Macrolide resistance in group A streptococci

D. Savoiaa,*, C. Avanzinia, K. Bosioa, G. Volpea, D. Carpib, G. Dottic and M. Zuccaa

a Department of Clinical and Biological Sciences, University of Torino; b Laboratory, Agnelli Hospital, Pinerolo (Torino); c Laboratory, Paediatric Hospital, Torino, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two hundred and twenty one Streptococcus pyogenes isolates collected from throat swabs of untreated children with uncomplicated pharyngotonsillitis living in two centres situated in the north of Italy were tested to evaluate their macrolide resistance phenotype. Isolates were also typed for T protein and assayed for opacity factor (OF) and protease production. Resistance to macrolides was found to be similar in the two centres. Fifty-one point two per cent of Torino strains and 43.5% of Pinerolo strains were not inhibited by erythromycin. Resistant strains belonged to one of three phenotypes: CR, constitutive resistance (37.9 and 42.5% in Torino and Pinerolo, respectively); IR, inducible resistance (40.9 and 17.5%); NR, new resistance phenotype (21.2 and 40%). All the resistant and some of the susceptible strains were analysed by pulsed-field gel electrophoresis and genomic patterns were defined on the basis of band size and number. Five DNA profiles were found among erythromycin-resistant strains: three patterns characterized the NR resistance phenotype and one each the IR and CR phenotypes. The distribution of resistant strains according to their genomic patterns appears to be related to the resistance phenotype and only in some cases to the T serotype of bacteria. We conclude that the S. pyogenes strains analysed are genetically heterogeneous and therefore the high rate of erythromycin resistance observed is not caused by the spread of a single clone nor is it related to a particular serotype.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pyogenes represents one of the major human pathogens responsible for various acute suppurative manifestations. An increase in the incidence and severity of invasive infections caused by this microorganism has been reported in the last few years, renewing interest in the field. In particular, strains of M1 and M3 serotypes have frequently been associated with recent cases of invasive diseases.1,2

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.4–8 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

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 50–1000 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The distribution of S. pyogenes T serotypes isolated in the two centres is shown in Table IGo. About one-third of the strains (46/129 for Torino and 26/92 for Pinerolo) could not be typed because of autoagglutination. However, the T1 type was more common in Torino, whereas T4 was more common in Pinerolo.


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Table I. Distribution of different phenotypic characters in S. pyogenes from Torino (129 isolates) and Pinerolo (92 isolates)
 
Of the isolates from Torino and Pinerolo 79.8 and 84.1%, respectively, produced large amounts of the protease enzyme. In particular we noted a higher proteolytic activity, as assayed by the casein plate assay, in T1 and B3264 strains (Table IGo).

Analysis of erythromycin resistance (Table IGo) 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 IGo). 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 IGo).

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 (FigureGo and Table IIGo). 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 IIGo).



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Figure. PFGE patterns of chromosomal DNA restriction fragments of S. pyogenes digested with SmaI. Lanes with patterns A and C represent isolates with NR resistance phenotype; lanes with pattern E represent isolates with IR phenotype; lanes with pattern D represent isolates with CR phenotype. Susceptible isolates are represented in lanes with patterns F and G.

 

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Table II. Correlation between genomic PFGE patterns and serotypes in isolates with different resistance phenotypes from two centres
 
Analysis of the heterogeneity indices (number of patterns/number of isolates) showed that erythromycin-suceptible isolates were more heterogeneous (0.44) than resistant isolates. Isolates belonging to the NR phenotype (0.2) were more heterogeneous than those belonging to IR and CR phenotypes (indices of 0.05 and 0.08, respectively).

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 IIGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we examined S. pyogenes isolates in 1997 from throat swabs of untreated children with uncomplicated pharyngotonsillitis living in two closely situated north Italian cities. The evaluation of T serotypes revealed a high percentage of non-typeable isolates but different types were in circulation in the two centres. The simultaneous evaluation of T type and OF production by bacteria allowed a presumptive correlation with M types.14 In particular, we noted the presence of more T1 OF– isolates, presumptively M1, in Torino, and more T4 OF+ isolates, presumptively M4, in Pinerolo. Many researchers1,15 have observed an association between the S. pyogenes M1 serotype and invasive diseases, but others20,21 have found clonal diversity or some genetic variability22 among isolates causing invasive diseases, favouring the hypothesis that host immunity is of major importance in pathogenesis. Supporting this hypothesis is the observation that T4 OF+ strains, some of which were responsible for scarlet fever, have now been isolated in the district of Pinerolo, while they were a large percentage of isolates in Torino in previous years.5

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.


    Acknowledgments
 
Part of this paper was presented in abstract form at the ASM Conference on Streptococcal Genetics, Vichy, France; April 26–29, 1998. This work was supported in part by a Piemonte Regione grant to K.B.


    Notes
 
* Correspondence address. Department of Clinical and Biological Sciences, University of Torino, S. Luigi Hospital, Regione Gonzole 10, 10043 Orbassano (Torino), Italy. Tel: +39-0116708127; Fax: +39-0119038639; E-mail: savoia{at}pasteur.sluigi.unito.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 27 January 1999; returned 4 April 1999; revised 4 May 1999; accepted 24 August 1999