Medicina Laboratorial - Ano 2016 - Volume 52 - Número 5
Expressão de fatores de patogenicidade por amostras de Enterococcus isoladas de pacientes com infecção de corrente sanguínea em Belo Horizonte, Minas Gerais, Brasil
Expression of pathogenicity factors by Enterococcus strains isolated from inpatients with bloodstream infection in Belo Horizonte, Minas Gerais, Brazil
Universidade Federal de Minas Gerais (UFMG), Minas Gerais, Brazil
Endereço para correspondência
Paula Prazeres Magalhães
Universidade Federal de Minas Gerais; Instituto de Ciências Biológicas; Departamento de Microbiologia, bloco C4, sala 204
Av. Antônio Carlos, 6.627; Pampulha
CEP: 31270-901; Belo Horizonte-MG, Brasil
Primeira Submissão em 03/05/2016
Última Submissão em 02/09/2016
Aceitado para sua publicação em 11/09/2016
Publicado em 20/10/2016
Neste trabalho, avaliou-se o perfil de patogenicidade de 35 amostras de Enterococcus isoladas de pacientes com infecção de corrente sanguínea (ICS) em diferentes hospitais de Belo Horizonte (MG), Brasil. E. faecalis foi a espécie mais prevalente (27/77,14%). A produção de gelatinase e citolisina, detectada em 14/40% das amostras, apresentou distribuição heterogênea. A produção de biofilme foi observada em 27 amostras (77,14%) e classificada como fraca (8/22,86%), moderada (18/51,42%) ou alta (1/2,86%). Este estudo contribui para o conhecimento do perfil de patogenicidade de amostras de Enterococcus isoladas de ICS, buscando auxiliar na compreensão da virulência das amostras circulantes no Brasil.
Palavras-chave: Enterococcus; virulência; gelatinases; proteínas hemolisinas; biofilmes.
In this investigation, we addressed the pathogenicity profile of 35 Enterococcus samples isolated from inpatients with bloodstream infections (BSI) at hospitals in Belo Horizonte (MG), Brazil. The most prevalent species was E. faecalis (27/77.14%). The ability to produce gelatinase and cytolysin was detected in 14/40% of the samples and presented a heterogeneous distribution. Biofilm production was observed in 27 strains (77.14%) and classified as weak (8/22.86%), moderate (18/51.42%), or strong (1/2.86%). This study adds to knowledge of the pathogenicity profile of Enterococcus strains isolated from BSI, aiming to increase understanding about virulence of the circulating strains in Brazil.
Keywords: Enterococcus; virulence; gelatinases; hemolysin proteins; biofilms.
Bloodstream infections (BSI) are associated with high morbidity and mortality rates worldwide(1). Cases related to Candida spp., Pseudomonas aeruginosa and Enterococcus spp. are frequently fatal(2).
Bacteria of the Enterococcus genus are members of the indigenous intestinal microbiota. Initially, the group was not recognized as having great clinical relevance, but has lately emerged as an important nosocomial pathogen. These microorganisms can synthesize the enzymes gelatinase and cytolysin, as well as adhesion proteins, which confer capacity of biofilm formation to the samples. These abilities contribute to their capacity to survive for long periods in surfaces and can help explain their dissemination in hospital settings(3, 4).
The objective of this study was to confirm the identification of Enterococcus samples isolated from BSI patients in hospitals of Belo Horizonte (MG), Brazil, to the species level. Another objective was to assess the expression of pathogenicity factors that are important for the infectious process associated with the microorganism.
MATERIAL AND METHOD
Thirty-five Enterococcus samples isolated during 2008 and 2009 from blood cultures of inpatients with BSI in different hospitals of Belo Horizonte, Minas Gerais, Brazil, were included in the study. The project was approved by the Ethics Research Committees of the participant hospitals and of Universidade Federal de Minas Gerais (UFMG) (ETIC 114/08).
The species-level biochemical and physiological identification (Vitek® 2) of bacteria was confirmed by polymerase chain reaction (PCR), using the conditions and the starters described by Dutka-Malen et al.(5). Moreover, the samples identified as E. casseliflavus by the physiological and biochemical method were tested for pigment production in tryptic soy agar (TSA) with 5% horse blood (6).
In order to assess gelatinase production, bacteria were inoculated into nutrient agar with 3% gelatin; enzyme production was identified by the presence of clear zones around the colonies(7). In order to investigate cytolysin production, samples were inoculated into TSA with 5% horse blood, and the enzyme was detected by the presence of hemolysis zones(7).
The assay to assess biofilm production was based on modifications in a protocol described by López-Salas et al.(4). The inoculum was adjusted to the turbidity of a 0.5 McFarland standard, and then was diluted 1:40 in tryptic soy broth with 2% glucose. Suspensions were inoculated into 96-well microplates and incubated. Then, plates were processed and stained with crystal violet solution. Excess crystal violet was removed, the stain adhering to the microplate was solubilized, and the optical density of the solution was measured at 570 nm. The samples were classified as biofilm producers (absorbance > 0.5) or non-biofilm producers (absorbance ≤ 0.5); production, in its turn, was classified as strong (absorbance > 2), moderate (1 < absorbance ≤ 2), or weak (0.5 < absorbance ≤ 1).
The distribution of Enterococcus species, considering the method of genotypic identification, was E. faecalis – 27 samples (77.14%), E. faecium – five samples (14.29%), E. casseliflavus – two samples (5.71%), and E. gallinarum – one sample (2.86%) (Figure A). There was agreement of 97.14% (34/35) in species-level identification between the genetic technique and the physiologic and biochemical method. Both samples of E. casseliflavus produced pigment, forming yellow colonies. PCR result was considered definitive.
Figure − Distribution of Enterococcus species (A) and evaluated production of gelatinase (B), cytolysin (C) and biofilm (D) by the samples included in the study
Gelatinase production was detected in 14 (40%) samples of E. faecalis (Figure B). Capacity for cytolysin production was also detected in 14 (40%) samples, although with a distinct distribution (Figure C), and among which just two samples of E. faecalis were classified as beta-hemolytic. Five samples, all E. faecalis, were able to synthesize both enzymes.
Biofilm production was observed in most samples (27/35, 77.14%). Among them, 18 (51.42%) were included in the category of moderate production; one (2.86%) presented strong biofilm production; and eight (22.86%), weak production (Figure D).
Results indicated higher prevalence of E. faecalis species (77.14%), as well as high agreement between the genotypic (PCR) and automated phenotypic (Vitek® 2) methods. Considering the high accuracy of the employed genetic technique, identification by PCR was chosen to define the species(8, 9). Thus, a sample initially identified as E. casseliflavus was reidentified as E. gallinarum. The result indicates that the employed phenotypic method requires complementary tests, such as assessment of pigment production, to enable accurate species identification(6, 9). The data obtained in this study are in agreement with previous reports, which show elevated prevalence of E. faecalis in enterococci infections, including in Brazil(10, 11).
The pathogenicity factors of Enterococcus contribute to enhance fitness and persistence of the pathogen in the environment. Among them can be cited those related to adhesion (biofilm formation) and synthesis of enzymes, such as gelatinase and cytolysin/hemolysin(12). Knowledge about the presence of a specific pathogenicity factor and its real implication in enterococci infectious processes in human beings is still limited. In this regard, the present investigation is even more significant, once data on virulence of Enterococcus lineages circulating in Brazil are so far scarce(13).
The expression of gelatinase was observed in 40% of the studied samples. In the literature, values close to the ones of this investigation have already been reported(12, 14). Cytolysin activity was also observed in 40% of the samples. Values equal to or close to this have been reported in studies evaluating samples from diverse origins, including blood cultures(15, 16).
In this study, more than 75% of the samples were able to produce biofilm, predominantly included in the category moderate production. Among the five samples of E. faecium, two were classified as non-producers; and three, with low capacity for biofilm production. These results are similar to other already described in the literature, which also observed that most samples of E. faecalis were strong or moderate producers, while most samples of E. faecium were non-biofilm producers(17, 18).
The data provided by this study represent a contribution for the characterization of Brazilian samples of Enterococcus, considered of great relevance as infectious disease agent in our country.
1. Laupland KB. Incidence of bloodstream infection: a review of population-based studies. Clin Microbiol Infect. 2013; 19(6): 492-500. PubMed PMID: 23398633.
2. Al Mohajer M, Darouiche RO. Sepsis syndrome, bloodstream infections, and device-related infections. Med Clin North Am. 2012; 96(6): 1203-23. PubMed PMID: 23102485.
3. Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Ver Microbiol. 2012; 10(4): 266-78. PubMed PMID: 22421879.
4. López-Salas P, Llaca-Díaz J, Morfin-Otero R, et al. Virulence and antibiotic resistance of Enterococcus faecalis clinical isolates recovered from three states of Mexico. Detection of linezolid resistance. Arch Med Res. 2013; 44(6): 422-8. PubMed PMID: 23973462.
5. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol. 1995; 33(1): 24-7. PubMed PMID: 7699051.
6. Cartwright CP, Stock F, Fahle GA, Gill VJ. Comparison of pigment production and motility tests with PCR for reliable identification of intrinsically vancomycin-resistant enterococci. J Clin Microbiol. 1995 Jul; 33(7): 1931-3. PubMed PMID: 7665675.
7. Gonçalves A, Igrejas G, Radhouani H, et al. Detection of antibiotic resistant enterococci and Escherichia coli in free range Iberian Lynx (Lynx pardinus). Sci Total Environ. 2013; 456 57: 115-9. PubMed PMID: 23588135.
8. Fang H, Ohlsson AK, Ullberg M, Özenci V. Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates. Eur J Clin Microbiol Infect Dis. 2012; 31(11): 3073-7. PubMed PMID: 22706514.
9. Garcia-Garrote F, Cercenado E, Bouza E. Evaluation of a new system, VITEK 2, for identification and antimicrobial susceptibility testing of enterococci. J Clin Microbiol. 2000; 38(6): 2108-11. PubMed PMID: 10834961.
10. Conceição N, Oliveira CC, Silva PR, Ávila BGM, Oliveira AG. Trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital: a 4-year study. Rev Soc Bras Med Trop. 2011; 44(2): 177-81. PubMed PMID: 21468473.
11. Bender EA, Freitas AL, Reiter KC, Lutz L, Barth AL. Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil. Braz J Microbiol. 2009; 40: 693-700. PubMed PMID: 24031416.
12. Medeiros AW, Pereira RI, Oliveira DV, et al. Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil. Braz J Microbiol. 2014; 45(1): 327-32. PubMed PMID: 24948952.
13. Ruzon FI, Paula SB, Kanoshiki RL, et al. Virulence determinants in vancomycin-resistant Enterococcus faecium vanA isolated from different sources at University Hospital of Londrina, Paraná, Brazil. J Microbiol. 2010; 48(6): 814-21. PubMed PMID: 21221940.
14. Soares RO, Fedi AC, Reiter KC, Caierão J, d'Azevedo PA. Correlation between biofilm formation and gelE, esp, and agg genes in Enterococcus spp. clinical isolates. Virulence. 2014; 5(5): 634-7. PubMed PMID: 24782231.
15. Libertin CR, Dumitru R, Stein DS. The hemolysin/bacteriocin produced by enterococci is a marker of pathogenicity. Diagn Microbiol Infect Dis. 1992; 15(2): 115-20. PubMed PMID: 1572135.
16. Biswas PP, Dey S, Adhikari L, Sen A. Virulence markers of vancomycin resistant enterococci isolated from infected and colonized patients. J Glob Infect Dis. 2014; 6(4): 157-63. PubMed PMID: 25538454.
17. Anderson AC, Jonas D, Huber I, et al. Enterococcus faecalis from food, clinical specimens and oral sites: prevalence of virulence factors in association with biofilm formation. Front Microbiol. 2015; 6: 1534. PubMed PMID: 26793174.
18. Comerlato CB, Resende MCC, Caierão J, d'Azevedo PA. Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin. Mem Inst Oswaldo Cruz. 2013; 108(5): 590-5. PubMed PMID: 23903974.