Characterization of Aminoglycoside Resistance and Virulence Genes among Enterococcus spp. Isolated from a Hospital in China

This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as “other” Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

Generally, Enterococcus species are not recognized as dominant pathogens, and usually only cause infections in patients who have a severe underlying disease or are immunocompromised [2]. Penicillin, ampicillin, and aminoglycosides are the first-line drugs for the treatment of enterococcal infection [3]. However, enterococci rapidly acquire antibiotic resistance determinants, as shown by the increasing number of infections caused by HLAR Enterococcus species. The aim of present study was to determine the prevalence of HLAR strains, examine their antibiotic susceptibility phenotypes, screen for the presence of genes coding for AMEs (including aac(6')-Ie-aph(2''), aph(2'')-Ib, aph(2'')-Id, ant(3'')-I, ant(4')-Ia, ant(6')-Ia, and aph(3')-IIIa) and other virulence factors (including ace, asa1, cylA, efaA, esp, gelE, and hyl), and examine the relationships between AME-and virulence-encoding genes of enterococci isolated from clinical patients in China.

Bacterial Isolation and Identification
This study was performed between September 2013 and July 2014 at Zhejiang Province People's Hospital, Hangzhou, China, a 2000-bed tertiary hospital. Rectal swab or stool specimens were collected from all patients admitted to the 40-bed ICU, and all VRE strains isolated from other clinical samples collected from June 2011 to January 2014 were also examined. Duplicate isolates were discarded. Each enterococcal isolate was first identified using the VITEK-2 COMPACT fully automated microbiological system (bioMérieux, Inc., Durham, NC, USA). Genotyping was carried out by screening for the presence of aminoglycoside resistance and other virulence genes using a multiplex polymerase chain reaction (PCR) method.
The amplified PCR products were analyzed on 1% (w/v) agarose gels. DNA bands were visualized by staining with ethidium bromide and photographed under UV illumination.

Statistical Analyses
VRE data was managed using the WHONET version 5.6 software (The WHONET Team, Boston, MA, USA). SPSS software version 17.0 (IBM, Inc., Armonk, NY, USA) was used for statistical analyses. Differences in antimicrobial sensitivity profiles and the prevalence of resistance and virulence genes between HLAR and HLAS Enterococcus species were compared using the Chi-square test, with a p-value < 0.05 indicating statistical significance.

Antimicrobial Susceptibility
Of the 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 as "other" Enterococcus species. The antimicrobial susceptibility profiles of all of the isolates are shown in Table 2. HLAR for gentamicin, streptomycin, and both antibiotics was identified in 58.8%, 50%, and 34.4% of isolates, respectively. In the present study, resistance to either gentamicin or streptomycin (or both) was regarded as indicating HLAR. In total, 119 (74.4%) of the isolates were classified as HLAR. In contrast, very little resistance to linezolid or teicoplanin was detected amongst the 160 isolates. Amongst the HLAR strains, comparison of the two species showed that rates of resistance were significantly higher in E. faecium than in E. faecalis for all antibiotics examined, except ERY, LZD, and CHL. Among the HLAS strains, a significant difference was again observed for all antibiotics, except TET, CHL, TEC, and VAN. Comparison of HLAR vs. HLAS E. faecium isolates revealed that rates of antibiotic resistance were only significantly different for TEC and VAN, while a significant difference was only observed for erythromycin between the HLAR and HLAS E. faecalis isolates.

Discussion
In recent years, infections caused by multidrug-resistant Gram-positive organisms have become a significant cause of morbidity and mortality [13,14]. Among these Gram-positive organisms, Enterococcus species are the second most common nosocomial bloodstream pathogens isolated in the United States [15,16]. Enterococci have been considered nosocomial pathogens since the early 1970s, and high-level resistance to aminoglycosides has become a serious problem in most healthcare facilities [17]. Schouten, et al. [18] reported that high level resistance to gentamicin has been detected in all investigated European countries, with prevalence ranging from 1% to 48% (mean, 22.6% ± 12.3%) in 1997. Moreover, there were no geographic relationships among the studied countries. However, in the present study, HLAR was detected in 119 (74.4%) of the isolates, which is significantly higher than previously reported.  Notes: + = positive. * R (resistance); S (susceptible).
Previous studies by Creti, et al. [8] and Billström, et al. [10] showed that the virulence genes ace, asa1, cylA, efaA, esp, gelE, and hyl are present at varying levels in E. faecalis isolates. In the current study, esp was the most frequently identified virulence gene (50.6% of isolates), followed by hyl (28.8%). These findings differed from the results of Zou et al., who showed that gelE was the most prevalent virulence gene, and that hyl and cylA were not detected, while Vankerckhoven, et al. [20] detected esp in 65% of isolates, and gelE and cylA were not detected. Comparison of virulence genes amongst HLAR and HLAS strains in the current study showed that only esp was significantly more prevalent in HLAR isolates than in HLAS isolates. Other than hyl and esp, all tested virulence genes, including ace, cylA, efaA, gelE, and asa1, were significantly more prevalent in E. faecalis compared with E. faecium. These differences indicate that virulence genes are present at different levels between human and animal isolates [9], and that with the passage of time, Enterococcus species have acquired an increasing number of virulence genes.

Conclusions
In summary, enterococci have become a significant cause of hospital-acquired infections. Therefore, measures should be taken to manage infection caused by multidrug resistant Enterococcus species. More and more focus is being placed on mechanisms by many health care facilities to prevent such infections [16,21]. There are some programs to control such infections, including better stewardship of antimicrobial agents and better awareness of the source for pathogen transmission is hospital environment, and health care societies should be widely endorsed [16,22].