Enterococcus faecalis, Enterococcus faecium and other Enterococcus clinical isolates. Tryniszewska E, SieÅko A, Ojdana D, Majewski P, Sacha P, Wieczorek P.
Differences in virulence and resistance between biofilm-producing Enterococcus faecalis, Enterococcus faecium and other Enterococcus clinical isolates Tryniszewska E, Sieńko A, Ojdana D, Majewski P, Sacha P, Wieczorek P Department of Microbiological Diagnostics and Infectious Immunology, Faculty of Pharmacy, Medical University of Bialystok, Waszyngtona 15A Str., 15-269 Bialystok, Poland Introduction
Results and discussion
Today, Enterococcus spp. are the fourth most common etiological factor in nosocomial infections in Europe (Orsi & Ciorba, 2013). Although these cocci are members of the normal flora of the human gastrointestinal tract, they often infect the bloodstream, surgical sites, and urinary tract, due to their multiresistance to many antimicrobials (Amyes, 2007). Enterococcus have an intrinsic resistance to cephalosporins, lincosamides, and low levels of aminoglycosides, and they can easily acquire resistance, most prominently to glycopeptides and aminoglycosides (high level resistance), by means of mutations or as a result of transfer and incorporation of genes located on mobile genetic elements (Orsi & Ciorba, 2013). Moreover, these bacteria have the ability to form strong biofilm structure, and to produce several virulence factors, such as enterococcal surface protein (esp), aggregation substance (as), collagen adhesin (ace), hialuronidase (hyl) and gelatinase (gelE) (Di Rosa et al., 2006; Sava et al., 2010). The majority of nosocomial enterococcal infections are caused by Enterococcus faecalis and Enterococcus faecium species. However, today we observed increasing prevalence of infections caused by other rarely isolated species, e. g. E. avium, E. gallinarum, E. durans, E. casseliflavus (Tan et al., 2010). The aim of this study was to compare the antibiotic resistance and the presence of selected virulence genes between biofilmproducing E. faecalis, E. faecium and other Enterococcus strains.
The present study focused on determining the prevalence of biofilm-forming ability among 3 enterococcal groups: E. faecalis, E. faecium and other clinical isolates, and on comparison of the antibiotic resistance and the prevalence of selected virulence traits between biofilm-positive (BIO+) strains from these groups. Interestingly, we have found that the ability to form biofilm occurred in 4 (13.3%) E. faecalis, 18 (90%) E. faecium and 8 (57.1%) rarely isolated strains: 5 E. avium, 1 E. durans, 1 E. casseliflavus and 1 E. gallinarum (statistically significant difference, p = 0.026). In the next step of our research, only biofilm-positive (BIO+) strains were chosen for further investigation. A comparison of antibiotic resistance between E. faecalis, E. faecium and other isolates showed that all E. faecalis strains were susceptible to tested ß-lactams, while 37.5% of other strains and all E. faecium isolates were resistant. These results strongly overlap with results recently published by us (Sieńko et al., 2015) and other researchers (Billstrom et al., 2008; Tan et al., 2010). Resistance to gentamicin was detected in 75% E. faecalis, 55.5% E. faecium and 25% other strains; to streptomycin – in 25%, 83.3% and 50%, respectively. Our previous findings showed that more E. faecium isolates were resistant to aminoglycosides: 76% to gentamicin, and 91.4% to streptomycin (Sieńko et al., 2015). Resistance to glycopeptides occurred only in the case of 4 (22.2%) E. faecium isolates; two strains from rare group, E. gallinarum and E. casseliflavus, showed intrinsic resistance to vancomycin. Similar results were obtained by other authors (Contreras et al., 2008; Ten et al., 2010). Tigecycline and linezolid had the highest activity against all studied isolates (100% susceptibility), including those resistant to glycopeptides and aminoglycosides. Comparative analysis of the prevalence of virulence genes among E. faecalis, E. faecium and other strains revealed that esp gene was found in all E. faecium, 75% E. faecalis and 37.5% other strains. Similar proportions were seen by other researchers (Heikens et al., 2007; Fisher & Phylips, 2009;; Tsikrikonis et al., 2012; Top et al., 2013). These findings indicate that this gene has connection with biofilm-forming ability, especially in E. faecium strains. ace gene occurred in all E. faecium, 25% of E. faecalis and 37.5% of unusual isolates; hyl in 83.3%, 0% and 37.5%, respectively. as, gelE and cyl genes were detected only in E. faecalis strains. The exact resistance and virulence patterns among all tested BIO+ strains are shown in table 2. No predominant profile among each group was identified, not only due to small sample size, but also because of high interindividual variability of examined traits among tested Enterococcus groups. However, we have found that E. faecium isolates showed the greatest variety of virulence and resistance determinants, while the lowest variety was exhibited by unusual strains group. Moreover, all of E. faecium strains carried resistance to three or more antibiotics and had the ability to hemolyze. Different results were seen in recent research of Tsikrikonis et al. (2012), who detected only 1.9% of hemolysin-producing E. faecium clinical isolates. We also found that one E. faecalis isolate and 3 other isolates were susceptible to all tested antibiotics. This findings indicate that unusual Enterococcus biofilmproducing strains have lower resistance and virulence potency than E. faecalis and E. faecium.
Experimental procedures Tests were performed on 64 enterococcal isolates: 30 E. faecalis, 20 E. faecium, and 14 other: 5 E. avium, 3 E. casseliflavus, 3 E. gallinarum, 3 E. durans, randomly selected from the collection of the Department of Microbiological Diagnostics and Infectious Immunology (Medical University of Bialystok, Poland). Strains were isolated from clinical specimens from patients hospitalized at the University Hospital in Bialystok (Poland) from December 2013 to January 2015. Isolates were recovered from various clinical materials, mostly blood, peritoneal fluid, BAL, faeces, urine, and pus. Most of collected isolates were gathered from the intensive care unit and a hematology clinic. Identification and susceptibility testing The identification and susceptibility testing of study isolates were conducted on the automated VITEK 2 system (bioMérieux, France) . Biofilm production The tube method (Oliveira & Cunha, 2010) and Congo red agar (CRA) method (Cabrera-Contreras et al., 2013) were used to assess biofilm-forming ability. Each experiment was repeated three times for each strain. Strains that demonstrated the ability to produce biofilm by both methods were considered as biofilm positive (BIO+) isolates (Fig. 1 a,b).
b)
a)
c)
Table 2. Characteristics of resistance and virulence patterns among E. faecalis BIO+ (n = 4), E. faecium (n = 18) and other Enterococcus BIO+ (n = 8) strains. AMP, ampicillin; IMP, imipenem; CN, gentamicin; S, streptomycin; VA, vancomycin; TEI, teicoplanin; esp, surface protein; as, aggregation substance; gelE, gelatinase; hyl, hialuronidase; ace, collagen adhesin; cyl, cytolysin.
Fig. 1. Testing the biofilm-forming and hemolysis ability; a) Congo Red Agar metod; b) tube method; c) hemolysis on blood agar plates
Hemolysin production Hemolysin production was determined on Columbia blood agar with 5% sheep blood (OXOID, United Kingdom) (Fig. 1 c). PCR detection of virulence genes PCR assays were performed to detect the following virulence genes: gelE, ace, hyl, esp, as, and cyl. The primers are listed in Table 1. Table 1. PCR primers, annealing temperatures, and product sizes for detection of virulence genes.
References:
9.
1. 2. 3.
10.
4. 5. 6. 7. 8.
Orsi GB, Ciorba V 2013. Vancomycin resistant enterococci healthcare associated infections. Ann Ig 25:485–492. Amyes SG (2007) Enterococci and streptococci. Int J Antimicrob Agents 29(3): 43-52. Di Rosa R, Creti R, Venditti M, D'Amelio R, Arciola CR, Montanaro L, Baldassarri L (2006) Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium. FEMS Microbiol Lett 256(1): 145-50. Sava IG, Heikens E. Huebner J (2010) Pathogenesis and immunity in enterococcal infections. Clin Microbiol Infect 16(6): 533-40. Tan CK, Lai CC, Wang JY, Lin SH, Liao SH, Huang YT, Wang CY, Lin HI et al., Bacteriemia caused by non-faecalis and non-faecium enterococcus species at a Medical center in taiwan, 2000 to 2008. J Inf 2010: 34 – 43 Oliveira A, Cunha MDL (2010) Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci. BMC Res Notes 3: 260 Cabrera-Contreras R, Morelos-Ramirez R, Galicia-Camacho AN, Melendez-Herrada E (2013) Antibiotic resistance biofilm production in Staphylococcus epidermidis strains, isolated from a Tertiary Care Hospital in Mexico City. ISRN Microbiol 2013: 1–5. Camargo ILBC, Gilmore MS, Darini ALC (2006) Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycinsensitive Enterococcus faecium isolates from Brazil. Clin Microbiol Infect 12: 1123-1130.
11. 12. 13. 14.
Zou LK, Wang HN, Zeng B, Li JN, Li XT, Zhang AY, Zhou YS, Yang X, Xu CW, Xia QQ (2011) Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China. New Microbiol 34: 73-80. Sieńko A, Wieczorek P, Majewski P et al., Comparison of antibiotic resistance and virulence between biofilm-producing and non-producing clinical isolates of E. faecium. (2015) Act Biochim Pol 62:859-866 Billström H, Å. Sullivan A, Lund B (2008) Cross-transmission of clinical Enterococcus faecium in relation to esp and antibiotic resistance. J Appl Microbiol 105(6): 2115-2122. Heikens E, Bonten MJ, Willems RJ (2007) Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162. J Bacteriol 189(22): 8233-8240. Fisher K, Phillips C (2009) The ecology, epidemiology and virulence of Enterococcus. Microbiology 155: 1749–1757. Top J, Willems R, Blok H, de Regt M, Jalink K, Troelstra A, Goorhuis B, Bonten M (2007) Ecological replacement of Enterococcus faecalis by multiresistant clonal complex 17 Enterococcus faecium. Clin Microbiol Infect 13(3): 316-9
15. Tsikrikonis G, Maniatis AN, Labrou M, Ntokou E, Michail G, Daponte A, Stathopoulos C, Tsakris A, Pournaras S. Differences in biofilm formation and virulence factors between clinical and fecal enterococcal isolates of human and animal origin. Microb Pathog. 2012;52:336–343
