|Year : 2013 | Volume
| Issue : 2 | Page : 108-113
Molecular and phenotypic characterization of hospital-associated and community-associated isolates of Enterococcus spp.
Labib Z Azza, Mahmoud B Ahmed, Al Ragehy A Nahed, Zahran A Wafaa, Elmasry A Eman
Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menufia, Egypt
|Date of Submission||05-Feb-2013|
|Date of Acceptance||22-Jul-2013|
|Date of Web Publication||31-Jan-2014|
Elmasry A Eman
MD,907, Fifth Compound, First District, 6 October City, Giza
Source of Support: None, Conflict of Interest: None
The aim of our study was to estimate the extent of spread of enterococcal infection as well as vancomycin-resistant enterococci (VRE) colonization at Menofia University Hospitals. and also to delineate occurrence and species prevalence, characterize VRE phenotypes and genotypes by multiplex PCR, and detect esp virulence genes in the enterococcal isolates from different sources.
Enterococci are part of normal intestinal flora of both humans and animals; however, they have also emerged as significant community-acquired pathogens and are a leading cause of nosocomial infection.
Patients and methods
In this study, patients were classified into three groups. Group 1 included 195 hospitalized patients. Group 2 included 65 patients from group 1 and group 3 included 50 healthy individuals to detect VRE colonization. Culture was performed using selective media for enterococci (bile esculine agar); detection of the antibiotic susceptibility pattern for enterococcal isolates and detection of VRE were performed. Detection of vanA and vanB genes in VRE using multiplex PCR and PCR detection of the esp gene were also performed.
The most commonly isolated Enterococcus spp. were Enterococcus faecium (48%) followed by Enterococcus faecalis (32%), Enterococcus durans (12%), Enterococcus avium, and Enterococcus gallinarum (4%). Of the 36 enterococcal isolates, 22 (61.1%) were identified as VRE by minimum inhibitory concentration. The Van A genotype was more common than van B. Of the 36 enterococcal isolates, 16 (77.82%) isolates had the esp gene. A high percentage (10.7%) of hospitalized patients showed colonization with VRE. The occurrence of the esp gene was higher in VRE clinical isolates of group I patients than isolates in stool specimens of patients in group II.
E. faecium is more common than E. faecalis as a nosocomial pathogen and it also has a great ability to show drug resistance. Emergence of multidrug-resistant enterococci to high-level aminoglycoside and vancomycin is an alarming situation. Fortunately, all VRE isolates are susceptible to linezolid. The Van A genotype is the predominant genotype among different species. A higher rate of gastrointestinal tract colonization by VRE is detected in hospitalized patients.
Keywords: Enterococcal colonization, enterococcal infection, vancomycin-resistant enterococci
|How to cite this article:|
Azza LZ, Ahmed MB, Nahed AA, Wafaa ZA, Eman EA. Molecular and phenotypic characterization of hospital-associated and community-associated isolates of Enterococcus spp. Menoufia Med J 2013;26:108-13
|How to cite this URL:|
Azza LZ, Ahmed MB, Nahed AA, Wafaa ZA, Eman EA. Molecular and phenotypic characterization of hospital-associated and community-associated isolates of Enterococcus spp. Menoufia Med J [serial online] 2013 [cited 2020 Mar 30];26:108-13. Available from: http://www.mmj.eg.net/text.asp?2013/26/2/108/126138
| Introduction|| |
Enterococci are part of normal intestinal flora of both humans and animals; however, they have also emerged as significant community-acquired pathogens and are a leading cause of nosocomial infection  . During the past decades, an increase in the prevalence of enterococcal infections such as bacteremia and urinary tract infections along with the emergence of multidrug resistance, particularly vancomycin-resistant enterococci (VRE), has been reported worldwide  . Despite the fact that Enterococcus faecalis has been observed as the predominant species in clinical infections, an increase in the prevalence of Enterococcus faecium has been observed recently. Six glycopeptide resistance phenotypes (VanA to VanE and VanG) have been described in enterococci. The VanA and VanB phenotypes are the most frequently encountered phenotypes  . Control of VRE has become an increasing burden on healthcare resources. An accurate, rapid diagnostic test has the ability to considerably reduce the spread of this organism. Current techniques used for their diagnosis include phenotypic methods and molecular methods  . Monitoring the antibiotic resistance of enterococci isolated from clinical specimens is a useful tool to obtain information about the prevalence of VRE and will be essential for controlling the spread of bacterial resistance  . The Esp gene is a surface protein that acts as an adhesion factor and is involved in biofilm synthesis. In addition, the fact that it can be transmitted with the vanA resistance gene is believed to contribute toward the wide spread of pathogenic VRE  . The aim of our study was to estimate the extent of spread of enterococcal infection as well as VRE colonization at Menofia University Hospital. Also, this study aimed to delineate occurrence and species prevalence, characterize VRE phenotypes and genotypes by multiplex PCR, and detect esp virulence genes in the enterococcal isolates.
| Patients and methods|| |
This study was carried out at Menoufia University Hospitals and Microbiology and Immunology Department. Samples were taken after patient consent.
Patients were classified into three groups.
Group 1 included 195 (95 men and 100 women) patients from several wards and ICUs with nosocomial infections. Their ages ranged from 19 to 64 years.
Group 2 included 65 patients from group 1; they were screened for gastrointestinal tract (GIT) colonization of VRE.
Group 3 included 50 healthy individuals matched by age and sex to detect fecal carriage of VRE.
A total of 220 clinical samples were collected from 195 patients of group 1 (77 urine, 63 pus, 50 blood, and 30 sputum samples) and processed according to standard microbiological methods  . Clinical samples from group 1 and stool samples from groups 2 and 3 were cultured on nutrient agar, blood agar, and MacConkey agar (Oxoid, Hampshire, UK) and incubated aerobically at 37°C for 24 h. Colonies were identified according to the standard microbiological methods.
Identification of enterococci and VRE isolates and susceptibility tests
Selective culture was performed on bile esculine agar for all colonies suspected to be enterococci. Enterococci were identified on the basis of cultural characteristic, morphology, and biochemical tests  . Identification of Enterococcus spp. was carried out using the API test kit (BioMèrieux, France). Antibiotic sensitivity tests to all enterococcal isolates were carried out using the disc diffusion method and interpreted according to the method described by Clinical Laboratory Standards Institution  . Disk diffusion screening tests using gentamicin (120 μg) and streptomycin (300 μg) for high-level aminoglycoside resistance were interpreted according to the Clinical Laboratory Standards Institution  . All enterococcal isolates were tested for vancomycin susceptibility using the agar screen method and confirmed by the broth dilution method, which determined the minimum inhibitory concentration (MIC)  . Phenotypic classification of VRE was performed according to vancomycin and teicoplanin MICs  .
Detection of vanA and vanB genes in VRE using multiplex PCR
Vancomycin resistance genotypes (vanA and vanB) were detected by amplifying the respective genes by multiplex PCR. The oligonucleotide primers chosen for amplification of the vanA and vanB genes are shown in [Table 1]  .
Rapid DNA extraction method was performed  . Each PCR reaction mixture (50 μl) consisted of 10 μl 5× Taq Master Mix, 0.2-1 μmol/l each primer, 2-50 ng template DNA, and was then filled up to 50 μl PCR-grade H2O. Enterococcal vanA and vanB genes were amplified by predenaturation of the reaction mixture for 4 min at 95°C; this was followed by 30 cycles at 94°C for 1 min, 45°C for 45 s, and 72°C for 1 min, and a final elongation for 7 min at 72°C; these reactions were performed in a GeneAmp PCR System 9600 (Perkin-Elmer Cetus Corp., Norwalk, Connecticut, USA)  . The amplified PCR products were detected by agarose-gel electrophoresis according to the method described by Cho et al. 
Detection of the esp gene in VRE strains using PCR
PCR amplification of the esp gene was performed using primers Esp 11 (5-TTGCTAATGCTAGTCCACGACC-3) and Esp 12 (5-GCGTCAACACTTGCATTGCCGAA-3). The PCR reaction mixture consisted of 250 ng of DNA; 0.2 μl each of 2-deoxyadenosine 5-triphosphate, 2-deoxycytosine 5-triphosphate, 2-deoxyguanosine 5-triphosphate, and 2-deoxythymidine 5-triphosphate; 2.5 mmol/l MgCl2; and 2.5 U of AmpliTaq DNA polymerase in 1×reaction buffer. The samples were subjected to initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation (94°C for 45 s), annealing (63°C for 45 s), and extension (72°C for 1 min)  . The PCR product bands (450 bp) were visualized by ethidium bromide staining.
| Results|| |
This study included 195 patients (group 1) admitted to different wards and ICUs of MUH and with nosocomial infections; their mean age was 28.02 ± 17.43 years. Of these, 220 specimens were obtained and 212 clinical isolates were recovered. In this study, 36 enterococci were isolated and accounted for 16.9% of nosocomial infections isolates. The most common isolated species of enterococci was E. faecium 12 isolates (48%), followed by E. faecalis eight isolates (32%) and three isolates of Enterococcus durans (12%), whereas only one isolate of Enterococcus avium and Enterococcus gallinarum was detected. The antimicrobial susceptibility patterns of isolated Enterococcus spp. are summarized in [Table 2]. All VRE isolates were susceptible to linezolid. Four (33.3%) of E. faecium isolates had high-level gentamicin resistance (HLGR) and eight (66.6%) showed high-level streptomycin resistance (HLSR), whereas three (37.5%) of E. faecalis showed HLGR and six (50%) showed HLSR. Of 36 enterococcal isolates, 22 (61.1%) were identified as VRE by MIC. E. faecium was the most commonly encountered enterocccal species (36.3%) showing vancomycin resistance, followed by E. faecalis (22.7%), whereas The E. durans resistance rate was 4.5% [Table 3]. Enterococci and VRE were isolated commonly from urine specimens. There was a statistically significant difference between urine specimens and other types of specimens in terms of the isolation of VRE. Phenotypic classification of VRE was performed according to vancomycin and teicoplanin MICs  . The most common phenotype among the enterococcal isolates was Van A, followed by Van B. Ten of 36 (27.7%) enterococcal isolates were the Van A phenotype, six (16.6%) were the Van B phenotype, three were the Van C phenotype, and three (8.3%) were the Van D phenotype. According to the distribution of Enterococcus spp. among VRE phenotypes, of 10 van A-resistant phenotype, four isolates (40%) were E. faecium. In terms of the van B phenotype, two (33.33%) were E. feacium and two (33.33%) were E. faecalis [Table 4].
|Table 2: Antimicrobial susceptibility of different Enterococcus spp. (n = 36) isolated from group I|
Click here to view
|Table 4: Distribution of different VRE phenotypes according to Enterococcus spp|
Click here to view
This study investigated the prevalence of van A and van B genes among enterococcal isolates. The Van A genotype was more common than van B among the 36 strains as eight (22.2%) isolates were the van A genotype, four (11.1%) were the van B genotype, and one isolate (2.7%) had both van A and van B resistance genes. Four E. faecium isolates had the van A gene and one had the van B gene [Table 5]. In our work, Van A and Van B phenotypes were mostly consistent with their corresponding van genotypes.
|Table 5: Distribution of van A and van B genes among Enterococcus spp. obtained from group I|
Click here to view
In terms of the distribution of the esp gene among enterococcal isolates, of the 36 enterococcal isolates, 16 (77.82%) isolates had the esp gene. There was a statistically significant difference (P< 0.05) between the occurrence of the esp gene in VRE (14/22, 63.63%) and its presence among vancomycin-sensitive enterococci (2/14, 14.29%). Also, six (50%) of VR E. faecium isolates had the esp gene at a higher rate compared with VR E. faecalis (37.5%) [Table 6].
|Table 6: Distribution of the esp gene among different VRE and VSE enterococcal species|
Click here to view
Seven (10.7%) of 65 of patients in group II showed colonization with VRE. Only one of those seven patients in group II was esp gene positive. In group III, only one patients showed colonization with VRE and was esp gene negative.
| Discussion|| |
Enterococci have become the major reservoir of antibiotic-resistant genes and VRE is a major cause of nosocomial infections  . This study was carried out at Menoufia University Hospitals and included 195 hospitalized patients. In this study, 36 enterococci were isolated and accounted for 16.9% (36 of 220) of nosocomial infection isolates. Similar results were obtained by Arias et al.  , who found that enterococci represent 15.6% of nosocomial isolates. According to the API identification system, our study showed that the most common isolated Enterococcus spp. was E. faecium (48%), followed by E. faecalis (32%). Several studies also supported our finding that E. faecium was the most commonly isolated enterococci , . Recently, an increase in the prevalence of E. faecium has been observed. This shift can likely be explained in part by the emergence of VRE and E. faecium being the dominant species among them  . Our finding showed that teicoplanin and rifampicin were the most active agents against E. faecium. In this study, 61.1% of enterococcal isolates were identified as VRE by MIC. Almost the same finding was reported by Worth et al.  , who found that the rate of VRE in infected patients was 61.66%. The emergence of VRE had considerably affected the treatment of the conditions caused by this organism. For these types of cases, newer antibiotics, such as linezolid and tigecycline, are useful  . All VRE isolated in this study were susceptible to linezolid, indicating that it is an appropriate therapeutic option. This study showed that high-level aminoglycoside resistance in enterococci is slightly high and HLGR is a possible risk and could be a major problem. Comparable results were obtained by Galindo et al.  , who reported that overall HLGR in E. faecalis was 12/30 (40%) and HLSR was 28/40(70%). Another study by Shinde et al.  reported that high-level resistance to gentamicin was observed in 44.4% of isolates as opposed to 41.6% (15/36) in our isolates. The presence of HLGR is predictive of the loss of synergy between gentamicin and a cell-wall-active agent such as ampicillin or vancomycin. This study investigated the prevalence of van A and van B genes encoding vancomycin resistance among enterococcal isolates. The Van A genotype was more common than van B and only one isolate had both van A and van B genes. Sharifi et al.  also reported that the vanA gene (89.58%) was the dominant gene. Also Shinde et al.  reported that the most prevalent glycopeptide resistance gene was van A (92.9% of VRE strains). Our results showed that vanA was found in four (33.3%) isolates of E. faecium, two (25%) isolates of E. feacalis, and one isolate of E. gallinarum. Van B was found in one (8.3%) isolate of E. faecium, two (25%) isolates of E. feacalis, and one isolate of E. durans. Comparable to our findings, the results of Alm El-Din and El-Mahdy  showed that the van A gene was found in four (80%) isolates of E. feacalis, two (100%) isolates of E. faecium, and one isolate of E. gallinarum, and the van B gene was found in two (25%) of the isolates of E. feacalis, one (12.5%) isolate of E. faecium. Our results showed that Van A and Van B phenotypes were consistent with corresponding resistance between van A and van B genotypes among different species of VRE isolates. Our results showed that the esp gene was detected in 63.63% of VRE isolates and 14.29% of vancomycin-sensitive enterococci isolates with a statistically significant difference (P< 0.05). The presence of the esp gene was reported to be highly associated with biofilm formation and with VRE  . Similar to our results, Sun et al.  also reported that the esp gene was isolated with a higher incidence in E. faecium isolates than in E. faecalis. In terms of the origin of esp-positive enterococcal isolates, a significant relationship was found between esp isolates of clinical specimens compared with GIT colonization isolates from group II and group III. The same results were reported by Shankar et al.  , who found that clinical isolates more frequently express the surface protein ESP. In this study, screening for VRE was carried out on 65 stool specimens of hospitalized patients and 50 controls. A high percentage (10.7%) of hospitalized patients showed colonization with VRE. The rate of VRE colonization varies widely in different studies  . In group III of healthy controls out of 50, only one showed colonization with the van A phenotype and was esp negative. Similar results were reported by Young et al.  , who found that VRE were detected in seven of the 54 fecal samples (13%). Higher rates were detected by D'Azevedo et al.  , who reported that vancomycin-resistant isolates were detected in 37 (45.7%) of 81 fecal cultures.
| Conclusion|| |
E. faecium is more common than E. faecalis as a nosocomial pathogen and it also has considerable ability to show drug resistance. Emergence of multidrug-resistant enterococci to high-level aminoglycoside and vancomycin is an alarming situation. Fortunately, all VRE isolates are susceptible to linezolid. The Van A genotype is the predominant genotype among different species. A higher rate of GIT colonization by VRE is detected in hospitalized patients.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
|1.||Olawale KO, Fadiora SO, Taiwo SS. Prevalence of hospital - acquired enterococcal infections in two Primarycare hospitals in Osogbo Nigeria Afr. J Infect Dis 2011; 5 :40-46. |
|2.||Cetinkay Y, Falk P, Mayhall CG. Vancomycin-resistant enterococci. Clin Microbiol Rev 2000; 13 :686-707. |
|3.||Song JY, Hwang IS, Eom JS, Cheong HJ, Bae WK, Park YH, et al. Prevalence and molecular epidemiology of vancomycin-resistant enterococci (VRE) strains isolated from animals and humans in Korea. Korean J Intern Med 2008; 20 :55-62. |
|4.||Ryu H, Henson M, Elk M, Toledo-Hernandez C, Griffith J, Blackwood D, et al. Development of quantitative PCR assays targeting 16S rRNA gene of Enterococcus spp. and their application to the identification of Enterococcus species in environmental samples. Appl Environ Microbiol 2013; 79 :196-204. |
|5.||Cho HH, Sung JY, Kwon KC, Lim JS, Koo SH. Antimicrobial resistance and multilocus sequence typing of vancomycin-resistant Enterococcus faecium isolated from the Chungcheong Area. Korean J Clin Microbiol 2011; 14 :60-66. |
|6.||Cheesbrough M. District laboratory practice in tropical countries. Part 2. Chapter 7. UK: Cambridge University Press; 2004. pp. 105-115. |
|7.||Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. 8th ed.. Wayne, PA: Clinical and Laboratory Standards Institute; 2011. |
|8.||Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-resistant enterococci. Clin Microbiol Rev 2000; 13 :686-707. |
|9.||Clark NC, Cooksey RC, Hill BC, Swenson JM, Ternover FC. Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Antimicrob Agents Chemother 1993; 37 :2311-2317. |
|10.||Giridhara Upadhyaya PM, Ravikumar KL, Umapathy BL. Review of virulence factors of enterococcus: an emerging nosocomial pathogen. Indian J Med Microbiol 2009; 27 :301-305. |
|11.||Arias CA, Reyes J, Zuniga M, Cortes L, Rico C, Panesso D. Multicenters surviellance of antimicrobial resistance in Enterococcous and Staphylococcous from columbian hospitals. J Antimicrob Chemother 2003; 51 :59-68. |
|12.||Worth LJ, Slavin MA, Vankerckhove V, Goossens H, Grabsch EA, Thursky KA. Virulence determinants in vancomycin-resistant Enterococcus faecium vanB: clonal distribution, prevalence and significance of esp and hyl in Australian patients with haematological disorders. J Hosp Infect 2008; 68 :137-144. |
|13.||Cuzon G, Naas T, Fortineau N, Nordmann P. Novel chromogenic medium for detection of vancomycin - resistant Enterococcus faecium and Enterococcus faecalis. J Clin Microbiol 2008; 46 :2442-2444. |
|14.||Galindo JA, Tejada YG, Silvia GC, Olga MS, Reyes EA. High-level aminoglycosied resistance Enterococcus spp in a tertary care hospital in Mexico. Electron J Biomed 2005; 2 :40-45. |
|15.||Shinde RS, Koppikar GV, Oommen S. Characterization and antimicrobial susceptibility pattern of clinical isolates of Enterococci at a tertiary care hospital in Mumbai, India. Ann Trop Med Public Health 2012; 5 :85-88. |
|16.||Sharifi Y, Hasani A, Ghotaslou R, Varshochi M, Akbar H, Mohammad A, Morteza M. Survey of virulence determinants among vancomycin resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens of hospitalized patients of North west of Iran. Open Microbiol J 2012; 534 :34-39. |
|17.||Alm El-Din RA, El-Mahdy HS. Molecular characterization of enterococcus strains isolated from cases of neonatal sepsis in neonatal intensive care unit. Afr J Microbiol Res 2012; 6 :7206-7211. |
|18.||Sun H, Wang H, Xu Y, Jones RN, Costello AJ, Liu Y, et al. Molecular characterization of vancomycin-resistant Enterococcus spp. clinical isolates recovered from hospitalized patients among several medical institutions in China. Diagn Microbiol Infect Dis 2012; 74 :399-403. |
|19.||Shankar N, Lockatell C, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE. Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection. Infect Immun 2001; 69 :4366-4372. |
|20.||Young HL, Ballard SA, Roffey P, Grayson ML. Direct detection of vanB2 using the Roche LightCycler vanA/B detection assay to indicate vancomycin-resistant enterococcal carriage - sensitive but not specific. J Antimicrob Chemother 2007; 59 :809-810. |
|21.||D′Azevedo PA, Dias CA, Lemos SK. Antimicrobial susceptibility among Enterococcus isolates from the city of Porto Alegre, RS, Brazil. Braz J Microbiol 2008; 35 :199-204. |
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]