Abstract

Staphylococcus aureus is a frequent cause of infections worldwide. Methicillin-resistant S. aureus (MRSA) is one of the main causes of Gram-positive infections, and methicillin-susceptible strains (MSSA) primarily colonize and infect community hosts. Multiple virulence factors are involved, with toxins playing a significant role in several diseases. In this study, we assess the prevalence of toxin genes in 89 S. aureus clinical isolates (31 MRSA and 58 MSSA). We evaluated the discriminatory power of the association of internal transcribed spacer-PCR (ITS-PCR) and 3’- end coa gene ( coa-PCR) when compared with other more commonly used and costly techniques. The isolates showed a high level of genetic diversity, and toxins were found in all the isolates. While most toxin classes displayed no statistically significant correlations and were equally distributed in isolates regardless of their resistance status, classic enterotoxins ( sea-see) showed a positive correlation with MSSA isolates. The combination of coa-PCR with ITS-PCR showed a discriminatory index of 0.84, discriminating 22 genotypes that agree with previously determined data by PFGE and MLST. This association between the two PCR-based methods suggests that they can be useful for an initial molecular epidemiological investigation of S. aureus in hospitals, providing significant information while requiring fewer resources.

Keywords:
Staphylococcus aureus ; MRSA; MSSA; genotyping; exotoxins

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) are major pathogens associated with serious infections in both hospitals and communities worldwide ( Tong et al., 2015Tong SY, Davis JS, Eichenberger E, Holland TL and Fowler VG (2015) Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603-661. ; Arias et al., 2018Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, Ibarra G, Rios R, Munita JM, Salles MJ et al. (2018) Prospective cohort multicenter study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine latin american countries. Antimicrob Agents Chemother 61:e00816-17.; Junie et al., 2018Junie LM, Jeican II, Matroş L and Pandrea SL (2018) Molecular epidemiology of the community-associated methicillin-resistant Staphylococcus aureus clones: A synthetic review. Clujul Med 91:7-11. ; Lakhundi and Zhang, 2018Lakhundi S and Zhang K (2018) Methicillin-resistant Staphylococcus aureus: Molecular characterization, evolution, and epidemiology. Clin Microbiol Rev 31:e00020-18.). In Latin American countries like Brazil, Bolivia, Chile, and others, more than 50% of S. aureus isolates are already categorized as MRSA and can be considered resistant to most β-lactams ( Lee et al., 2018Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A and Harbarth S (2018) Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers 4:18033.).

Exotoxins produced by S. aureus can cause diarrhea that is either related or not to the use of antibiotics, gastroenteritis, or food poisoning ( Ortega et al., 2010Ortega E, Abriouel H, Lucas R and Gálvez A (2010) Multiple roles of Staphylococcus aureus enterotoxins: Pathogenicity, superantigenic activity, and correlation to antibiotic resistance. Toxins (Basel) 2:2117-2131.; Pinchuk et al., 2010Pinchuk IV, Beswick EJ and Reyes VE (2010) Staphylococcal enterotoxins. Toxins 2:2177-2197.). This pathogen is also connected to severe illnesses like Toxic Shock Syndrome (TSS) and Scalded Skin Syndrome (SSS), which are regarded as superantigens and caused by toxin production ( Ladhani et al., 1999Ladhani S, Joannou CL, Lochrie DP, Evans RW and Poston SM (1999) Clinical, microbial and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome. Clin Microbiol Rev 12:224-242.; Dinges et al., 2000Dinges MM, Orwin PM and Schlievert PM (2000) Exotoxins of Staphylococcus aureus. Clin Microbiol Rev 13:16-34.). The group of superantigens includes staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST), and exfoliative toxins (ET) ( Portillo et al., 2013Portillo BC, Moreno JE, Yomayusa N, Álvarez CA, Cardozo BEC, Pérez JAE, Díaz PL, Ibañez M, Mendez-Alvarez S, Leal AL et al. (2013) Molecular epidemiology and characterization of virulence genes of community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus isolates in Colombia. Int J Infect Dis 17:e744-e749.).

For epidemiological purposes, it is crucial to identify the distribution of clinical isolates, and genotyping has emerged as a key tool in medical investigations to identify strain origin, clonal relatedness, and outbreak epidemiology ( Olive and Bean, 1999Olive DM and Bean P (1999) Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 37:1661-1669.; Arias et al., 2018Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, Ibarra G, Rios R, Munita JM, Salles MJ et al. (2018) Prospective cohort multicenter study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine latin american countries. Antimicrob Agents Chemother 61:e00816-17.). Genotyping methods often involve applying different molecular techniques based on PCR, sequencing, or genomic macrorestriction ( Rodríguez-Noriega et al., 2010Rodríguez-Noriega E, Seas C, Gúzman-Blanco M, Mejía C, Alvarez C, Bavestrello L, Zurita J, Labarca J, Luna CM, Salles MJC et al. (2010) Evolution of methicillin-resistant Staphylococcus aureus clones in Latin America. Int J Infect Dis 14:e560-6.).

By combining these methods, strains can be classified into different lineages, or clones. Some clones of S. aureus are known as epidemic clones, meaning they are descendants of the same ancestor and are widely distributed among different countries. Some of these lineages, such as the Brazilian Epidemic clone (BEC), the Pediatric clone (PC), and the Cordobes/Chilean clone, have already been identified as native to Latin America and are useful for describing the various genetic backgrounds of S. aureus ( Zurita et al., 2016Zurita J, Barba P, Ortega-Paredes D, Mora M and Rivadeneira S (2016) Local circulating clones of Staphylococcus aureus in Ecuador. Braz J Infect Dis 20:525-533.).

Since few data have been published in Brazil, particularly in the Northeast region, our goal was to investigate the exotoxin gene profile for MRSA and MSSA isolates from patients admitted to hospitals in Recife city, Pernambuco state, Brazil. Additionally, we aimed to evaluate the ability of coagulase gene typing ( coa-PCR) and ribosomal 16S-23S internal transcribed spacer (ITS-PCR) to distinguish isolates and clones from clinical onsets in comparison to techniques more frequently used for molecular epidemiology studies of S. aureus, such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, and Staphylococcal Cassette Chromosome mec (SCC mec) genotyping.

Material and Methods

Ethics statement

The project was approved by the Oswaldo Cruz Foundation Health Research Ethics Committee, Aggeu Magalhães Institute, IAM/Fiocruz, Brazil (CEP: 0024.0.095.000-07).

Bacterial isolates and DNA extraction

In this investigation, 89 S. aureus isolates were obtained on spontaneous demand from hospitals in Recife that treat patients from different parts of Pernambuco ( ^{Table S1} Table S1 - Clinical specimens and oxacillin/cefoxitin resistance of Staphylococcus aureus obtained from hospitals in Recife, Brazil. ). Prior to the present study, these isolates were typed using PFGE, MLST, spa, phenotypic identification of resistance to cefoxitin, and SCC mec genotyping ( Andrade-Figueiredo and Leal-Balbino, 2016Andrade-Figueiredo M and Leal-Balbino TC (2016) Clonal diversity and epidemiological characteristics of Staphylococcus aureus: High prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil. BMC Microbiol 16:115. ).

Genomic DNA was extracted using the automated NucliSens-easyMAG (bioMérieux, Durham, NC), and PCR for toxigenic genes and molecular typing methods, designed for the present work, were performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA).

Detection of staphylococcal toxins genes

Using the primers given in Table 1, we examined the toxic shock syndrome toxin gene ( tst), two exfoliative genes ( eta and etb), and 14 staphylococcal enterotoxin (se) genes.

Two multiplex PCR were performed, one for sea-see genes and another for tst, eta and etb genes. Uniplex PCRs were designated for seg to seo genes. PCR uniplex assays were prepared to a final volume of 50 µL, containing 40 ng chromosomal DNA, 0.8 mM of deoxynucleotide triphosphates, 1X PCR buffer, 1.5 mM MgCl₂, 1 U Taq DNA polymerase (Promega, Madison, WI, USA) and 20 µM of each oligonucleotide. Amplification happened as follows: 95 ºC for 2 min, then 30 cycles of 95 ºC for 1 min, 55 ºC for 1 min and 72 ºC for 1 min. Products were separated by electrophoresis through 1.5% agarose gel.

Multiplex assays were prepared to a final volume of 50 µL, containing 40 ng chromosomal DNA, 1 mM of deoxynucleotide triphosphates, 1X PCR buffer, 3 mM MgCl₂, 1.5 U Taq DNA polymerase (Promega, Madison, WI, USA) and 20 µM of each oligonucleotide. Amplification consisted of denaturation 95 ºC for 2 min, then 30 cycles of 95 ºC for 1 min, 60 ºC for 1 min and 72 ºC for 2 min. Products were separated by electrophoresis through 1.5% agarose gel.

The following S. aureus strains were used as positive controls: FRI722 and FRIS6 (for sea and seb genes, respectively), FRI361 ( sec, sed, seg, sei and sej genes), and FRIMN8 ( tst gene), provided by Food Research Institute (Madison, Wiscosin, EUA), 1SB and 3SB ( sek, sel and sem), MRSA41 ( sen and seo) and CR6 ( seh) (Aggeu Magalhães Institute, laboratory collection, Recife, PE, Brazil).

Toxin profiling was done by converting the toxin gene data into a binary matrix. For each isolate, a concatenation of these data yielded a binary profile resembling a barcode sequence. ^{Table S2} Table S2 - Toxin gene profiling and data collection. Sheet 1 = toxin profile arrangement and counting; Sheet 2 = toxin profiles and toxin gene PCR findings for all isolates; Sheet 3 = MRSA isolates results; Sheet 4 = MSSA isolates results. has a detailed listing of these data.

coa-PCR

The coagulase gene typing was performed as previously described ( Aarestrup et al., 1995Aarestrup FM, Dangler CA and Sordillo LM (1995) Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis. Can J Vet Res 59:124-128.). PCR assays were prepared to a final volume of 50 µL, containing 40 ng of chromosomal DNA, 0.8 mM of deoxynucleotide triphosphates, 1X PCR buffer, 2.5 mM MgCl₂, 1 U Taq DNA polymerase (Promega, Madison, WI, USA), and 20 µM of each oligonucleotide. Amplification was made up of an initial denaturation of 95 ºC for 2 min, then 30 cycles of 95 ºC for 1 min, annealing at 55ºC for 1 min and extension at 72 ºC for 1 min. Products were submitted to electrophoresis through 1.5% agarose gel. S. aureus strain ATCC 25923 was used as a positive control.

ITS-PCR

Amplification of the 16S-23S intergenic spacer region (ITS-PCR) employed a single pair of primers, as previously described ( Jensen et al., 1993Jensen MA, Webster JA and Straus N (1993) Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 59:945-952.). PCR assays were prepared to a final volume of 50 µL, containing 40 ng chromosomal DNA, 1 mM of deoxynucleotide triphosphates, 1X PCR buffer, 3 mM MgCl₂, 1.5 U Taq DNA polymerase (Promega, Madison, WI, USA) and 20 µM of each oligonucleotide (Jensen et al., 1993). Amplification was made up of 95 ºC for 5 min, then 30 cycles of 95 ºC for 1 min, 55 ºC for 1 min and 72 ºC for 2 min. Electrophoresis was performed using a 2% agarose gel. For quality assurance, S. aureus strains ATCC 33591 and ATCC 25923 were employed.

Sequencing

A random sample of purified PCR products from toxigenic genes, ITS-PCR and coa-PCR (Purelink PCR purification kit, Invitrogen, Carlsbad, CA, USA) were chosen for DNA sequencing using the Big Dye Terminator Kit v3.1 and an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) in order to confirm only the specificity of the amplified genes. The PureLink PCR purification kit was provided by Invitrogen, Carlsbad, California, USA (Applied Biosystems, Foster City, CA, USA). The nucleotide sequences obtained were compared with the S. aureus sequence database in the GenBank through BLAST (http://www.ncbi.nlm.nil.gov).

Discriminatory analysis

In order to compare the discriminatory index of typing methods, we used the formula described by Hunter and Gaston (1988Hunter PR and Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: An application of Simpson’s index of diversity. J Clin Microbiol 26:2465-2466.): DI = 1 - [1 / N(N - 1)] Σs nj(nj - 1), where N is the total number of isolates in this population, s is the total number of different types, and nj is the number of isolates representing each type. This formula is based on the probability that two unrelated strains taken from the population sample will be placed in different types of groups.

Statistical analysis

We investigated any connections between the presence of toxins and the genotypes identified by Coagulase/ITS-PCR, as well as between the presence of toxins and the resistance status (either MRSA or MSSA). We used the Jamovi software’s Pearson’s correlation test, and only results with a p value of 0.05 or lower were considered statistically significant ( Jamovi, 2023Jamovi (2023) Software version 2.3, 3, https://www.jamovi.org (accessed 29 May 2023).
https://www.jamovi.org... ).

Results

Toxigenic profiles of Staphylococcus aureus isolates

None of the 89 isolates carried sed, see, or etb, but all isolates were positive for at least three of the investigated genes. The most frequent toxin gene was seg, presented in 88/89 (99%) isolates, followed by sem 81/89 (91%), seo 80/89 (90%), sen 78/89 (88%) and sei 74/89 (83%), all members of the enterotoxin gene cluster ( egc). Six isolates (Sa08, Sa32, Sa80, Sa82, Sa87, and Sa88) were positive for the tst gene, and only one (Sa17) harbored eta. In one isolate (Sa87) from an ICU patient in Hospital 2, 12 of the 17 genes under investigation were found.

Statistically, however, the only relevant correlation happened between the MSSA group and classical enterotoxins. 19 MSSA isolates carried a sum of 21 genes, while MRSA isolates carried only one ( sea, in isolate Sa86 - toxin profile 33) ( Table 2).

All strains, regardless of susceptibility, carried at least two egc-related genes. A complete set of the egc, consisting of seg, sei, sem, sen, and seo, was found in 63/89 (71%), of which 22 are MRSA and 41 MSSA.

Isolates were subdivided into 45 genetic profiles based on their toxigenic content, and these genotypes were called toxin profiles ( ^{Table S2} Table S2 - Toxin gene profiling and data collection. Sheet 1 = toxin profile arrangement and counting; Sheet 2 = toxin profiles and toxin gene PCR findings for all isolates; Sheet 3 = MRSA isolates results; Sheet 4 = MSSA isolates results. ). The 31 MRSA isolates exhibited 16 profiles, while the 58 MSSA isolates were associated with 39, with both groups sharing 10 of these toxin profiles. The toxin profile 22 - representing a complete egc in addition to genes sej, sek, and sel - was the most prevalent genotype, occurring in 10 isolates (10/89 - 16%; 5 MRSA and 5 MSSA). For both groups, toxin profile 22 was also the most frequent, followed by 17, which had four representatives in each group and corresponds to a complete egc in addition to gene sek.

PCR-based typing methods

Coagulase genotyping analysis revealed five coagulotypes, according to the amplified segment, denominated as C1= ~730 bp (corresponding to 4 repeats), C2= ~810 bp (corresponding to 5 repeats), C3= ~890 bp (corresponding to 6 repeats), C4= ~970 bp (corresponding to 7 repeats) and C5 = ~650 bp (corresponding to 3 repeats). S. aureus ATCC 25923 amplified a fragment of ~810 bp ( Figure 1). In ITS-PCR reactions were observed 3 to 8 fragments with approximate sizes of 380 to 650 bp. Based on the amplification patterns, isolates were classified into 15 types, designated in this study as R1-R15 ( Figure 2).

The association between coa-PCR and ITS-PCR (C/R) analysis revealed 22 genotypes ( Table 3). The discriminatory index obtained by this combination was 0.84, similar to the index obtained by MLST (0.86) and spa-typing (0.89) but lower than the index obtained by PFGE (0.99). The 22 genotypes and the toxins under investigation did not show any statistically significant correlations.

Twenty-six isolates (26/89 - 29%) exhibited the genotype C/R-9. Of these isolates, 21 (21/26 - 80,8%) exhibit ST (multilocus sequence type profile) 5, in which 10 MRSA isolates are related to the Pediatric clone (PC, also called USA800), harboring the following molecular characteristics: ST5, spa types t002 (5 isolates), t267, t6787, spa non-typeable (3 isolates), SCC mecIV, and grouped into PFGE clusters C or D. Four MSSA isolates within ST5 (t002/t10548/t1277/t214) and 3 MSSA isolates ST1635/t002 are also related to PC and were grouped together into PFGE cluster F and within genotype 9. One additional MRSA isolate (Sa01) was related to USA800/PC, even though with another ST (the newly described ST2381), grouped into PFGE cluster C, and also was classified as genotype 9.

One MRSA isolate (Sa81, ST105/t002-SCC mecII) and 1 MSSA isolate (Sa03, ST5/t2164) were individually related to the New York/Japan clone, or USA100 (ST5-SCC mecII) in a PFGE dendrogram previously obtained ( Andrade-Figueiredo and Leal-Balbino, 2016Andrade-Figueiredo M and Leal-Balbino TC (2016) Clonal diversity and epidemiological characteristics of Staphylococcus aureus: High prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil. BMC Microbiol 16:115. ). These STs within genotype C/R-9 (ST105, ST1635, and ST2381) belong to the same clonal complex of ST5 isolates (CC5) since they have six matching loci.

Genotype C/R-16 consists of only one isolate, a MSSA with ST5 and in PFGE cluster F, and therefore, also related to PC/USA800 ( Table 3). Genotypes C/R-9 and C/R-16 differ only in their ITS-PCR profiling. While R1 (ITS-PCR profiling for genotype C/R-9) has 5 bands, R9 (ITS-PCR profiling for genotype C/R-16) has 4, and 3 of these bands are exactly at the same height. We can therefore suggest that both of these genotypes are related.

The genotype C/R-4 was observed in 23 isolates (23/89 - 26%), of which 17 (17/23 - 74%) are MRSA ST239/t037-SCC mecIII, related to the Brazilian epidemic clone (BEC); 13 of these isolates were grouped into PFGE clusters A (7 isolates) or B (6 isolates). Two MSSA isolates with the genotype C/R-4 were also ST239/t037. In addition, 3 MSSA isolates that exhibit ST333 (t084 [2 isolates], spa non-typeable) and 1 MSSA isolate, ST15 and spa non-typeable, were genotyped C/R-4 and grouped together into PFGE cluster G ( Table 3).

The genotype C/R-11 was observed in 6 MSSA isolates (6/89 - 7%), in which 5 ST30 (t318 [4 isolates] and t021 [1]), and 1 isolate ST285/t021 are related to the Oceania Southwest Pacific clone (OSPC, also called USA1100 - ST30-SCC mecIV) and were grouped in PFGE cluster H. Similarly, the genotype C/R-12 was observed in 4 MSSA isolates (4/89 - 4%) MSSA isolates, of which 3 are ST30 (t433 [2 isolates] and t1001 [1]) and 1 (Sa4) is ST and PFGE non-typeable. These isolates are also related to the OSPC clone and were grouped into cluster H ( Table 3). ST30 and ST285 belong to the same clonal complex (CC30), as they have four or more similar MLST loci.

The genotype C/R-3 was observed in 6 isolates (6/89 - 7%), harboring STs ST239, ST25, ST30, ST333, and ST71. Two of these isolates were related to international clones: Sa73, classified as ST239/t037-SCC mecIII is related to the BEC clone (PFGE cluster B); and Sa65 (ST30) is related to OSPC (cluster H).

All 5 MSSA isolates (5/89 - 6%) within ST1 exhibited the genotype C/R-6, of which 3 (ST1/t127) are related to the USA400 clone (ST1-SCC mecIV) and grouped into PFGE cluster E.

Three isolates (3/89 - 3%) were genotype C/R-20, of which two (Sa72 [ST669/t359] and Sa49 [ST97/t267]) were also related to the USA400 clone and grouped into cluster E. The genotype C/R-10 was observed in two (2/89 - 2%) MSSA isolates that exhibit a new ST (ST2382) and spa type t189.

Discussion

Staphylococcus aureus is responsible for a broad spectrum of diseases in humans due to its ability to express several virulence factors, including enterotoxins, toxic shock syndrome toxin, and exfoliative toxins. Among them, SEs are the major cause of staphylococcal food poisoning ( Argudin et al., 2010Argudin MÁ, Mendoza MC and Rodicio MR (2010) Food poisoning and Staphylococcus aureus enterotoxins. Toxins (Basel) 2:1751-1773.). Ferry et al. (2005Ferry T, Thomas D, Genestier AL, Bes M, Lina G, Vandenesch F and Etienne J (2005) Comparative prevalence of superantigen genes in Staphylococcus aureus isolates causing sepsis with and without septic shock. Clin Infect Dis 41:771-777.) described that S. aureus strains that cause sepsis, with or without shock, harbor at least one superantigen-encoding gene. Several diseases, including infectious endocarditis and food poisoning, have already been linked to egc ( Johler et al., 2015Johler S, Giannini P, Jermini M, Hummerjohann J, Baumgartner A and Stephan R (2015) Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins. Toxins (Basel) 7:997-1004.; Stach et al., 2016Stach CS, Vu BG, Merriman JA, Herrera A, Cahill MP, Schlievert PM and Salgado-Pabón W (2016) Novel tissue level effects of the Staphylococcus aureus enterotoxin gene cluster are essential for infective endocarditis. PLoS One 11:e0154762.).

Different rates of toxigenic genes have been found in S. aureus clinical isolates from multiple countries, according to a number of investigations ( Song et al., 2016Song M, Shi C, Xu X and Shi X (2016) Molecular typing and virulence gene profiles of enterotoxin gene cluster ( egc)-positive Staphylococcus aureus isolates obtained from various food and clinical specimens. Foodborne Pathog Dis 13:592-601. ; De Carvalho et al., 2019De Carvalho SP, De Almeida JB, De Freitas LM, Guimarães AMS, Do Nascimento NC, Dos Santos AP, Campos GB, Messick JB, Timenetsky J and Marques LM (2019) Genomic profile of brazilian methicillin-resistant Staphylococcus aureus resembles clones dispersed worldwide. J Med Microbiol 68:693-702.). In our study, an elevated frequency of S. aureus clinical isolates comprising toxigenic genes was observed, especially those with a complete egc. All isolates related to the USA800/PC clone contained a complete egc, as well as other toxins, except for two isolates (Sa1 and Sa76) which exhibited only part of this cluster. According to Monecke et al. (2011Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D et al. (2011) A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant S taphylococcus aureus. PLoS One 6:e17936.), CC5 isolates carry the enterotoxin gene cluster, although partial deletions have been observed.

Only a few studies have examined the frequency of toxigenic genes in S. aureus in Brazil, particularly in the North and Northeast regions and especially in clinical specimens ( Vasconcelos et al., 2011Vasconcelos NG, Pereira VC, Araujo Junior JP and da Cunha ML (2011) Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil. J Appl Microbiol 111:749-762.). In one study, 14% of MRSA strains from a university general hospital in Recife during 2002-2003 were related to the USA800 clone and harbored egc. Additionally, approximately 70% of MRSA strains were related to BEC, and none of them had toxigenic genes ( De Miranda et al., 2007De Miranda OP, Silva-Carvalho MC, Ribeiro A, Portela F, Cordeiro RP, Caetano N, Vidal CFL and Figueiredo AMS (2007) Emergence in Brazil of methicillin‐resistant Staphylococcus aureus isolates carrying SCC mecIV that are related genetically to the USA800 clone. Clin Microbiol Infect 13:1165-1172.).

Only the classic staphylococcal enterotoxins ( sea-see) were statistically associated with the resistance status of the isolates, and MSSA had the highest frequency of these superantigens. According to earlier research, MRSA makes up no more than 10% of colonizing strains, which suggests that MSSA is very common in colonizing and/or community-acquired infections ( Mehraj et al., 2016Mehraj J, Witte W, Akmatov MK, Layer F, Werner G and Krause G (2016) Epidemiology of Staphylococcus aureus nasal carriage patterns in the community. Curr Top Microbiol Immunol 398:55-87.). The predominance of these genes in MSSA isolates raises concerns for community-associated infections because the classical enterotoxins are strongly associated with food poisoning ( seb), lethal sepsis ( sec), and infective endocarditis ( sec) ( Salgado-Pabón et al., 2013Salgado-Pabón W, Breshears L, Spaulding AR, Merriman JA, Stach CS, Horswill AR, Peterson ML and Schlievert PM (2013) Superantigens are critical for Staphylococcus aureus infective endocarditis, sepsis, and acute kidney injury. mBio 4:e00494-13.; Ahmad-Mansour et al., 2021Ahmad-Mansour N, Loubet P, Pouget C, Dunyach-Remy C, Sotto A, Lavigne J and Molle V (2021) Staphylococcus aureus toxins: An update on their pathogenic properties and potential treatments. Toxins (Basel) 13:677.).

The eta gene was found in only one isolate, and the low rates of isolates carrying the eta and etb genes responsible for SSS are in accordance with other studies, which have also demonstrated the low frequency of these genes in S. aureus isolates ( Becker et al., 1998Becker K, Roth R and Peters G (1998) Rapid and specific detection of toxigenic Staphylococcus aureus: Use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol 36:2548-2553., 2003Becker K, Friedrich AW, Lubritz G, Weilert M, Peters G and Eiff CV (2003) Prevalence of genes encoding pyrogenic toxin superantigens and exfoliative toxins among strains of Staphylococcus aureus isolated from blood and nasal specimens. J Clin Microbiol 41:1434-1439.).

The tst gene, responsible for TSS, was observed in six isolates. Two of which were related to USA800 (MRSA Sa82 and MSSA Sa87), agreeing with the results of Durand et al. (2006Durand G, Bes M, Meugnier H, Enright MC, Forey F, Liassine N, Wenger A, Kikuchi K, Lina G, Vandenesch F et al. (2006) Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxin 1 gene responsible for hospital-and community-acquired infections in France. J Clin Microbiol 44:847-853.) and Takano et al. (2008Takano T, Higuchi W, Zaraket H, Otsuka T, Baranovich T, Enany S, Saito K, Isobe H, Dohmae S, Ozaki K et al. (2008) Novel characteristics of community-acquired methicillin-resistant Staphylococcus aureus strains belonging to multilocus sequence type 59 in Taiwan. Antimicrob Agents Chemother 52:837-845.), and one isolate was related to USA600 (Berlin clone, BC, ST45-SCC mecIV), in accordance with the observations of Tenover et al. (2008Tenover F, McAllister S, Fosheim G, McDougal L, Carey R, Limbago B, Lonsway D, Patel J, Kuehnert M and Gorwitz R (2008) Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004. J Clin Microbiol 46:2837-2841. ) and King et al. (2016King JM, Kulhankova K, Stach CS, Vu BG and Salgado-Pabón W (2016) Phenotypes and virulence among Staphylococcus aureus USA100, USA200, USA300, USA400, and USA600 clonal lineages. mSphere 1:e00071-16.). Monecke et al. (2011Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D et al. (2011) A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant S taphylococcus aureus. PLoS One 6:e17936.) described that a similar CC45-MRSA-SCC mecIV strain, isolated in Australia, carries sec, sel, tst, and arginine catabolic mobile elements (ACME). Additionally, Portugal, Australia (WA MRSA-4), and Germany have all reported occasional cases of ST45-MRSA-SCCmecV. Most isolates tested in the study harbor tst, sek, and seq ( Aires-de-Sousa et al., 2008Aires-de-Sousa M, Correia B and de Lencastre H (2008) Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospital: Surveillance over a 16-year period. J Clin Microbiol 46:2912-2917.; Monecke et al., 2011Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D et al. (2011) A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant S taphylococcus aureus. PLoS One 6:e17936.).

The isolates from our study were from different clinical specimens. It is interesting to note that one isolate (MSSA Sa08, tst positive) was obtained from vaginal secretion. TSS was initially associated with the use of superabsorbent tampons in women with S. aureus tst producers on vaginal secretion; however, later, cases of non-menstrual TSS in the community and hospitals became prevalent ( Fitzgerald et al., 2001Fitzgerald JR, Sturdevant DE, Mackie SM, Gill SR and Musser JM (2001) Evolutionary genomics of Staphylococcus aureus: Insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc Natl Acad Sci U S A 98:8821-8826.; Durand et al., 2006Durand G, Bes M, Meugnier H, Enright MC, Forey F, Liassine N, Wenger A, Kikuchi K, Lina G, Vandenesch F et al. (2006) Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxin 1 gene responsible for hospital-and community-acquired infections in France. J Clin Microbiol 44:847-853.).

In the current study, we found significant genetic diversity among S. aureus isolates as well as a high frequency of toxigenic genes. Molecular typing techniques can be used to understand this diversity and how these strains are related. In some circumstances, where speed is required to identify a local outbreak and design containment plans, PCR-based genotyping approaches, such as coagulase gene typing and ITS-PCR, are fast and offer significant discriminatory power. According to Hunter and Gaston (1988Hunter PR and Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: An application of Simpson’s index of diversity. J Clin Microbiol 26:2465-2466.), a discriminatory index greater than 0.90 can be interpreted as reliable and is thus desirable. However, even though PFGE has shown greater discriminatory power ( DI 0.99), the combination of PCR-based typing methods proved to be a useful and inexpensive procedure for conducting epidemiological surveys of S. aureus on a local or regional scale, even with a DI of 0.84.

The analysis of PCR- coa identified five different amplicons. The 3´end coding region of the coa gene contains a series of repeating 81bp DNA sequences that differ in the number of tandem repeats. Since this region exhibits polymorphism, it is useful as a typing method ( Aarestrup et al., 1995Aarestrup FM, Dangler CA and Sordillo LM (1995) Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis. Can J Vet Res 59:124-128.).

We chose ITS-PCR as one of our genotyping methods in this study because of its practicality, cost-effectiveness, and alignment with the specific research objectives ( Liu et al., 2008Liu Y, Liu C, Zheng W, Zhang X, Yu J, Gao Q, Hou Y and Huang X (2008) PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. Int J Food Microbiol 125:230-235. ). This technique has several advantages, such as simplicity, speed, and the ability to discern between closely related strains based on variations within the ITS region ( Boom et al., 1990Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM and van der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28:495-503. ; Gray et al., 2014Gray TJ, Kong F, Jelfs P, Sintchenko V and Chen SC-A (2014) Improved identification of rapidly growing Mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis. PLoS One 9:e102290.). However, it is important to recognize that there are other genotyping techniques, such as ribotyping, that provide extensive information about the ribosomal RNA sequence and rely on the sequencing or hybridization of ribosomal RNA segments ( Bouchet et al., 2008Bouchet V, Huot H, Goldstein R (2008) Molecular genetic basis of ribotyping. Clin Microbiol Rev 21:262-273.).

On the other hand, ITS-PCR focuses specifically on the amplification and analysis of a specific genomic region, the ITS region of the ribosomal RNA operon. This targeted approach allows efficient screening of large collections of strains and can provide valuable information about the genetic diversity and lineage of strains based on variations within the ITS region ( Ryberg et al., 2011Ryberg A, Olsson C, Ahrne S and Monstein HJ (2011) Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates. J Microbiol Methods 84:183-188.; Lian and Zhao, 2016Lian D-S and Zhao S-J (2016) Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease. Clin Chem Lab Med 54:707-738.). By employing ITS-PCR in conjunction with other molecular typing techniques such as coagulase typing, PFGE, spa typing and SCC mec genotyping, we were able to obtain comprehensive information on the molecular epidemiology and clonal relationships between the S. aureus strains under investigation.

In this study, C/R-9 was the most frequent genotype observed among isolates. All 26 isolates in C/R-9 were associated with Clonal Complex 5, grouping all USA800/PC and USA100 clones. Thus, the results indicate that the C/R combination was able to distinguish USA800/PC and USA100 clones from other strains. Only one isolate related to the USA800 clone (Sa28 ST5/ newly described t10548, cluster F) exhibited a different genotype, C/R-16, but this genotype appears to be related to C/R-9.

The BEC represents a multidrug-resistant lineage described in Brazil in 1992, linked to hospital-acquired infections (HA-MRSA), which was widely distributed in Brazil and later in other countries. However, in the first ten years of the 2000s, there was an increase in “imported” clones, such as the Pediatric clone (PC), the New York/Japan Clone, and other less common lineages ( Andrade et al., 2020Andrade MM, Luiz WB, Souza RSO and Amorim JH (2020) The history of methicillin-resistant Staphylococcus aureus in Brazil. Can J Infect Dis Med Microbiol 7:1721936.).

Since then, BEC prevalence appears to be decreasing, while infections by community-acquired (CA-MRSA) clones, such as PC and OSPC, have been steadily increasing. Complete BEC substitution has already been reported in some hospitals ( Chamon et al., 2017Chamon RC, Ribeiro SS, Costa TM, Nouér AS and Santos KRN (2017) Complete substitution of the Brazilian endemic clone by other methicillin-resistant Staphylococcus aureus lineages in two public hospitals in Rio de Janeiro, Brazil. Braz J Infect Dis 21:185-189.; Monteiro et al., 2019Monteiro AS, Pinto BLS, Monteiro JM, Ferreira RM, Ribeiro PCS, Bando SY, Marques SG, Silva LCN, Nunes-Neto WR, Ferreira GF et al. (2019) Phylogenetic and molecular profile of Staphylococcus aureus isolated from bloodstream infections in northeast Brazil. Microorganisms 7:210. ), and CA-MRSA clones appear to be becoming more common across Brazil ( Romero and Cunha, 2021Romero LC and Cunha MLRS (2021) Insights into the epidemiology of community-associated methicillin-resistant Staphylococcus aureus in special populations and the community-healthcare interface. Braz J Infect Dis 25:101636.). Historically, there have been distinctions between the epidemiologies of HA-MRSA and CA-MRSA, with CA-MRSA typically being linked to more virulent infections and infecting patients who have few or no risk factors. However, these distinctions are becoming less clear today ( Lee et al., 2018Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A and Harbarth S (2018) Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers 4:18033.).

The genotype C/R-4 grouped 17 of 19 (89,5%) BEC and related clones, including all five BEC MRSA isolates from ICU (hospital 1), as well as distinguishing these from other epidemic/pandemic clones such as USA800/PC, USA100, USA1100, USA400, and USA 600. Only two BEC clones exhibited a diverse C/R pattern. The genotype C/R-4 also grouped 3 isolates within ST333 ( spa nontypeable, t084 [2 isolates], cluster G) and 1 isolate ST15 ( spa nontypeable, cluster G), both CC15.

All isolates within ST1 (Cluster E, CC1) were grouped into the genotype C/R-6 association. Coagulotype and ITS-PCR analyses were capable of distinguishing these isolates from others also clustered into PFGE cluster E and related to USA400 that exhibited ST669 and ST97, both STs from CC97. The genotypes C/R-11 and C/R-12 were able to group all isolates related to clone USA1100, except for isolate Sa65 (C/R-3).

We observed a more robust correlation between coa-PCR/ITS-PCR and PFGE/MLST patterns in MRSA isolates. The C/R association allowed us to observe the clonal spread of MRSA and MSSA within the main hospital analyzed (hospital 1). Patients from these isolates were dispersed throughout various hospital wings. Additionally, we discovered closely related isolates between hospital 1’s isolates and all four of hospital 2’s isolates.

No one genotyping technique that is now available is thought to be best for epidemiological studies. Every circumstance is unique, so it is important to assess the benefits and drawbacks of each technique both separately and collectively in order to choose the best methodology based on the targets and objectives outlined in each study.

Through combining coagulotype and ITS-PCR analysis, which showed a relationship with PFGE genotype and MLST as well as a minor correlation with spa typing, we found a high genetic diversity among the isolates in our study and observed clonal spread of MRSA and MSSA in hospital settings. It is important to emphasize that this specific association between techniques may be practical, quick, and affordable for initial epidemiological investigations in hospitals or local outbreaks, thus becoming an interesting strategy for countries and institutions with fewer resources.

We emphasize the need for further studies for epidemiological surveillance of MRSA and MSSA due to the change in S. aureus epidemiology and the growing threat of this pathogen to hospital and community environments.

Acknowledgements

This study was supported by research support from IAM/Fiocruz and INOVA/Fiocruz (VPPCB-008-FIO-18-2-81-30). In addition, the study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Brazil - Finance Code 001.

References

Internet Resources

Supplementary material

The following online material is available for this article:

Table S1 - Clinical specimens and oxacillin/cefoxitin resistance of Staphylococcus aureus obtained from hospitals in Recife, Brazil.

Table S2 - Toxin gene profiling and data collection. Sheet 1 = toxin profile arrangement and counting; Sheet 2 = toxin profiles and toxin gene PCR findings for all isolates; Sheet 3 = MRSA isolates results; Sheet 4 = MSSA isolates results.

Edited by

Associate Editor:

Publication Dates

History