Virulence genes and resistance to antibiotics of beta-hemolytic streptococci isolated from children in Chiapas , Mexico

Introduction: Among beta-hemolytic streptococci, Streptococcus pyogenes causes a wide variety of human disease including pharyngitis, necrotizing fasciitis and streptococcal toxic syndrome. Group A Streptococcus (GAS) uses a variety of virulence traits to colonize and then cause damage to the host; others species of beta-hemolytic streptococci are considered as emerging pathogens for humans. Despite its recognized virulence, only few studies have investigated virulence factors of GAS strains isolated in Mexico. Methodology: We conducted an epidemiological study to investigate the prevalence of GAS strains in child illnesses in Chiapas Mexico. Virulence genes encoding proteases, DNases, superantigens, as well as susceptibility to antibiotics were investigated. Results: During 2010, 2013 and 2014, beta-hemolytic streptococci (N=12) were isolated from cases of bacterial infections including pharyngitis and bacteremia, with a prevalence of 0.42, 0.04 and 0.20%, respectively. S. pyogenes was the most frequent species (33%) followed by S. agalactiae and S. dysgalactiae subsp. equisimilis (25%, each). Most GAS strains encoded genes for proteases: scpA, speB, spyCEP and mac (75%), followed by sdaD and sdaB (DNases) (50%), speA and speG (superantigens; 50 and 25%, respectively). The scpA gene was amplified in all S. agalactiae strains and in 35% of SDSE strains. Strains were all susceptible to beta-lactams, cephalosporins and quinolones. Conclusions: The present study provides evidence on the epidemiology of beta-hemolytic streptococci infecting children at the southeast Mexico, their virulence traits and sensitivity to first-line antibiotics.


Introduction
For almost 20 years (1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004), streptococcal pharyngitis and tonsillitis (International Code Diseases -ICD-numbers J02.0, J03.0) were located among the 20 major diseases which affected the Mexican population, mainly children of 1 to 9 years of age.From 2012 to 2014 the diseases reappeared with incidence rates exceeding the national rate (113.73,113.08 and 134.08 new cases for 100,000 inhabitants in 2012, 2013 and 2014, respectively), being Tamaulipas, Coahuila, Sonora and Zacatecas (Mexican northeast) and Guerrero and Oaxaca (Mexican southeast) the states presenting the largest quantity of new cases [1].This kind of infections are mainly caused by Streptococcus pyogenes or GAS (Group A Streptococcus) which causes direct infections on the skin, soft tissues, pharynx, bones, bacteraemia, toxin-mediated infections (scarlet fever, Streptococcal toxic shock syndrome, necrotising fasciitis) and some other affections mediated by the immune system [2].Other species that are potentially pathogenic for humans are the opportunistic Streptococcus agalactiae or Group B Streptococcus (GBS) which colonize the gastrointestinal and gastro urinary tracts in adults causing pneumonia, septicemia and meningitis in neonates, being a factor of morbimortality in pregnant women, elders and immunocompromised persons [3].S. dysgalactiae subsp.equisimilis (SDSE) causes infections in the pharynx and skin, as well as acute rheumatic fever and bacteremia favored by its ability of adding platelets, internalizing in the human endothelium [4,5].S. constellatus subsp.pharyngis colonizes the human pharynx causing pharyngitis [6].Pathogenicity among these species is measured by different factors as hemolysins, adhesins, proteases, DNAses and superantigens [7,8].Proteases may degrade antibodies (SpeB), chemotactic cytokines as IL-8 (SpyCEP), proteins from complement system (SCPA and SIC) or prevent phagocytosis (Mac) [8]; DNases (as Spd3, Sdc, SdaB and SdaD) degrade the neutrophil extracellular traps (NETs) allowing the bacteria to escape from phagocytosis, meanwhile superantigens (SpeG, SpeJ, SMEZ, SpeA, SpeC, SpeH, SpeI, SpeJ, SpeK, SpeL, SpeM, and SSA) induce the massive secretion of cytokines which increase the severity of infections [9].In a greater proportion, infections caused by GAS affect children and young people from developing countries, besides the fact that it is located amongst the group of neglected diseases [2].In Mexico, since 2010, the state of Chiapas (in the Mexican southeast) holds the first place of poverty [10] and for this reason, the child population may be more susceptible to this kind of infectious diseases.The purpose of this work was to investigate the prevalence, pathogenicity profile and antimicrobial susceptibility among strains of beta-hemolytic streptococci isolated from children living in a major city of South Mexico, Tuxtla Gutiérrez, Chiapas.
Bacteriological cultures were performed at the Microbiology and Pathology Laboratory of HEP, from tonsillar exudates, bronchial aspirates, blood, urine, abscess and ulcers, according to procedures from Spanish Society for Infectious Diseases and Clinical Microbiology [15][16][17][18].Identification at the species level was conducted with a Vitek 2 Compact (bioMérieux, Marcy l'Etoile, France) system, by using the Vitek 2 GP (Gram-positive) and Vitek 2 GN (Gram-negative) ID cards; the Vitek 2 AST-ST01 cards were used for the susceptibility test.Bacterial strains were cryopreserved in skim milk (10% v/v) at -80º C and placed at the UNICACH bacterial culture collection [19].S. pyogenes strain ATCC (American Type Culture Collection, Manassas, USA) 19615 was used as a control.This project was approved by the HEP Ethics Committee (approval ID DPEI/00394/13) offering the possibility to obtain different information as: age, sex, type of infection and treatment of the affected child.

Lancefield group's determination
The serological group was investigated with a commercial kit (Pastorex Strep, Biorad, Marnes La Coquette, France) according to the manufacturer´s specifications.

Data analysis
Age, as well as categorical variables such as sex, biologic material, Streptococus species, virulence genes and the antimicrobial susceptibility profile was analyzed through descriptive statistics.The Fisher´s exact test was used to evaluate the association between the non-parametric variables, establishing a statistical significance value if p ≤ 0.05.Analyses were conducted using the SPSS software (version 20; Armonk, New York: IBM Corp.).

Results
The average mean age of the affected patients was 5.6 years; most of these were boys (59.7%;N = 387), followed by girls (40.3%;N = 261).
Regarding beta-hemolytic streptococci, a total of 12 strains were isolated during 2010, 2013 and 2014 with a prevalence of 0.42, 0.04 and 0.20%, respectively.S. pyogenes strains were the most frequent pathogen isolated (N = 4; 33%) from all biological material tested (except for blood and urine), followed by S. agalactiae and S. dysgalactiae subsp.equisimilis (SDSE) strains (N = 3; 25% each) from urine and tonsillar exudates, respectively (Table 2).Serological analysis showed that strains of S. pyogenes belonged to the Lancefield group A, S. agalactiae to group B, SDSE to Lancefield group G, and S. constellatus subsp.pharyngis (N = 2) to Lancefield groups C and non-typeable, respectively.More than a half of the beta-hemolytic streptococci strains were isolated from patients suffering of primary infections such as tonsillitis and bacteraemia (N = 9; 75.0%), followed by one case of toxin-mediated infection (N = 1, 8.3%) (Table 3); due to the low isolation rate, an association between the Streptococcus species and these diseases could not be established (data not shown).
Regarding virulence genes carried by strains, most of S. pyogenes isolates (75%) carried genes encoding for scpA, speB, spyCEP and mac proteases, whereas 50% of strains encoded sdaD and sdaB DNases and speA superantigen while only 25% of strains carried the speG superantigen gene.All S. agalactiae strains carried the scpA gene but only 33% of the SDSE strains exhibited this gene (Figure 1).There was no significant association between any Streptococcus species with genes codifying for these virulence factors (Table 4).
Nonetheless, in Chiapas, the incidence rates caused by these diseases during the period of this study (2010, Table 4.Virulence genes of beta-hemolytic streptococci strains from Tuxtla Gutierrez, Chiapas, Mexico.2013 and 2014) were of 1.21, 12.48 and 46.72 new cases for 100,000 inhabitants, respectively, not exceeding national incidence rates (104.92,113.08 and 134.08, respectively); these trends may explain the low prevalence of beta-hemolytic streptococci reported here.In contrast, during the same years the state of Tamaulipas (Mexican northeast) had incidence rates for strep throat, 8 times greater (1151.93,1018.91 and 1061.95,respectively) in relation to national rates [1].Perhaps this disparity is related to the weather because S. pyogenes infections are more prevalent in regions with dry weather, such as Tamaulipas, in comparison to regions with rainy weather as the case for Chiapas [25].

GAS
In this study, S. pyogenes was the species with more prevalence among the studied population (Table 2).In 2005, at the global level, it was estimated that at least 18.1 million persons were affected by invasive diseases caused by this species; besides it showed an incidence of 1.78 million of new cases per year, plus more than 111 and 616 million cases of streptococcal pyoderma and pharyngitis, respectively [23].After S. pyogenes, S. agalactiae and SDSE strains were the ones that mostly caused UTI and pharynx colonization, respectively (Table 3).SDSE strains isolated during the current work belong to Group G Lancefield; in fact, 90% of SDSE strains appertain to this group, even though a minority may belong to Group C [7].SDSE has a virulence profile that causes a range of infections similar to GAS [26].One of the analyzed SDSE strains in the current work was isolated from blood of a patient with leukemia; it has been reported that malignancy, as well others underlying diseases constitute a predisposing factors in bacteremia caused by SDSE [27].A multicenter study done in Argentina reported 23 invasive infections (60.9% isolated from blood) caused by SDSE [26].
S. constellatus subsp.pharyngis belongs to the Lancefield group C streptococci, strains are mainly found in the human throat where can cause pharyngitis [6].Whereas it is not a primary pathogen, patients with underlying immune system diseases can carry this bacterium in the upper airways, such as it was demonstrated in the nasopharynx of patients included in this study (Table 3).
The genetic analysis of the Streptococcus species analyzed in the current work showed that S. pyogenes was the only one exhibiting greater repertoire of virulence genes (proteases, DNases and certain superantigens); this could explain the broad spectrum of diseases caused by this Gram-positive bacteria [2].
Interestingly, in all clinical isolates of GBS and a third of SDSE, we amplified the gene for the protease SCPA (Table 4).This may be due to the mechanism of horizontal gene transfer described between both species, as protease SCPB of GBS exhibits a high genomic and proteomic homology with the SCPA of GAS [28]; besides, this event seems to occur in Group G Streptococcus, as a copy of this gene has been described in this species [29].Along with the gene encoding SCPA protease, genes speB, spyCEP and mac were detected in most clinical isolates of S. pyogenes isolated in this work.The gene speB, encode a cysteine protease, SpeB, which promotes the dissemination of GAS in the affected tissues, but also plays a role modulating the host immune system and certain virulence factors of such Gram-positive [30].SpyCEP, which operon spyCEP was identified in this study, degrades IL-8 allowing S. pyogenes to be refractory to the destruction by neutrophils [31], while Mac/IdeS, encoded by mac, is a protease that degrades IgG [32].
Half of S. pyogenes isolates exhibited genes that encode for DNases, immunogenic proteins conferring bacterial protection against polymorphonuclear leukocytes through its action over the neutrophil extracellular traps (NETs) [33].Finally, some S. pyogenes isolates carried genes encoding SpeA and SpeG superantigens.This information is similar to the one obtained through a study made with 54 S. pyogenes strains isolated from children affected by pharingytis (Mexico City), where 61% (N = 33) showed the presence of speA gene.Besides, a variability in sic gene was found; such gene codifies a protease which inhibits the complement [34].Another study carried out in Mexico City analyzed 47 S. pyogenes strains isolated from a scarlet fever outbreak and more than a half (67.5%) exhibited the speA gene [35].
The strains analyzed in this study were susceptible to cephalosporins and beta-lactams.These findings are similar to the ones of a study made with 100 S. pyogenes strains from the "Hospital Infantil de la Ciudad de México" indicating that all the strains were sensitive to beta-lactams and clindamycin [36].In the same way, another work performed in Mexico City showed that 100% from 197 S. pyogenes isolates from adults presenting pharyngotonsillitis were sensitive to moxifloxacin, a third generation quinolone that can be employed as an alternative for the treatment of patients allergic to penicillin or for S. pyogenes strains resistant to macrolides [37].Another study reported that, between 1999 and 2000, all GAS strains isolated in Mexico (N = 467) and USA (N = 174) were susceptible to penicillin, ceftriaxone and vancomycin, while the resistance for erythromycin was low for both groups (4.9 and 5.2% for Mexico and USA, respectively).In this study, the resistance observed for such macrolide was 15.4%, and for this reason, it should be necessary to analyze the presence of certain genes as mefA and ermB in macrolide resistant strains, as well as its association with this phenotype [38].

Conclusions
The current study revealed a low prevalence of beta-hemolytic streptococci species among children who assist to the HEP (Tuxtla Gutiérrez, Chiapas) demonstrating that: 1) S. pyogenes is the most frequent species which causes direct infections and, 2) indicating that its virulence profile is mainly constituted by genes that encode for proteases.
It is important to note that GAS strains were susceptible to a wide range of antibiotics.This information is important in view that this was the first study analyzing virulence factors, and resistance to antibiotics, in Gram-positive bacteria isolated from children of Tuxtla Gutiérrez, Chiapas.The information will guide therapeutics.Future studies should also include strains causing disease in children living in marginalized municipalities of the state.We acknowledge the need of incorporating more sophisticated analysis such as the typification of emm genes to study the geographical distribution and its association with symptoms.We will likely identify, in Mexico, a high diversity of emm types, as reported in other developing countries [39].
The additional study of these molecular markers among beta-hemolytic streptococci strains from Tuxtla Gutiérrez, Chiapas and other geographical regions of Mexico will enable us to determine the origin of epidemic breakouts, characterize the strains and predict its pathogenic potential to implement preventive strategies such as the use of biological products directed to the central and northern population of Mexico, where it is usual to find strep throat incidence rates greater than the national average.Financial support for this work was granted to the academic group "Recursos fitogenéticos y sustentabilidad en Chiapas" by Universidad de Ciencias y Artes de Chiapas (grant number 150100 022 2.01PDI428.PY024.001),as well as "Apoyo al fortalecimiento y desarrollo del a infraestructura científica y tecnológica 2014, Consejo Nacional de Ciencia y Tecnología -CONACyT"-(grant number 226293).Authors are gratefully to Dr. Dalia Mendoza Guillén, from health center "Albania", Tuxtla Gutiérrez, Chiapas, for her clinical support.

Authors´contributions
JGJ: conceived the whole study, assessed methodology, analyzed data and wrote the manuscript;MIMO: performed molecular analysis of bacterial strains; AVS: performed molecular analysis of bacterial strains; LMLC: assessed methodology, and wrote the manuscript; JMFG: performed phenotypic analys as well as antimicrobial susceptibility tests of bacterial strains; JAGH: assessed clinical data, and provided intellectual support to the overall manuscript; JEV: Analyzed data and wrote the manuscript.

Table 1 .
Bacterial strains isolated from children of Chiapas, Mexico.

Table 2 .
Isolation source of beta-hemolytic streptococci strains from children in Tuxtla Gutierrez, Chiapas, Mexico.