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Association of group A streptococcal emm types with virulence traits and macrolide-resistance genes is independent of the source of isolation

¹Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy ²Dipartimento di Medicina di Laboratorio e Microbiologia, Università Campus Biomedico, Via Emilio Longoni 83, 00155 Rome, Italy ³Dipartimento di Scienze Neurologiche, Psichiatriche e Riabilitative dell'Età Evolutiva, Università ‘La Sapienza', Via dei Sabelli 108, 00185 Rome, Italy

Correspondence Roberta Creti roberta.creti{at}iss.it

Received February 3, 2005
Accepted June 15, 2005

Streptococcus pyogenes (group A streptococci; GAS) recovered from paediatric pharyngitis (101 isolates) and asymptomatic children (79 isolates) in the same geographical area and period, as well as isolates collected during an enhanced national surveillance programme for GAS invasive diseases (79 isolates), were screened for the incidence of the streptococcal pyrogenic exotoxin (spe) genes speA and speC, as well as the macrolide-resistance genes erm(B), erm(A) subclass erm(TR) and mef(A), and typed by emm sequencing. The speA gene was detected with comparable incidence among throat isolates (13.9 % of asymptomatic children and 16.8 % of pharyngitis isolates) and in 25 % of invasive cases; in contrast, speC incidence was, surprisingly, higher in paediatric populations (55.4 % in pharyngitis isolates and 65.8 % in asymptomatic children) than in invasive isolates (30 %; P < 0.0001). Macrolide resistance was detected in 26.6, 38.0 and 37.6 % of strains belonging to invasive, asymptomatic and pharyngitis populations, respectively. The different incidences of exotoxin and antibiotic-resistance genes among populations did not appear to have an intrinsic clinical significance, but may reflect the propensity of these traits to be associated with certain emm types independent of the source from which the strains were isolated. Further investigations with larger emm-type populations are warranted to confirm this.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The virulence of Streptococcus pyogenes (group A streptococci; GAS) is determined by a variety of structural molecules, enzymes and toxins. In particular, phage-encoded exotoxins, of which SpeA and SpeC are the first to have been described and studied, seem to play a critical role in the emergence of unusually virulent clones by horizontal gene transfer (Banks et al., 2002). Besides virulence traits, antibiotic resistance, in particular to macrolides, can enhance bacterial fitness and be responsible for treatment failure (Freeman & Shulman, 2002).

The aim of the present study was to investigate whether particular virulent clones, both antibiotic-resistant and exotoxin ‘producers', were related to a particular disease. To this end, the presence of speA, speC and macrolide-resistance erm(B), erm(A) subclass erm(TR) and mef(A) genes was investigated in GAS strains collected in the same geographical area and time period from paediatric pharyngitis cases (101 isolates) (Dicuonzo et al., 2001, 2002) or throat swabs of asymptomatic children (79 isolates) (Cardona & Orefici, 2000; Creti et al., 2004), as well as in isolates obtained during an enhanced national surveillance programme for GAS invasive diseases (79 isolates, mean patient age 47.1 ± 23.6 years) (Suligoi et al., 1998; von Hunolstein et al., 1997).

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial growth conditions.

Bacteria were grown on 5 % defibrinated sheep blood agar plates at 37 °C in 5 % CO₂ overnight.

DNA isolation and PCR.

Total bacterial DNA was prepared by a Chelex-based procedure using an InstaGene Matrix (Bio-Rad). Briefly, a large loop of overnight colonies was dissolved in a tube containing 1 ml sterile distilled water and then treated according to the manufacturer's instructions. An aliquot of the supernatant (5 µl) was used as template in a final volume of 25 µl PCR mixture. For spe gene amplification, single PCR reactions were performed each containing: 1x PCR buffer, 2 mM MgCl₂, 200 mM of each dNTP, 400 nM of each primer and 0.25 U Platinum Taq DNA polymerase (Invitrogen). For amplification of erm(B), erm(A) subclass erm(TR) and mef(A) genes, a single multiplex PCR reaction was run containing 1x PCR buffer, 1.5 mM MgCl₂, 200 mM each dNTP, 400 nM of each of the six primers and 0.25 U Platinum Taq DNA polymerase.

Samples were amplified on a DNA thermal cycler (MJ Research) by heating for 5 min at 95 °C, followed by 30 cycles of 95 °C for 60 s, 52 °C for 60 s (48 °C for the multiplex PCR) and 72 °C for 60 s (90 s for the multiplex PCR) and a final step of 72 °C for 10 min. PCR products were analysed by gel electrophoresis in a 2 % (w/v) agarose gel (UltraPure Agarose-1000; Invitrogen).

HinfI restriction enzyme digestion of the speA amplicon giving two fragments of 142 and 211 bp was performed for further confirmation of the specificity of the speA PCR product. The speB gene was always present and a speB-specific PCR was used to ascertain the quality of the template DNA.

Primers.

Oligonucleotide primers were synthesized by a custom primer service (Life Technologies) and have been described previously (Dicuonzo et al., 2002; Tyler et al., 1992).

emm typing.

Standard methods from the Centers for Disease Control (CDC) protocol for emm typing were used. According to the CDC guidelines, a sequence was considered to belong to a specific emm gene (or to a sequence type) allele when, over the first 160 bases of sequence, it had 95 % or greater identity with that of the reference emm gene.

Statistics.

Data were analysed using the STATISTICA program for Windows (StatSoft). Categorical data were compared using the S² test. Differences were considered significant when P < 0.05.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The speA gene was detected in 13.9 % of isolates from asymptomatic children, in 16.8 % of pharyngitis isolates and in 25 % of invasive cases, thus showing an increasing trend in more severe disease. In contrast, speC incidence, surprisingly, was higher in throat isolates (55.4 % of pharyngitis isolates and 65.8 % of asymptomatic children) than in invasive isolates (30 %) (Fig. 1a). Moreover, the difference in speC incidence among populations was statistically significant (P < 0.0001).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Incidence of speA and speC genes (a) and macrolide-resistance erm(B), erm(A) subclass erm(TR) and mef(A) genes (b) in the three GAS populations examined.

Erythrogenic toxin speA and speC gene distribution in different GAS populations has been intensively investigated and incidences have been found to be highly variable (DelVecchio et al., 2002; Descheemaeker et al., 2000; Hauser et al., 1991; Haukness et al., 2002; McCormick & Schlievert, 2000; Nandi et al., 2002; Tyler et al., 1992). While the role of the SpeA exotoxin in the pathogenesis and severity of GAS infection is well documented, less is known about the contribution of SpeC, although its presence and putative importance is always related to the development of severe invasive streptococcal disease (Brussow et al., 2004; Freeman & Shulman, 2002; McCormick & Schlievert, 2000). To our knowledge, a high frequency of the speC gene, but also associated with high speA gene levels, has only once been reported in pharyngitis GAS isolates (Haukness et al., 2002); it has been never observed in GAS isolates from asymptomatic children before the present study.

Overall, the abundance of the speC gene in pharyngeal GAS isolates was an impressive finding, but its significance remains unexplained. It has been noted that when GAS are co-cultured with human pharyngeal cells, they upregulate and secrete the SpeC toxin, but no further hypothesis on the role of SpeC in the infection process has been given (Broudy et al., 2001).

Macrolide resistance was detected at a higher incidence among non-invasive infections (38.0 and 37.6 % in asymptomatic and pharyngitis populations, respectively) than in invasive GAS isolates (26.6 %) (Fig. 1b). This finding could be explained by the increased possibility of non-invasive strains being involved in the acquisition of antibiotic-resistance determinants by interspecies recombination, as has been proposed for other streptococci such as Streptococcus pneumoniae (Pletz et al., 2005), as well as lower resistance in invasive isolates being due to greater patient age. Nevertheless, the overall macrolide resistance detected among the three GAS populations was among the highest in Europe and was in accordance with previous reports on the relevant spreading of macrolide-resistant GAS strains in Italy (Cocuzza et al., 1997; Albrich et al., 2004).

In order to determine whether particular virulent clones were circulating within GAS populations, the presence of speA, speC and macrolide-resistance genes of each strain was related to the emm type. In total, 259 GAS strains were investigated. A striking association of speA and speC genes as well as erm(B), erm(A) subclass erm(TR) and mef(A) genes with emm types was noted, which was independent of the source of isolation (Table 1). The association between spe genes and emm type in Streptococcus pyogenes has already been reported (Bessen et al., 1995, 1999; Miyoshi-Akiyama et al., 2003; Mylvaganam et al., 2000) and in our analysis it was more apparent for speA than for speC. The speA exotoxin gene was carried by seven out of 31 emm types analysed, mostly by emm-1 and emm-3 GAS strains. The speC gene seemed to be more randomly distributed, but it was also associated with specific emm types. The emm types always carrying the speC gene were emm-2, emm-5, emm-11 and emm-78 strains, although low numbers for each emm type made strong conclusions difficult (Table 1).

With regard to antibiotic resistance, among the wide range of emm types analysed, only a few carried macrolide-resistance genes. In particular, all emm-2 GAS isolates were resistant to macrolide antibiotics and carried the mef(A) determinant. When resistant, emm-4 and emm-9 strains were only mef(A) positive, while emm-12 strains could be either mef(A) or erm(B) positive. Most emm-22 and emm-89 strains were macrolide resistant and erm(B) positive; the erm(B) gene was also present in emm-5, emm-6, emm-12, emm-28 and emm-78 strains. The presence of the erm(A) gene was restricted to emm-5, emm-11, emm-27/77 and emm-50/62 strains (Table 1). Antibiotic resistance by methylation of the 23S rRNA gene mediated by the erm(B) gene was found to be the predominant mechanism in this study (Table 1).

Although evidence of non-random distribution of macrolide resistance and emm types was noted (Dicuonzo et al., 2002; Reinert et al., 2003; Zampaloni et al., 2003; Doktor et al., 2005), the present study was intended to further our knowledge on these aspects and it is the first report considering such a wide spectrum and number of emm-type GAS strains, notably isolated from different sources and circulating in the same period. The observation that speA and speC exotoxin gene acquisition and macrolide resistance was non-random and associated with emm genes could either indicate that a limited number of GAS clones were spreading across the populations examined (Doktor et al., 2005) or could reflect the existence of an ancestral emm-type clone that has kept its signature toxin gene and antibiotic profile (Schmitz et al., 2003). Recent reports have stressed the importance of prophages in the genotypic variation within isolates with the same emm type (Banks et al., 2002, 2004). Only few prophage genetic markers were considered in this study to analyse this aspect; nevertheless, it cannot be excluded that greater variation than observed could occur in a broader time range due to differences in prophage content.

These data could also indicate that M proteins function, directly or indirectly, as barriers to horizontal gene exchange (Schmitz et al., 2003). A phenotypic linkage between resistance to bacteriophage infection and M protein surface expression has been recognized for more than 20 years (Cleary & Johnson, 1977), but it is still little understood. Whether this could be extended to transmissible macrolide antibiotic-resistance determinants, such as the mef(A) gene, is at present only speculation but is intriguing nevertheless.

Our findings suggested that there was not a hypervirulent GAS strain. In this study, an emm type presented the same ‘virulence and antibiotic resistance string', even if it was isolated from a different niche and was responsible for either no symptoms or severe disease.

For this reason, statistical analysis could be inferred only on the significance of the incidence of the speC gene among populations, because the other determinants examined [speA, erm(B), mef(A) and erm(A) subclass erm(TR) genes] were not randomly distributed but were clearly associated with a few emm types, which in turn were variously represented among the populations under study (Table 1).

The most prevalent emm types in ‘throat’ populations were emm-12, emm-22, emm-4, emm-1, emm-89 and emm-5 (Creti et al., 2004; Dicuonzo et al., 2001); most carried the speC gene and were macrolide antibiotic resistant. The speA-restricted emm types such as emm-1 and emm-3 strains were more representative in invasive strains (von Hunolstein et al., 1997) and were macrolide susceptible. Antibiotic resistance in invasive strains was mostly related to emm-89/erm(B)-resistant strains (Orefici et al., 2003) (Table 1).

In conclusion, the overall prevalence of the erythrogenic toxin and macrolide-resistance genes observed in the present study depended on the different distribution and spread of emm types across the GAS populations under investigation with which they appeared to be associated. Further evaluation of larger bacterial populations is required to confirm this hypothesis.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work was supported by a grant from the Italian Ministry of Health, Project 1 % no. 4AI-F1 (G. O.), by the ISS grant no. C3MF (G O.), and by an ISS–NIH grant, no. 5303, from an ISS-NIH collaborative study on ‘Group A streptococcal infections and neurobehavioral disorders in children'.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES