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ORIGINAL ARTICLE
Year : 2017  |  Volume : 19  |  Issue : 2  |  Page : 104-109

Characterisation of Streptococcus pyogenes on the basis of pyrotoxin exotoxin genes in north India


1 Department of Medicine & Microbiology, AIIMS, New Delhi, India
2 Department of Microbiology, AIIMS, New Delhi, India
3 Department of Paediatrics, AIIMS, New Delhi, India
4 Department of Cardiothoracic and Vascular Surgery, AIIMS, New Delhi, India
5 Department of Cardiology, AIIMS, New Delhi, India

Date of Web Publication15-Nov-2017

Correspondence Address:
Bimal K Das
Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110029, Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomt.jomt_16_17

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  Abstract 


Introduction: Scarlet fever and toxic shock syndrome are two major clinical entities caused due to the action of pyrogenic exotoxin. We screened the clinical isolates of Streptococcus pyogenes for the presence of major exotoxin encoding genes SpeA, SpeB and SpeC.
Materials and Methods: Isolates of S. pyogenes obtained from clinical samples were characterised on the basis of pyrogenic exotoxin encoding genes SpeA, SpeB, and SpeC. The sample was cultured and identified by conventional biochemical test and further confirmed by latex agglutination test kit (Remel, USA). On confirmed group A streptococci isolates, deoxyribonucleic acid extraction and polymerase chain reaction were performed. Antimicrobial susceptibility testing and detection of inducible clindamycin resistance for all clinical isolates were performed using Clinical and Laboratory Standard Institute guidelines by Kirby–Bauer disk diffusion method.
Results: Thirty-one confirmed isolates of S. pyogenes were tested; out of which, the respective prevalence values of SpeB and SpeC genes were 80.6 and 35.5%. SpeA gene was detected in only single isolate. All the isolates were susceptible to penicillin, but resistance was seen with erythromycin, tetracycline and levofloxacin. Inducible clindamycin resistance was seen in two isolates.
Conclusion: The present study found that the genes encoding pyrogenic exotoxin is circulating in the S. pyogenes isolates present in north India and have the potential of causing scarlet fever that is not reported so far from the country. A significant percentage of drug resistance was noted for the alternative drugs required for the treatment of penicillin allergic patients. The recent upsurge in the cases of scarlet fever in south-east Asia warns us to keep a check on gene profile of these isolates in the community.

Keywords: Drug resistance, polymerase chain reaction, pyrogenic exotoxin, scarlet fever, Streptococcus pyogenes


How to cite this article:
Baidya A, Das BK, Kapil A, Ghosh A, Mathur P, Kabra SK, Chandra D, Juneja R. Characterisation of Streptococcus pyogenes on the basis of pyrotoxin exotoxin genes in north India. J Med Trop 2017;19:104-9

How to cite this URL:
Baidya A, Das BK, Kapil A, Ghosh A, Mathur P, Kabra SK, Chandra D, Juneja R. Characterisation of Streptococcus pyogenes on the basis of pyrotoxin exotoxin genes in north India. J Med Trop [serial online] 2017 [cited 2022 Jun 28];19:104-9. Available from: https://www.jmedtropics.org/text.asp?2017/19/2/104/218399




  Introduction Top


Streptococci are gram positive, catalase negative bacteria that tend to grow in pairs and chains. In developing country like India, beta-haemolytic group A streptococci plays important role in disease pathogenesis. It is associated with diseases such as pharyngitis, skin and soft tissue infection, rheumatic heart diseases, acute glomerulonephritis.

Group A streptococcus (GAS; Streptococcus pyogenes), pharyngitis in children assumes special significance due to the development of rheumatic fever/rheumatic heart disease. Streptococcal infections with an estimated death rate of over 500,000 individuals/year place GAS among major human pathogens, exceeded by human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Plasmodium falciparum and Streptococcus pneumoniae, and probably comparable to rotavirus, measles, Haemophilus influenzae type b, and hepatitis B.[1],[2] It is estimated that approximately seven sore throat episodes occur per child per year, with 13.5% of these being caused by GAS in India.[3]

The organism can be found in normal healthy individuals too, the common site being pharynx. During winter months in temperate climates, up to 20% of asymptomatic school age children may be group A streptococcal carriers.[4] Studies among children in India showed that the prevalence of a symptomatic throat carriage of group A streptococci ranges between 3.7 and 20%.[5],[6],[7],[8]

In recent years, S. pyogenes is frequently reported from invasive infections and toxic shock-like syndrome. In this study, we had screened GAS obtained from clinical samples for SpeA, SpeB and SpeC toxin-encoding genes responsible for scarlet fever and toxic shock syndrome. The SpeB gene is chromosomally encoded, whereas the exotoxin-encoding genes SpeA and SpeC are carried by lysogenic phages, and are easily disseminated to other GAS.

There is no documented case of scarlet fever from India though in past few years, south-east Asia had suffered many outbreaks of the same. The present study has looked in the prevalence of major pyrogenic exotoxin producing genes in clinical isolates of group A streptococci obtained from throat swab and pus samples in a tertiary care centre in north India.

Antimicrobial susceptibility was also performed according to Clinical and Laboratory Standard Institute (CLSI) guidelines.

Although all GAS strains remain sensitive to penicillin, erythromycin is the drug of choice in penicillin allergic patients. Resistance to erythromycin has been reported from many parts of the world. Erythromycin resistance was first reported in streptococci in 1959.[9] There are two main mechanisms of macrolide resistance in the streptococci. One is an active efflux mechanism encoded by the mefA/E genes that affects only macrolides.[9] The second major mechanism is the modification of the same or adjacent binding site on the ribosome targeted by macrolides and lincosamides.


  Materials and methods Top


The study was conducted in the period from July 2012 to May 2014 at the department of Microbiology in All India Institute of Medical Sciences, New Delhi. The samples were obtained from skin and soft tissue infections (pus), upper respiratory tract infections (throat swab) and patients suffering from physical trauma. Throat swabs were collected from both paediatric and adults patients.

Culture and isolation

The samples were seeded on culture media (blood agar, chocolate agar and MacConkey agar). The MacConkey agar was seeded for pus samples and incubated aerobically for 24 h at 37°C. Throat swabs were cultured on blood agar and chocolate agar and incubated in candle jar for 24 h. The clinical samples that showed beta-haemolytic colonies on blood agar were further confirmed to be group A streptococci by both conventional biochemicals and latex agglutination.

Identification of bacterial isolates

The beta haemolytic colonies were identified by performing catalase test (3%), bacitracin disk (0.04 U) test, pyrolidonyl arylamidase (PYR) test and confirmed by latex agglutination test (Remel, USA).

Deoxyribonucleic acid isolation and polymerase chain reaction

Deoxyribonucleic acid (DNA) was isolated from strains of S. pyogenes using standard laboratory protocol. Bacterial culture that was grown in nutrient broth were centrifuged at 4500 rpm for 5 min and pellet that was obtained was resuspended in 0.1 ml of molecular biology-grade water and were centrifuged at 13,000 rpm for 10 min. Pellets were resuspended in 40 μl of water, subjected to boiling at 100°C in a water bath for 10 min, cooled on ice, and centrifuged at 13,000 rpm for 10 s. the extracted DNA was stored at −20°C. Polymerase chain reaction (PCR) was made to happen using published primers [Table 1][10],[11] for genes SpeA, SpeB, and SpeC.
Table 1: PCR primers and amplicon size[10],[11]

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The standard PCR reaction mixture contained: 1× reaction buffer containing 5 μl of extracted DNA, 200 μM of each Deoxynucleotide Triphosphates (dNTPs), 1.5 mM MgCl2, 0.4 pM of each primers and 0.04 U/μl of Taq DNA polymerase and 10.8 μl of sterile double distilled water milli-Q (d/w MQ). The PCR was standardised.

Agarose gel electrophoresis

Amplicons were visualised on 1.8% agarose gel. The agarose gel (Sigma Aldrich Corp., St. Louis, MO, USA) was prepared in 0.5× TBE (1× TBE − Tris buffer + Ethylenediaminetetraacetic acid (EDTA) = 10 mM Tris, pH = 8 and 1 mM EDTA), and the amplicons were loaded with the help of 2 μl of 6× loading buffer in the wells. Electrophoresis was performed for about 1 h at a constant voltage of 80 V in running buffer containing 0.5× TBE and ethidium bromide (0.5 μg/ml) (Sigma Aldrich Corp., St. Louis, MO, USA). After electrophoresis gels were visualised in a Gel Doc XR+ gel documentation system [Figure 1] (Bio-Rad Laboratories, Inc., USA).
Figure 1: Gel electrophoresis topmost run for SpeA (309 bp), middle layer for SpeC (584 bp), lowermost layer for SpeB (170 bp). NC = negative control, PC = positive control, s1–s13 = samples.

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Antibiotic susceptibility testing

Direct colony suspension equivalent to 0.5 McFarland standard solution of organism (GAS) was prepared using overnight culture on sheep blood agar. Mueller–Hinton agar plate containing 5% sheep blood was used for the disk diffusion method. Antibiotic disks of penicillin (10 U), erythromycin (15 μg), clindamycin (2 μg), vancomycin (30 μg), tetracycline (30 μg), linezolid (30 μg), levofloxacin (5 μg) was tested according to CLSI guidelines by Kirby–Bauer method. Inducible clindamycin resistance (D-zone test) was induced on all isolates that were resistant to erythromycin but susceptible to clindamycin.[12]


  Results Top


A total of 31 confirmed isolates of S. pyogenes were obtained from the above mentioned samples in the study period. Overall prevalence of SpeB gene was 80.6% followed by SpeC that was found to be 35.5%. SpeA gene was detected only in a single isolate [Table 2].
Table 2: Superantigens genes in GAS isolates from various clinical samples

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All the isolates were susceptible to penicillin but resistance was seen with erythromycin, tetracycline and levofloxacin. Inducible clindamycin resistance was seen in two isolates [Table 3].
Table 3: Result of antimicrobial susceptibility testing of 31 GAS isolate

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  Discussion Top


Group A streptococci have a worldwide distribution. GAS is the most predominant cause of pharyngitis and is associated with the non-suppurative sequelae, that is, rheumatic fever and acute glomerulonephritis. The organism also causes scarlet fever and toxic shock syndrome in many parts of the world due to the action of pyrogenic exotoxin. This pyrogenolic exotoxins are superantigens that are potent immunostimulators and bind simultaneously to the major histocompatibility complex class II molecules and the T-cell receptor[13] that results in activation of a large number of T cells leading to increased secretion of proinflammatory cytokines.

There is no case report of scarlet fever from India. In this study, we looked for the genes that are more commonly associated with scarlet fever and toxic shock syndrome, that is, SpeA, SpeB, SpeC in the clinical isolates from patient suffering from the symptomatic GAS infection. The SpeB gene is chromosomally encoded, whereas the exotoxin-encoding genes SpeA and SpeC are carried by lysogenic phages, and are easily disseminated to other GAS.

In a study performed by Charmaine and Menon[14] in school children in south India, GAS isolates were obtained from asymptomatic school children and children suffering from tonsillitis and pyoderma. But none of the isolate was obtained from an adult. Another study was conducted by Nandi et al.[3] and Sagar et al.[15] on isolates obtained from patients with pharyngitis or rheumatic fever/rheumatic heart disease. None of the isolates were from the patient with skin and soft tissue infections or trauma patients.

Here in our study, we included isolates from both paediatric and adult patients. The samples included were from pharyngitis, bronchoalveolar lavage (BAL), pus from skin and soft tissue infections, and trauma patients. In the present study, we found that the prevalence of SpeA gene is 3.2%, SpeB gene is 80.6%, and SpeC gene is 35.5%.

In a study conducted by Mathur et al.[16] strains of all beta-hemolytic streptococci from north India and south India were included in this study. The prevalence of pyrogenic exotoxin genes among this study was 7.5% SpeA, 97% SpeB, and 24% SpeC. In their study, all the isolates from throat swab showed the presence of SpeB gene, in contrast, in our study only 80% of the isolates from throat swab showed this gene.

Studies from various parts of the world found the prevalence of Spe genes (SpeA, SpeC) much higher than that in India [Table 4]. These are the areas where scarlet fever and streptococcal toxic shock syndrome occur as a complication of GAS infection.
Table 4: Studies from various countries showing prevalence of superantigens genes

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Scarlet fever outbreaks are increasingly recognised in the last decade. Scarlet fever outbreaks have been reported in Vietnam (2009, over 23,000 cases); Guernsey, the United Kingdom (2011, six cases); Valencia, Spain (2011, 40 cases); Shanghai, China (2011, over 40 cases); Guangdong, China (2011, over 841 cases); Northwest Territories, Canada (2012, over 100 cases); Kansas, USA (2012); and Hong Kong (2011, 1534 cases).[27],[28] There is a recent outbreak of scarlet fever in United Kingdom (7198 cases) and south-east Asia including Hong Kong, Shanghai, Thailand, Macau and Singapore.[29] More than 900 cases of scarlet fever were recorded in Hong Kong during 2011. High resistance rates against macrolides were also observed for the outbreak in mainland China.[30] The epidemic in Hong Kong alerted the health authorities in Macau and also in nearby Thailand, where authorities are making all effort to assuage public fear in spite of the increasing number of cases that had already been reported.[31]

Though there is no case report of scarlet fever from India. There is a recent outbreak of GAS reported in a tertiary care hospital reported by Mathur et al.[32] in an high dependency unit (HDU) having Spe genes, SpeB, SpeC, SpeG, SpeF and SmeZ and lack SpeA gene. The strains were of emm type 58.

In the present study, we found that though in India, there is no documented case of scarlet fever, but the genes responsible for the production of pyrogenic exotoxin (superantigen − responsible for scarlet fever and toxic shock syndrome) are present in the clinical GAS isolates causing symptomatic infection. Pyrogenic exotoxin A, the most potent toxin of GAS, has low prevalence (4%) in the clinical isolates from India. This toxin is encoded by phage-mediated gene SpeA. SpeB gene is chromosomally mediated and has high prevalence (80.6%) among the clinical isolates. This gene encodes for a cysteine protease enzyme that cleaves or degrades host serum proteins such as human extracellular matrix, immunoglobulins, complement components, and even GAS surface and secreted proteins. Destruction of both host and bacterial proteins makes SpeB the key virulence factor in GAS pathogenesis. SpeC has the prevalence (35.6%) in clinical isolates of GAS. It encodes for pyrogenic exotoxin C that is moderately potent and is phage-encoded. Because the SpeA and SpeC are phage-mediated genes, they can be transmitted to other strains of GAS.

The genes of pyrogenic exotoxin are not only associated with GAS but are also found in some strains of group C streptococci, and group G streptococci.[33],[34] This again shows that the genes can be transferred between strains.

In the present study, all the clinical isolates of GAS were found susceptible to penicillin, vancomycin and linezolid that were similar to earlier studies. Erythromycin resistance was found in 33% of isolates, among which the inducible clindamycin resistance was found in 20% of isolates tested by D-test.[12] We also found GAS isolates to be resistant to tetracycline 35.5% and levofloxacin 6.5%.

The present study highlights the need to characterise group A streptococci strains by molecular methods based on Spe genes because the organism is virulent and can cause outbreak of disease like scarlet fever. There is a recent upsurge in the cases of scarlet fever caused by the action of pyrogenic exotoxin, a superantigen in south-east Asia warning us to keep a check on gene profile of the circulating GAS isolates in the community.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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