Characterization of group A streptococci causing invasive diseases in Sri Lanka

Abstract Group A β haemolytic streptococcus (GAS) or Streptococcus pyogenes is a human pathogen that causes an array of infections, including pharyngitis, cellulitis, impetigo, scarlet fever, toxic shock syndrome, and necrotizing fasciitis. The present study characterizes 51 GAS isolates from invasive infections in Sri Lanka, focusing on resistance profiles, genetic determinants of resistance, and virulence markers. Isolates were tested for sensitivity to penicillin, erythromycin, clindamycin, and tetracycline. The presence of erm(A), erm(B), and mef(A) was detected in erythromycin-resistant isolates, while tet(M) was detected in the tetracycline-resistant isolates. PCR was used to identify SpeA, SpeB, SpeC, SpeF, SpeG, smez, and ssa as virulence markers. Selected GAS isolates were emm-typed using the updated CDC protocol. All 51 isolates were susceptible to penicillin. The number of isolates non-susceptible to erythromycin was 16. The commonest resistance determinant identified was erm(B) (11/16). Tetracycline non-susceptibility was found in 36 (70.6 %) isolates and 26 of them contained the tet(M) gene. Thirteen (25.5 %) isolates were resistant to both tetracycline and erythromycin, while 12 (23.5 %) isolates were sensitive to both antibiotics. The commonest virulence markers detected among the isolates were SpeB (44, 86.3 %), SpeG (36, 70.6 %), and SpeF (35, 68.6 %), while SpeJ (15, 29.4 %), SpeA (10, 19.6 %), and ssa (5,9.8 %) were less common. The emm types were diverse. In conclusion, the GAS isolates studied showed resistance to erythromycin and tetracycline, while retaining universal susceptibility to penicillin. Additionally, these isolates exhibited diverse genetic backgrounds, displaying varying patterns of virulence genes and emm types.


INTRODUCTION
Group A β haemolytic streptococcus (GAS) or Streptococcus pyogenes is a successful human pathogen.Despite being identified as a human pathogen for many years, many aspects of the bacteria and the disease burden remains to be studied globally, particularly in the Global South.
Conditions such as improved quality of living ha led to the reduction of some GAS-related diseases, such as rheumatic heart diseases.However, there have been increasing reports of invasive GAS (iGAS) diseases [1].
The pathogenicity of GAS is dependent on their virulence markers.M proteins are a major virulence determinant of GAS.Other recognized virulence markers include GAS superantigens (SAgs).There are different types of SAgs in GAS.These include streptococcal pyrogenic exotoxins (SPEs), streptococcal mitogenic exotoxins (SMEs), and streptococcal superantigens (SSAs).Some of these are chromosomally encoded, while others are associated with bacteriophages [2].Their distribution has been identified to be varied [3].Other virulence factors include opacity factor, streptolysin O and F, C5a protease, hyaluronidase, and streptokinase [2].
GAS have different subtypes.Traditionally, they have been subtyped based on the M protein, which is also an important virulence marker.Over 50 different serotypes were first identified using the M typing method [4].However, the typing of GAS isolates has now changed based on the advent of molecular techniques.The most commonly implemented method is emm typing.The emm gene encodes for the M protein and sequences of its 5′ end have been used globally to type GAS isolates [5].So far, more than 200 emm types and more than 750 sub-types have been identified.The predominant types are shown to vary across different regions of the world [5].However, uncommon types have been shown to increase globally in different parts of the world [6].There is also evidence of persistence and expansion of clones associated with antibiotic resistance [7,8].In some countries, specific emm types are associated with particular disease types [9].However, there is also evidence that many emm types could be responsible for the same disease entity [10].Thus, there is a need to understand more about the pathogenicity of different emm types in different locales.
In Sri Lanka too, there is an apparent increase in iGAS diseases (personal communications with clinical microbiologists).A literature search with the keywords group A streptococcus, Streptococcus pyogenes and Sri Lanka fails to identify any literature describing the epidemiology or biology of GAS within Sri Lanka.Further, emm subtypes from Sri Lanka are not available within the emm databases to the best of our knowledge.Given that emm type-based, M protein-targeting vaccines are being developed against GAS, identifying the types present within a country, particularly those responsible for invasive diseases, is important.
This study aimed to characterize GAS isolates obtained from patients with invasive diseases from selected sites in Sri Lanka by identifying their resistance profiles, genetic markers of resistance, and selected virulence markers.Further, typing of selected isolates was also performed through emm typing.

METHODOLOGY Isolates
Fifty-one isolates of GAS collected from the National Hospital, Kandy, Provincial General Hospital, Polonnaruwa, Teaching Hospital, Peradeniya, and District General Hospital, Kegalle, Sri Lanka were characterized for this study (Table 1), after obtaining ethical approval from the Ethics Review Committee, Faculty of Allied Health, Sciences, University of Peradeniya, Sri Lanka (AHS/ERC/2021/101).Isolates were obtained from blood cultures or deep tissue samples.In the case of deep tissue samples, they were obtained from patients with a diagnosis of necrotizing fasciitis or any other clinically diagnosed invasive skin and soft tissue infection.Data related to the isolates were obtained through the request forms forwarded by the co-investigators at the respective hospitals.

Laboratory procedure
All isolates were taken from storage at -80 °C and passaged twice on blood agar before further analysis was performed.The isolates were identified as GAS via bacitracin susceptibility testing as well as Lancefield grouping with latex agglutination (Oxoid, UK).

Identification of resistance markers
The presence of erm(A), erm(B), and mef(A) was identified in erythromycin-resistant isolates, while tet(M) was identified in the tetracycline-resistant isolates using previously described protocols [13].

Molecular epidemiology
Isolates from 2017 were emm-typed using the updated CDC protocol [14].

Data analysis
Data were entered into an MS Excel database and analysed via SPSS (version 21) (Supplementary File 1).Percentages were calculated for the year of isolation, origin, antibiotic susceptibility patterns, and presence of genetic markers and virulence factors.
The potential association between resistance to tetracycline and erythromycin was assessed using a Chi-square test for association.

Antibiotic susceptibility patterns and resistant determinants
All 51 isolates were susceptible to penicillin, which is in agreement with the well-established norm [15].Four (7.8 %) isolates were resistant to clindamycin.Erythromycin non-susceptibility (either intermediately sensitive or resistant) was seen in 16 (31.4%) isolates and tetracycline non-susceptibility was found in 36 (70.6 %) isolates (Table 2).
Thirteen (25.5 %) isolates were non-susceptible to both tetracycline and erythromycin, while 12 (23.5 %) isolates were sensitive to both antibiotics.Three isolates (5.9 %) were resistant to erythromycin alone, while 23 (45.1 %) were resistant to tetracycline alone.In contrast to penicillin susceptibility, resistance to other classes of antimicrobial drugs has shown a gradually increasing trend among GAS isolates globally [16].Over the past few decades, increasing macrolide resistance of GAS has been reported.Globally, resistance rates have been higher in some countries, such as PR China [17] and India [18], while it has been lower in some other countries, such as Japan and Taiwan, ROC [19].
Of the 16 erythromycin non-susceptible isolates, 11 had the erm(B) gene, 3 had the mef(A) gene, and 1 isolate had the erm(A) gene.Of the 36 isolates non-susceptible to tetracycline, 26 contained the tet(M) gene.Co-occurrence patterns for the resistant determinants are given in Table 3. Erm(B) has been identified as the commonest resistant determinant for erythromycin resistance in many studies [17,18,20].
A recent study conducted in Central Africa reported 80 % tetracycline resistance among 76 GAS clinical isolates, which is parallel to the findings of our study [21].Ayer et al. identified 90 % (n=72) of the GAS isolates with tet(M) gene [22].Further, they determined the relationship between resistance to tetracycline and macrolides in GAS.According to their findings, the tet(M) was found in association with erm(B) in 71 % of the total erm(B)-positive isolates.Similarly, we identified that 7 of the 13 isolates that were non-susceptible for both erythromycin and tetracycline were positive for both erm(B) and tet(M).
Resistance to tetracycline is naturally conferred by ribosome protection genes, such as tet(M) and tet(O) in Gram-positive bacteria.Tetracycline resistance genes can be located on mobile genetic elements that carry macrolide resistance genes, which enable the co-occurrence of resistance to both classes of antibiotics [23].This is the likely reason for the concurrent occurrence of the two genes.
Considering the antibiotic susceptibility patterns, while universal susceptibility to penicillin is encouraging, the higher rates of resistance to tetracycline and erythromycin is a concern that the wider clinical community should be aware of, since erythromycin is a commonly used alternative for GAS infections in the presence of penicillin allergy.
In other countries too, SpeB, SpeG, and SpeF have been found to be commonest virulence determinants [24].Streptococcal pyrogenic exotoxin B (SpeB) has been identified to be the commonest secreted protein present among a majority of clinically identified GAS isolates.Its function is a subject of ongoing debate, as contradictory findings have been identified by different groups [25].However, there are more than sufficient data currently to suggest that it plays an important contributory role in causing infections [26].Therefore, identifying it as the commonest virulence marker is expected.Streptococcal pyrogenic exotoxin G (SpeG) is chromosomally encoded but is not found in all isolates [27].However, many studies have identified it to be present in a majority of isolates [24,27].SpeF has also been found in higher concentrations among antibiotic-resistant GAS isolates in India [28].Superantigens (SpeA, SpeC, SpeH, SpeJ, SpeZ, smez) are associated with invasiveness in different studies [29,30].
Research evidence supports that the phage-encoded speA and speC genes are associated with invasive infection.A high rate of speA and speC gene carriage has frequently been identified among streptococcal toxic shock syndrome-associated isolates compared with those isolated from superficial infections [29,30].
An Italian study that characterized GAS clinical isolates reported SpeB as the commonest virulence gene, which is in agreement with the present study.SpeC, SpeH, and smez were also identified among their isolates [31].However, a Finish study aiming to identify superantigen profiles of GAS isolates from patients with bacteraemia has identified SpeA and SpeC in 20 and 30 % of the strains, respectively, as the commonest virulence determinants [29].These were less prevalent in our study.In a recent study by Lintges et al., superantigens were found to be more important for the invasiveness of GAS, with SpeA, SpeJ, and SpeZ having the greatest invasive potential [32].
Ssa is a phage-encoded superantigen that may co-occur with SpeA.In recent studies, ssa has been associated with scarlet fever, while it has also been found in association with other isolates [17,33].Our isolate cohort showed a lower prevalence of both ssa  *Of these, 12 were sensitive to both antibiotics, 7 were resistant to tetracycline, and 1 was resistant to both antibiotics.Percentages were not calculated as only the nonsusceptible isolates were tested.
SpeA.Our isolates were from invasive diseases that are mostly of musculoskeletal origin rather than scarlet fever.This could be a reason for the low prevalence of ssa and SpeA.
The overall patterns of virulence factors are in keeping with the clinical picture patients from whom the isolates were obtained.However, to understand the action and clinical significance of these virulence factors fully, a wider study, including both invasive and non-invasive isolates as well as a carriage component, is warranted.

emm types
Diverse group of emm types were identified for the 15 isolates that were typed (Table 4).
The M protein gene (emm) encodes the cell surface M virulence protein responsible for at least 100 Streptococcus pyogenes M serotypes.While we could not perform emm typing on all isolates, emm types 9, 92, 68, and 232 were identified in more than one isolate.A systematic review that focused on global emm type distribution of group A streptococci has reported that the emm typing related epidemiological data from high-income countries were predominant while data from low-income countries were deficient [5].Further, a significant diversity can be identified among the emm types across different geographical areas [34].

Limitations
This is the first study in Sri Lanka to characterize the GAS causing invasive diseases with profiling of genetic determinants of resistance, virulence genes, and emm types.However, there are some limitations in the present study.The availability of partial funding led us to identify a limited number of resistant determinants, virulence markers (six), and limited emm typing, which does not describe the full picture of the isolates present.We lack clinical data for a meaningful analysis of associations with clinical outcomes.While the lack of clinical data is a major drawback, since this is the first time that GAS isolates are being described from Sri Lanka, we consider that the higher antibiotic resistance rates, higher presence of resistance markers, and the presence of diverse emm types indicate a broad spread of genetically diverse group A streptococci in the country, which could pose a treatment challenge and warrants regular surveillance.As highlighted by Steer et al. in their systematic review [5], there is a lack of global epidemiological data on GAS from developing countries.Therefore, despite being a small study, our data add value to the global literature.

CONCLUSION
We have demonstrated that while the susceptibility to penicillin remains, resistance to erythromycin and tetracycline is prevalent in GAS isolates in Sri Lanka.Further, these isolates are from diverse backgrounds with different patterns of virulence genes and emm types.This preliminary study indicates that there is a necessity to monitor invasive bacterial isolates such as GAS globally, particularly from countries with poor data generation, as this would benefit the background work in the search for potential vaccine types and in the treatment of patients.

Fig. 1 .
Fig. 1.Co-occurrence pattern of virulence determinants (two isolates did not contain any of the selected virulent factors).

Table 2 .
Antibiotic sensitivity patterns of the study isolates

Table 3 .
Co-occurrence patterns for the resistant determinants