Trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital: a 4-year study

Introduction: In the past two decades members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. This study prospectively analyzed the distribution of species and trends in antimicrobial resistance among clinical isolates of enterococci in a Brazilian tertiary hospital from 2006-2009. Methods: Enterococcal species were identified by conventional biochemical tests. The antimicrobial susceptibility profile was performed by disk diffusion in accordance with the Clinical and Laboratory Standards Institute (CLSI). A screening test for vancomycin was also performed. Minimal inhibitory concentration (MIC) for vancomycin was determined using the broth dilution method. Molecular assays were used to confirm speciation and genotype of vancomycin-resistant enterococci (VRE). Results: A total of 324 non-repetitive enterococcal isolates were recovered, of which 87% were E. faecalis and 10.8% E. faecium . The incidence of E. faecium per 1,000 admissions increased significantly (p < 0.001) from 0.3 in 2006 to 2.3 in 2009. The VRE rate also increased over time from 2.5% to 15.5% (p < 0.001). All VRE expressed high-level resistance to vancomycin (MIC >256μg/ mL) and harbored vanA genes. The majority (89.5%) of VRE belonged to E. faecium species, which were characteristically resistant to ampicillin and quinolones. Overall, ampicillin resistance rate increased significantly from 2.5% to 21.4% from 2006-2009. Resistance rates for gentamicin, chloramphenicol, tetracycline, and erythromycin significantly decreased over time, although they remained high. Quinolones resistance rates were high and did not change significantly over time. Conclusions : The data obtained show a significant increasing trend in the incidence of E. faecium resistant to ampicillin and vancomycin.

Enterococci are widespread in nature and are normal constituents of the human gastrointestinal tract, but nowadays they have been recognized as important pathogens, especially among hospitalized patients.Enterococci may cause a range of different disorders, such as urinary tract infections, intraabdominal abscesses, wound infections, endocarditis and bacteraemia 1 .According to the SENTRY Antimicrobial Surveillance Program, enterococci are the fourth most common pathogen of bacteremia in North America and the fifth in Europe 2 .In Brazil, they are the eighth agent of bacteremia overall and the third among the Grampositive cocci 3 .
Intrinsic or acquired resistance to various commonly used antimicrobial agents is a remarkable characteristic of enterococci 4 .Acquired resistance to glycopeptides (vancomycin and teicoplanin), penicillins and aminoglycosides (high-level resistance) are the most clinically important, because therapeutic options in these cases are limited.
Six types of acquired vancomycin resistance have been reported in enterococci; however, the most prevalent are VanA and VanB, in which the genes encoding resistance are associated with mobile genetic elements that allow resistance to spread clonally and laterally 5 .The VanC type confers an intrinsic nontransferable low-level resistance to vancomycin that has been observed primarily in Enterococcus gallinarum and Enterococcus casseliflavus.
Clinical vancomycin-resistant enterococci (VRE) isolates were first recognized in the 1980s in Europe and USA 4 .Approximately ten years later, the first VRE were isolated in Brazil, in the States of Paraná and São Paulo, located in the southern and southeastern regions of the country, respectively 6,7 .Although these VRE isolates belong to Enterococcus faecium species, until recently in Brazil, Enterococcus faecalis was the predominant VRE commonly reported in hospitals in the State of São Paulo [8][9][10][11][12][13] .Despite the increasing number of reports regarding the evolution of antimicrobial resistance of enterococci in different countries, published data on this subject are still sporadic in Brazil.In this study, our group analyzed the distribution of species and trends in antimicrobial resistance among enterococci recovered from clinical specimens in a Brazilian tertiary hospital over a four-year period.The genotypes of VRE isolates were also determined.

Study design
A prospective study was conducted from 2006 to 2009 in the hospital of the Triangulo Mineiro University Hospital of the Federal University (Universidade Federal do Triângulo Mineiro, UFTM).This hospital, located in the State of Minas Gerais in southeastern Brazil, is a 294-bed tertiary-care teaching hospital with a 40-bed intensive care unit (ICU) and a full range of medical specialties.Over the study period, an average of 11,000 patients were admitted annually.All enterococci isolates recovered from hospitalized patients were included in the study, but only the first isolate from each patient was used.

Phenotypic identification of Enterococcus species
Isolates were identified at genus level by Gram staining, cellular morphology, absence of catalase production, hydrolysis of L-pyrrolidonyl-β-naphthylamide (PYR test), hydrolysis of esculin in presence of bile salts (bile-esculin test) and tolerance to 6.5% NaCl.Species identification was determined based on tests of carbohydrate fermentation, arginine hydrolysis, mobility, yellow pigment production and growth in 0.04% tellurite 14 .

Susceptibility testing
The antimicrobial susceptibility profile was performed using the disk diffusion method.The antimicrobials agents tested were: vancomycin (30µg), teicoplanin (30µg), ampicillin (10µg), penicillin (10U), streptomycin (300µg), gentamicin (120µg), norfloxacin (10µg), ciprofloxacin (5µg), chloramphenicol (30µg), tetracycline (30µg), and erythromycin (15µg).Beta-lactamase production was tested with chromogenic nitrocefin disk (Becton, Dickinson and Company, Cefinase TM , USA), in accordance with the manufacturer's instructions.A screening test for vancomycin was performed on BHI agar supplemented with 6μg/mL of this drug for all enterococcal isolates.For isolates resistant according to the screening test, the minimal inhibitory concentration (MIC) for vancomycin was determined using the broth dilution method.All susceptibility tests were performed and interpreted according to guidelines established by the Clinical and Laboratory Standards Institute (CLSI) 15 .Staphylococcus aureus ATCC (American Type Culture Collection) 25923, S. aureus ATCC 29213, and E. faecalis ATCC 29212 were used for quality control.

Molecular testing
Bacterial DNA was extracted from the enterococcal isolates that were phenotypically resistant to vancomycin using the QIAamp® DNA Mini kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's guidelines.Detection of vancomycin resistance genes was performed by a multiplex polymerase chain reaction (PCR) assay, in accordance with procedures described 16 by Woodford et al.Species identification of VRE isolates was confirmed by PCR, as described previously 17 .PCR products were analyzed by electrophoresis on 1.5% agarose gels and stained by ethidium bromide.

Statistical analysis
To evaluate the trend in antimicrobial resistance among enterococci over time, the χ 2 -test for trend was performed using Epi Info (CDC, Atlanta, GA) statistical software (version 3.5.1).The significance level was set at p ≤ 0.05.

Ethical considerations
The present study was approved by Research Ethics Committee of the UFTM.
Figure 1 shows the incidence of E. faecalis, E. faecium and other enterococcal species per 1,000 patient admissions from 2006 to 2009.A significant increasing trend in the incidence of E. faecium (p < 0.001) from 0.  All VRE isolates uniformly harbored vanA genes, as demonstrated by PCR.They were resistant to teicoplanin and expressed highlevel resistance to vancomycin (MIC >256μg/mL).The two VREfs showed the same antimicrobial susceptibility profile characterized by resistance to norfloxacin, ciprofloxacin, chloramphenicol, tetracycline, and erythromycin, and susceptibility to ampicillin, penicillin, streptomycin, and gentamicin.All VREfm were resistant to ampicillin, penicillin, norfloxacin, ciprofloxacin, and erythromycin and susceptible to streptomycin, gentamicin, chloramphenicol, and tetracycline.
Enterococcus gallinarum (n = 1) and E. casseliflavus (n = 4) isolates were susceptible to vancomycin by the disk diffusion method, but grew on agar screening with 6µg/mL of vancomycin, while the MIC observed for this drug was 8µg/mL.These five isolates were not considered as VRE in this study.
As demonstrated in Table 2, ampicillin and penicillin resistance rates increased from 2006 to 2009 from 2.5% to 21.4% and 23.8% to 35.9%, respectively, but only the increasing rate of ampicillin was statistically significant (p < 0.001).Among the 282 E. faecalis and 35 E. faecium isolates, 4 (1.4%) and 25 (77.4%) of them, respectively, were resistant to ampicillin and to penicillin.However, 63 (22.3%)E. faecalis isolates were resistant to penicillin, but remained susceptible to ampicillin.Beta-lactamase producing isolates were not detected.
Although there are at least 30 species of the genus Enterococcus, both E. faecalis and E. faecium are the most common species causing human infections 1,14 .Similarly, in this study, E. faecalis was the most prevalent species followed by E. faecium, while the other species were rarely recovered from clinical specimens.Nevertheless, a significant increasing trend in the incidence of E. faecium per 1,000 patient admissions from 0.3 in 2006 to 2.3 in 2009 was verified, approximately an eight-fold increase.In a study conducted in a hospital in Greece, the authors reported a similar increased incidence of E. faecium infections (0.7 in 2002 to 2.4 in 2007), although that hospital is larger than ours, with more than 60,000 admissions annually 18 .
The vancomycin-resistant enterococci rate also increased in our institution, by almost the same proportion as the incidence of E. faecium incidence.Most (89.5%) of the VRE isolates were E. faecium exhibiting the vanA genotype.In a retrospective study conducted at a tertiary Brazilian hospital located in the State of São Paulo, Furtado et al. also showed an increase in the rate of VRE over time from 9.5% in 2000 to 14.7% in 2001 and to 15.8% in 2002.However, the authors did not identify the VRE species or genotypes 19 .According to more recent data from the SENTRY Program, the percentage of VRE in Brazil increased from 6.9% in 2003 to 31.1% in 2008 and the majority (68.5%) of these isolates were E. faecium 20 .These VRE rates are much higher than that observed in other Latin America countries and in our hospital.
In the last two decades, the importance of E. faecium as a nosocomial pathogen has increased throughout the world due to

FIGURE 1 -
FIGURE 1 -Incidence of Enterococcus faecalis (gray bars), Enterococcus faecium (black bars) and others species of Enterococcus (white bars) per 1,000 patient admissions from 2006 to 2009.*χ 2 for trend p < 0.001.as E. faecium (VREfm).The first two VREfs were isolated in 2006.Thereafter, no other VREfs were isolated during the study period.The first VREfm appeared at the end of 2008, since then an increasing number of VREfm were noted in 2009.All VRE isolates uniformly harbored vanA genes, as demonstrated by PCR.They were resistant to teicoplanin and expressed highlevel resistance to vancomycin (MIC >256μg/mL).The two VREfs showed the same antimicrobial susceptibility profile characterized