the gel was visualized and photographed under UV light.
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Results |
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Analysis of erythromycin resistance (Table I) shows that about half of the isolates, 66/129 (51.2%) in Torino and 40/92 (43.5%) in Pinerolo, were resistant to this drug. Moreover, OF-negative S. pyogenes strains, 62/129 (48.1%) and 53/92 (57.6%) in the two centres, respectively, demonstrated a statistically significant (P < 0.01) greater susceptibility to this drug (Table I
). The percentage of strains with the CR phenotype was similar in the two centres (19.4 and 18.5%, respectively). In Torino there were more isolates with the IR phenotype, while in Pinerolo more had resistance linked to an efflux pump, the NR phenotype (Table I
).
Fifty-eight isolates with different T types and different erythromycin resistance phenotypes were studied by PFGE. Distinct patterns were revealed within each of the three resistance phenotypes. Seven distinct electrophoretic patterns were visualized and designated with the letters from A to G (Figure and Table II
). According to Tenover et al.,19 each pattern was characterized by a difference of more than seven fragments from the others. Patterns that are closely or possibly related to the seven main profiles are considered subtypes and are designated with small letters after capital letters (-a, differs in one band from the main type, -b, differs in two bands, etc). We found that the DNA of bacteria with the NR phenotype shows three patterns (A, B and C) and two subtypes (Ab and Cb), whereas isolates with the IR or CR phenotype show only one pattern (E and D, respectively) and three subtypes (Eb, Ec, Ed, Da, Db, Dc). The susceptible isolates tested were grouped into two different patterns (F and G) and three subtypes (Ab, Eb and Ec) which were common also to NR or IR phenotype isolates (Table II
).
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Some T serotypes of S. pyogenes were related to particular PFGE profiles, for example those with T protein 2, 4 or 6 had pattern A, but the genotypic discrimination did not seem in general related to the T serotype (Table II).
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Discussion |
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Evaluation of bacterial susceptibility to erythromycin, one of the drugs more frequently used to treat infections, revealed a high rate of resistance, 51.2 and 43.5% of the strains tested from Torino and Pinerolo. Also other authors6,7 observed an increase in erythromycin resistance in Italy and a great variability between different centres. In particular Cocuzza et al.23 reported macrolide resistance levels of 3 and 30% in strains isolated in 1996 in Catania and Milano, respectively. The majority (37.9 and 42.5% in Torino and Pinerolo, respectively) of all resistant S. pyogenes examined demonstrated constitutive MLS resistance. However, in Torino there was a prevalence of inducible-resistant isolates (40.9%), while in Pinerolo there were many with the NR phenotype.
Most of the isolates assayed produced large amounts of protease, an enzyme that is produced in larger amounts among invasive isolates and those associated with mortality.1,15 The OF group A streptococci, which include some streptococcal M types (1, 3, 5, 6, 14, 18, 19, 24), are particularly associated with rheumatic fever.1,24 The observation that there were fewer OF isolates and that the majority of these appeared more susceptible to erythromycin could suggest that the chances of more severe and invasive diseases are low after correct therapy. Hence, it would seem important to monitor the resistance of these bacteria in the future, and also to evaluate the involvement of more virulent serotypes.
The analysis of the genetic characters of erythromycin-resistant and of some susceptible isolates demonstrated that there is a good correlation between the resistance phenotype and the PFGE pattern. This discrimination was related to particular S. pyogenes T serotypes in several cases but not in all. Single & Martin16 have shown that there are differences between isolates within the same M type and that these appear to represent clonal distinctions. The erythromycin-susceptible strains were more heterogeneous in genomic pattern in comparison with the resistant strains, particularly those with MLS resistance. From this data it is not possible to establish whether the differences observed in genotypic characteristics also reflect different antigenic properties or some virulence factors. Our results, in agreement with those of other Italian authors,6,7 show that there is considerable genetic heterogeneity among the S. pyogenes strains examined. The high rate of erythromycin resistance observed in group A streptococci is not caused by the spread of a single clone or related to a particular serotype, suggesting the importance of epidemiological surveillance of S. pyogenes infections and continuous monitoring of the resistance characters of these microorganisms.
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Acknowledgments |
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Notes |
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References |
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Received 27 January 1999; returned 4 April 1999; revised 4 May 1999; accepted 24 August 1999