Distribution of species and antimicrobial resistance ...

Science of the Total Environment 615 (2018) 1428–1437

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Distribution of species and antimicrobial resistance among enterococci isolated from the fecal microbiota of captive blue-fronted parrot (Amazona aestiva) in Rio de Janeiro, Brazil Andréa de Andrade Rangel de Freitas a, Adriana Rocha Faria a,b, Tatiana de Castro Abreu Pinto a, Vânia Lúcia Carreira Merquior b, Daniel Marchesi Neves c, Rodrigo de Cerqueira da Costa d, Lúcia Martins Teixeira a,⁎ a

Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Departamento de Microbiologia, Imunologia e Parasitologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Centro de Triagem de Animais Silvestres do Rio de Janeiro (CETAS-RJ), Seropédica, RJ, Brazil d Zoológico do Rio de Janeiro (RIO-ZOO), Rio de Janeiro, RJ, Brazil b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Five enterococcal species were recovered from the fecal microbiota of captive Amazona aestiva. • A major clonal group was identified among Enterococcus hirae, the predominant species. • A considerable proportion (48.0%) of the bacterial strains was multidrugresistant. • A diversity of antimicrobial resistance genes was detected.

a r t i c l e

i n f o

Article history: Received 20 March 2017 Received in revised form 31 August 2017 Accepted 1 September 2017 Available online 19 October 2017 Editor: D. Barcelo Keywords: Enterococcus Amazona aestiva Enterococcus hirae Enterococcus faecalis Antimicrobial resistance Genetic diversity

a b s t r a c t Enterococcal strains recovered from fecal samples of captive blue-fronted parrots (Amazona aestiva) assisted at two wild animal screening centers in Rio de Janeiro, Brazil, were identified as Enterococcus hirae (the predominant species; 75.3%), followed by Enterococcus faecalis (17.3%), Enterococcus casseliflavus (4.8%), Enterococcus gallinarum (1.7%), and Enterococcus hermanniensis (0.9%). All strains were susceptible to linezolid and teicoplanin. Rates of nonsusceptibility (including resistant and intermediate categories) to other 16 antimicrobials tested varied from 69.3% to 0.4%, A considerable proportion (48.0%) of the strains was multidrug-resistant and diverse genetic determinants associated with antimicrobial resistance were identified. Tetracyclineresistant strains carried the tet(M) and/or tet(L) genes. Macrolides resistance was associated with the erm(B), erm(A) and mefA genes, while 43.2% of the isolates were negative for the investigated genes. High-level resistance to gentamicin associated with the aac(6′)-le-aph(2″)-la gene was detected in one E. faecalis strain. The two strains presenting high-level resistance to streptomycin were negative for the ant(6′)-Ia, ant(3′)-Ia, ant(9′)-Ia and ant(9′)-Ib genes. The vat(D) gene was found in all the 47 quinupristin/dalfopristin resistant strains identified as non-E. faecalis. Analysis of PFGE profiles of E. hirae strains after restriction with SmaI demonstrated the occurrence of five clonal groups. The predominant E. hirae clone was distributed among birds in the two institutions, suggesting that this clone was well adapted to the host and environments investigated. The four clonal

⁎ Corresponding author at: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, CCS Bloco I, Rio de Janeiro, RJ 21941-902, Brazil. E-mail address: [email protected] (L.M. Teixeira).

https://doi.org/10.1016/j.scitotenv.2017.09.004 0048-9697/© 2017 Elsevier B.V. All rights reserved.

A.A.R. Freitas et al. / Science of the Total Environment 615 (2018) 1428–1437

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groups identified among E. faecalis were composed by small numbers of strains and, generally, restricted to birds in the same sector. The occurrence of enterococcal strains exhibiting antimicrobial resistance traits and carrying genetic determinants that represent potential threats to the health of both humans and animals, in the intestinal microbiota of A. aestiva, highlights the need for additional monitoring studies to elucidate the population structure and the dynamics of transmission of these microorganisms among animals, humans and the environment. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Enterococci are common inhabitants of the gastrointestinal tract of humans and a variety of animal hosts, and are also frequently found in man-made products, including fermented foods and dairy products, as well as environmental sources (Martín et al., 2009; Ahmad et al., 2011; Byappanahalli et al., 2012; Silva et al., 2012; Prichula et al., 2016; Roberts et al., 2016; Prieto et al., 2016). Although enterococci are commensals in the gut microbiota, in the last decades some of the species have gained a peculiar medical relevance, considering their role as causes of infections, mainly urinary tract and wound infections, bacteremia and endocarditis (Arias and Murray, 2012; Higuita and Huyck, 2014; Pãosinho et al., 2016). The role of these microorganisms as important opportunistic pathogens can be explained, at least in part, by the continuing progress of medical care toward more intensive and invasive medical therapies for human diseases (Higuita and Huyck, 2014). Other factors are the increasing resistance to antimicrobials expressed by clinical isolates, attributed to the continuing use of these drugs in human and veterinary medicine, in conjunction with the notable plasticity of the enterococcal genome (Prieto et al., 2016). Indeed, the treatment of enterococcal infections can be difficult. Enterococcal species are intrinsically resistant to many antimicrobial agents (Arias and Murray, 2012; Higuita and Huyck, 2014; Prieto et al., 2016), and have a remarkable capacity to acquire additional antimicrobial resistance traits, through acquisition of genes or mutations, highly limiting the therapeutic options (Prieto et al., 2016). In recent time, isolates harboring antibiotic resistance genes have emerged and disseminated not only in human sources but also in many environments (Liu et al., 2012), raising the interest in tracing the occurrence and characteristics of enterococcal isolates in various non-human habitats. As a consequence of the stimulus elicited by the evidence of exogenous acquisition of enterococci, molecular epidemiological studies have been performed to obtain insights into the dissemination of enterococcal clones in different hosts and environments, especially among strains expressing characteristics that represent threats to humans. The use of molecular typing techniques, such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), has greatly contributed to a better understanding on the epidemiology of these microorganisms (Poulsen et al., 2012; Prieto et al., 2016). However, MLST schemes are still restricted to Enterococcus faecalis and Enterococcus faecium, the two major enterococcal species recovered from human sources (Arias and Murray, 2012). Therefore, despite some difficulties in data exchange due to the lack of standard protocols and the relative slowness of the technique requiring several steps to obtain the results, PFGE remained as one of the most suitable techniques for molecular typing of enterococci, as it is potentially applicable to studies involving any of the species, including those that are not frequently isolated (Chadfield et al., 2005; Velkers et al., 2011). Epidemiological studies focusing on the presence of members of the genus Enterococcus in wild birds populations have shown that these microorganisms are part of the intestinal microbiota of birds and can act as reservoirs of genes associated with resistance to antimicrobials that play an important role in the treatment of enterococcal infections (Marrow et al., 2009; Radimersky et al., 2010; Radhouani et al., 2010; Silva et al., 2012; Radhouani et al., 2012; Oravcova et al., 2013; Oravcova et al., 2015; Roberts et al., 2016). The presence of antimicrobial-

resistant microorganisms in the intestinal tract of wildlife, especially bird populations, may represent a potential threat to both human and animal health since these animals provide a quite efficient pathway for spreading microorganisms and their respective genetic determinants (Radhouani et al., 2014). Among the 27 species of the Amazona genus found in Central and South America, Amazona aestiva (frequently referred as the bluefronted parrot) is a relatively wide-spread species, occurring in northeastern Brazil, eastern Bolivia, northern Argentina, and southern Paraguay (Seixas and Mourão, 2002). A. aestiva is mainly a granivorous and monogamous species that lives in large groups and one of the world's most popular species kept in captivity, perhaps because of its color patterns, size, and ability to imitate the human voice (Forshaw and Cooper 1989). However, when illegal owners realize that these animals are incompatible with their expectations and lifestyle, or when traders are arrested, trafficked animals are often considered unfit for release and sent to local zoos and wild animal triage centers, where these animals are frequently present in large numbers (Giovanini, 2002; Freitas et al., 2015). Considering the scarcity of data on the occurrence and epidemiology of enterococci recovered from animals in Brazil, the purpose of the present study was to investigate the occurrence, species distribution, antimicrobial resistance, and genetic characteristics of enterococci isolated from the intestinal microbiota of A. aestiva, by using phenotypic and genotypic methods.

2. Materials and methods 2.1. Blue-fronted Amazon parrot population sampling and bacteria isolation A total of 88 fecal samples were individually collected from the cloaca of blue-fronted parrots (A. aestiva), during the period from July to December 2010 in the state of Rio de Janeiro, Brazil. The animals were receiving veterinary assistance in two major wild animal screening centers: the Rio de Janeiro Zoo (Zoológico do Rio de Janeiro, RIO-ZOO, that contributed with samples from 37 birds) which is located in the urban area of Rio de Janeiro city, and the Wild Animal Screening Center (Centro de Triagem de Animais Silvestres do Rio de Janeiro, CETAS-RJ, that contributed with samples from 51 birds) which is located in the environmental preservation area of Rio de Janeiro state named National Forest Mario Xavier. The CETAS-RJ is an agency responsible for receiving animals handed by the population and/or seized from animal trafficking. These animals are taken to these centers with a recovery purpose and are later sent to zoos. Moreover, the birds included in the study were adults, although their gender and precise age were not determinate. Birds that have received treatment with antibiotics were not included in the study. The feeding of birds kept in both institutions consisted of commercial food for parrots, supplemented with fruits and vegetables. At the CETAS-RJ, the birds were kept in three different aviaries (aviary A, aviary B and aviary C). At the RIO-ZOO, birds were kept in different sectors of the institution that involved visiting areas of the park (sector 1, aviary A and sector 1, aviary B) and areas closed to the public (sector 2, aviary A; sector 2, aviary B; sector 2, aviary C; sector 2, aviary D; sector 3, aviary A; sector 4, aviary A).

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Fecal samples were obtained from the cloaca of each parrot by using swabs with Amies transport medium (Copan, Italy). After specimen collection, each cloacal swab was homogenized in 1 mL of Skim milk, tryptone, glucose and glycerin (STGG) broth (O'Brien et al., 2001). An aliquot of 100 μL was inoculated in Enterococcosel broth (B-D Microbiology Systems, USA) and another 100 μL aliquot was inoculated in Enterococcosel broth supplemented with vancomycin (6 mg/L). After incubation at 37 °C for 24 h, broth cultures that turned black, indicating the growth of a bile-esculin positive microorganism and thus suggesting the presence of enterococci, were streaked on Enterococcosel agar plates. After incubation at 37 °C for 24 h, up to five colonies resembling enterococci were randomly selected from each culture on Enterococcosel agar for further identification. For that, each selected colony on Enterococcosel agar was streaked on a blood-agar plate (Columbia agar supplemented with 5% sheep blood; Plast Labor, Brazil). Cultures were then screened for their characteristics as observed after Gram staining and catalase production testing. Gram-positive, catalase negative cocci were tested for growth in Heart Infusion Broth (HIB; Difco, USA) containing 6.5% NaCl and for hydrolysis of Lpyrrolidonyl-β-naphthylamide (PYR) and leucine-β-naphthylamide (LAP). Strains with positive results for these tests were identified to the species level by using a set of phenotypic tests based on the scheme proposed by Teixeira et al. (2015), including: pigment production, arginine hydrolysis, utilization of pyruvate, motility and acid production from the following carbohydrates (L-arabinose, mannitol, methyl-αglucopyranoside (MGP), D-raffinose, sucrose, sorbitol and sorbose). Enterococcal strains were further identified to the species level by PCR, using primers and conditions for the detection of different species-specific genes as proposed by Jackson et al. (2004), and the genus-specific tuf gene (Ke et al., 1999). DNA was obtained by thermal and enzymatic lysis, according to the protocol described by Beall et al. (1998) with modifications. Briefly, bacterial cells were centrifuged, resuspended in 90 μL TE buffer (10 mM Tris [pH 8], 1 mM EDTA) containing 0,1 mg of lysozyme (Sigma, EUA) and 5 U of mutanolysin (Sigma, EUA). After incubation at 37 °C for 30 min, the samples were heated at 100 °C for 3 min and centrifuged for 30 s. The generated supernatant was used as the source of template DNA for all amplification reactions used in the study. The identification of two E. hermanniensis strains was further investigated by sequencing of the 16S rRNA genes according to Carvalho et al. (2004). Briefly, DNA was amplified by using primers fL1 and rL1 previously described (Segonds et al., 1999). Amplification conditions were 94 °C for 5 min; 35 cycles at 94 °C for 15 s, 50 °C for 15 s, and 72 °C for 90 s; and finally a single extension of 72 °C for 5 min followed by a 4 °C hold. Excess deoxynucleoside triphosphates and primers were inactivated by using the ExoSAP-IT method (U.S. Biochemical Corp., Cleveland, Ohio). Cycle sequencing was performed with Big Dye terminator chemistry (Applied Biosystems, Foster City, Calif.). Sequences were subjected to a Blast search against GenBank. 2.2. Antimicrobial susceptibility testing The strains were tested for their susceptibility to 18 different antimicrobial agents by the disk diffusion method according to the CLSI guidelines (CLSI, 2009; 2016). The following antimicrobials (all from Oxoid Ltd., England) were tested: ampicillin (AMP), ciprofloxacin (CIP), chloramphenicol (CHL), enrofloxacin (ENR), streptomycin (STR), erythromycin (ERY), fosfomycin (FOS), gentamicin (GEN), levofloxacin (LEV), linezolid (LZD), nitrofurantoin (NIT), norfloxacin (NOR), penicillin (PEN), quinupristin/dalfopristin (QID), rifampicin (RIF), tetracycline (TET), teicoplanin (TEC), and vancomycin (VAN). Minimum inhibitory concentration (MIC) values for linezolid and vancomycin were determined by using the E-test gradient strip method (AB Biodisk, Solna, Sweden). Gentamicin and streptomycin MICs were determined by an agar dilution method using Mueller-Hinton agar supplemented with streptomycin (concentration range: 0.25 to 2048 mg/L) or gentamicin (concentration range: 0.25 to 512 mg/L). Interpretation

of the results was based on the CLSI recommendations (CLSI, 2009, 2016). For data analysis, intermediate and resistant strains were considered in a single category and classified as nonsusceptible. Multidrug resistance was considered when a strain was nonsusceptible to three or more classes of antimicrobials. 2.3. Detection of genes associated with antimicrobial resistance Enterococcal strains that showed resistance or reduced susceptibility to certain antimicrobials were tested by PCR for detection of the following antimicrobial resistance-associated genes: erm(A), erm(B) and mef(A) (in erythromycin-resistant strains); tet(K), tet(M), tet(L) and tet(O) (in tetracycline-resistant strains); aph(2″)-Ic, aph(2″)-Id, aph(3″)-IIIa, aph(2″)-Ib, ant(4′)-Ia and aac(6′)-aph(2″) (in high level gentamicin-resistant strains); ant(6)-Ia, ant(3)-Ia, ant(9)-Ia, ant(9)-Ib (in high level streptomycin-resistant strains); vat(D) and vat(E) (in quinupristin/dalfopristin-resistant strains); vanA, vanB, vanC, vanD, vanE and vanG (in vancomycin-resistant strains). Primers and conditions for PCR were as reported previously (Rende-Fournier et al., 1993; Swenson et al., 1995; Sutcliffe et al., 1996; Satake et al., 1997; Werner and Witte, 1999; Soltani et al., 2000; Ng et al., 2001; Vakulenko et al., 2003; Depardieu et al., 2004; Mahbub et al., 2005; Leelaporn et al., 2008). Positive and negative controls were used in all PCRs. 2.4. Analysis of chromosomal DNA restriction profiles by PFGE Strains representative of the two major enterococcal species recovered from A. aestiva (Enterococcus hirae, n = 74; and E. faecalis, n = 18) were selected for pulsed-field gel electrophoresis (PFGE) analysis based on differences on their antimicrobial resistance profiles and in order to test at least one strain isolated from each bird. The preparation of DNA in agarose plugs and enzyme restriction digests were performed on the basis of previous recommendations (Teixeira et al., 1997; Mondino et al., 2003) with a few modifications. The bacterial lysis step after embedding cells in agarose included an overnight treatment in 0.05% lysozyme before incubation in presence of proteinase K. Each plug was treated with the specific restriction enzyme buffer (New England Biolabs, USA) for 2 h at 25 °C for the balance of the reaction and, thereafter, incubated with 20 U of SmaI restriction enzyme (New England BioLabs) for 18–24 h at 25 °C. The fragments were resolved by PFGE in 1.2% SeaKem Gold agarose (Lonza, USA) gels in 0.5× TBE on a CHEF-DR III electrophoresis unit (Bio-Rad Laboratories, USA). The following parameters were used: running time, 22 h; temperature, 12 °C; voltage gradient, 6 V/cm; initial pulse time, 5 s; final pulse time, 35 s; included angle, 120 °C. Restriction profiles were analyzed by using the BioNumerics 7.5 software platform (Applied Maths, Belgium). The Dice correlation coefficient was used for calculating the percentages of similarity among the profiles and the unweighted pairgroup method with arithmetic mean (UPGMA) was used for clustering analysis, applying average linkages with 2% tolerance. The similarity cut-off of 90% was applied to define clonal relationships. 3. Results 3.1. Bacteria isolation and identification Enterococci were isolated at similar rates from fecal samples collected from birds in both sites investigated: CETAS-RJ (72.5%, 37/51) and RIO-ZOO (75.6%, 28/37). Overall, 231 enterococcal strains were recovered from 65 (73.9%) of the 88 fecal samples included in the study. E. hirae was the most frequent enterococcal species (75.3%) detected in A. aestiva intestinal microbiota, followed by E. faecalis (17.3%), Enterococcus casseliflavus (4.8%), Enterococcus gallinarum (1.7%) and Enterococcus hermanniensis (0.9%). E. hirae was the predominant species among birds from both institutions, being detected in 31 (83.8%) birds from the CETAS-RJ and 22 (78.6%) birds from the RIO-ZOO. Table 1


All the strains were susceptible to linezolid and teicoplanin. Seventeen (7.3%) strains, all identified as E. hirae and originating from different birds (twelve birds from CETAS-RJ and five birds from RIO-ZOO), were susceptible to all 18 antimicrobials tested. The higher percentages of nonsusceptible strains were observed for the following antimicrobials: enrofloxacin (69.3%), rifampicin (45.0%), and nitrofurantoin (37.7%), quinupristin/dalfopristin (37.7%), ciprofloxacin (26.0%), fosfomycin (17.3%) and erythromycin (16.0%). Low percentages of nonsusceptible strains were observed for ampicillin and gentamicin (0.4% each), streptomycin (0.8%), vancomycin (1.3%), levofloxacin (2.6%), chloramphenicol (3.0%), tetracycline (4.3%), penicillin (6.5%) and norfloxacin (7.8%). Vancomycin nonsusceptibility was only detected among strains belonging to the intrinsically vancomycin-resistant species E. gallinarum. Among the 231 enterococcal strains tested, 136 (58.9%) presented resistance to one or more of the antimicrobial agents tested. Resistance to rifampicin was detected in 81 (35.0%) strains, being 59.2% (48/81) identified as E. hirae; 38.3% (31/81) as E. faecalis and 2.5% (2/81) as E. casseliflavus. Overall, E. hirae strains were more frequently associated with resistance to nitrofurantoin (28.2%, 49 strains) and rifampicin (27.6%, 48 strains), whereas resistance to other drugs was less common: ciprofloxacin (two strains, 1.1%), chloramphenicol (one strain, 0.6%), enrofloxacin (three strains, 1.7%), erythromycin (two strains, 1.1%), fosfomycin (two strains, 1.1%), levofloxacin (one strain, 0.6%), norfloxacin (three strains, 1.7%), penicillin (thirteen strains, 7.5%), quinupristin/dalfopristin (three strains, 1.7%) and tetracycline (one strains, 0.6%). E. faecalis strains were associated with resistance to quinupristin/dalfopristin (38 strains, 95.0%) and rifampicin (31 strains, 77.5%) while resistance to ampicillin (1 strains, 2.5%), ciprofloxacin (2 strains, 5.0%), chloramphenicol (2 strains, 5.0%), erythromycin (7 strains, 17.5%), streptomycin (2 strains, 7.5%), norfloxacin (6 strains, 15.0%), and tetracycline (5 strains, 12.5%) was less frequent. Nine (81.8%) strains of E. casseliflavus showed decreased susceptibility to ciprofloxacin and enrofloxacin while decreased susceptibility to quinupristin/dalfopristin was observed in eight (72.7%) strains. Nonsusceptibility to quinupristin/dalfopristin was detected in E. hirae, E. casseliflavus and E. gallinarum strains (41.4%, 9.2% and 3.4%, respectively). Resistance to ampicillin was found in one E. faecalis strain,

Table 1 Distribution of enterococcal species isolated from fecal samples obtained from captive Amazona aestiva, according to the two institutions investigated in Rio de Janeiro, Brazil. Species

E. hirae E. faecalis E. casseliflavus E. gallinarum E. hermanniensis Total

Total

Number (%) of enterococcal isolates according to the institution CETAS-RJ

RIO-ZOO

104 (80.0) 21 (16.1) 3 (2.3) – 2 (1.5) 130 (100)

70 (69.3) 19 (18.8) 8 (7.9) 4 (4.0) – 101 (100)

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174 (75.3) 40 (17.3) 11 (4.8) 4 (1.7) 2 (0.9) 231 (100)

shows the distribution of the different enterococcal species identified among the strains recovered from fecal samples obtained from birds at both CETAS-RJ and RIO-ZOO. Forty-five (19.48%) strains showed atypical physiological reactions in one or more conventional physiological tests, as confirmed after repeat testing by three times, which led to uncertainty regarding their identifications. For these strains, the final identifications were stablished by PCR-based tests. The atypical physiological profiles were observed in E. hirae (13 strains were negative for production of acids from raffinose), E. faecalis (25 strains positive for MGP and one strain negative for arginine testing), E. casseliflavus (4 strains positive for sorbose testing) and E. hermanniensis (2 strains positive for pyruvate and sorbitol testing). The 16S rRNA gene sequences obtained for the two strains identified as E. hermanniensis showed 99.70% and 99.40% of sequence identity to that of the type strain of E. hermanniensis (GenBank accession number GQ337028), respectively. 3.2. Antimicrobial susceptibility All the strains were evaluated for their susceptibility to 18 antibiotics representing twelve classes. The overall results of antimicrobial susceptibility testing obtained for 231 strains are shown in Table 2, and the percentages of nonsusceptible strains according to the different enterococcal species are shown in Table 3.

Table 2 Occurrence of nonsusceptibility to different antimicrobial agents among enterococcal strains isolated from the fecal microbiota of captive Amazona aestiva in Rio de Janeiro, Brazil. Antimicrobiala

Number (%) of strains according to the institution and the category of nonsusceptibility CETAS-RJ (n = 130)

RIO-ZOO (n = 101)

Total (n = 231)

Category of nonsusceptibilityb

AMP CIP CLO ENR ERI FOS GEN LEV LZD NIT NOR PEN QID RIF STR TEC TET VAN

I

R

I

R

I

R

NAc 29 (22.3) 2 (1.5) 75 (57.7) 14 (10.8) 20 (15.4) NA 2 (1.5) 0 17 (13.0) 0 NA 19 (18.8) 15 (11.5) NA 0 0 0

1 (0.8) 3 (2.3) 3 (2.3) 3 (2.3) 7 (5.4) 2 (1.5) 0 0 0 38 (29.2) 6 (4.6) 13 (10.0) 21 (16.1) 49 (37.7) 2 (1.5) 0 6 (4.6) 0

NA 27 (26.7) 2 (2.0) 82 (81.2) 9 (8.9) 18 (17.8) NA 3 (3.0) 0 21 (20.8) 0 NA 26 (20.0) 8 (7.9) NA 0 1 (1.0) 2 (2.0)

0 1 (1.0) 0 0 7 (6.9) 0 1 (1.0) 1 (1.0) 0 11 (10.9) 12 (11.9) 2 (2.0) 21 (20.8) 32 (31.7) 0 0 3 (3.0) 1 (1.0)

NA 56 (24.2) 4 (1.7) 157 (68.0) 23 (9.9) 38 (16.4) NA 5 (2.2) 0 38 (16.4) 0 NA 45 (19.5) 23 (9.9) NA 0 1 (0.4) 2 (0.9)

1 (0.4) 4 (1.7) 3 (1.3) 3 (1.3) 14 (6.1) 2 (0.9) 1 (0.4) 1 (0.4) 0 49 (21.2) 18 (7.8) 15 (6.5) 42 (18.2) 81 (35.0) 2 (0.8) 0 9 (3.9) 1 (0.4)

a Antimicrobial: AMP, Ampicillin; CIP, Ciprofloxacin; CLO, Chloramphenicol; ENR, Enrofloxacin; ERY, Erythromycin; STR, Streptomycin; FOS, Fosfomycin; GEN, Gentamicin; LEV, Levofloxacin; LZD, Linezolid; NIT, Nitrofurantoin; NOR; Norfloxacin; PEN, Penicillin; QID, Quinupristin/dalfopristin; RIF, Rifampicin; TEC, Teicoplanin; TET, Tetracycline; VAN, Vancomycin. b Category of nonsusceptibility: I, intermediate, R, resistant. c NA: Not applicable.

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Table 3 Distribution of antimicrobial nonsusceptible strains among the different enterococcal species isolated from the fecal microbiota of captive Amazona aestiva in Rio de Janeiro, Brazil. Isolates

CNSa

E. hirae (n = 174) E. faecalis (n = 40) E. casseliflavus (n = 11) E. gallinarum (n = 4) E. hermanniensis (n = 2)

I R I R I R I R I R

Percentage of nonsusceptible strains according to the category of nonsusceptibility and the antimicrobialb AMP

CIP

CLO

ENR

ERI

FOS

GEN

LEV

LZD

NIT

NOR

PEN

QID

RIF

STR

TEC

TET

VAN

NAc 0 NA 2.5 NA 0 NA 0 NA 0

17.8 1.1 40.0 5.0 81.8 0 0 0 0 0

0.6 1.1 5.0 5.0 0 0 0 0 0 0

60.3 1.7 97.5 0 81.8 0 75.0 0 50.0 0

1.1 1.1 45.0 17.5 27.3 45.4 0 0 0 0

19.0 1.1 0 0 36.4 0 0 0 50.0 0

NA 0 NA 2.5 NA 0 NA 0 NA 0

1.1 0.6 2.5 0 18.2 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

21.8 28.2 0 0 0 0 0 0 0 0

0 1.7 0 15.0 0 81.8 0 0 0 0

NA 7.5 NA 5.0 NA 0 NA 0 NA 0

19.0 1.7 5.0 95.0 72.7 0 50.0 25.0 0 0

9.8 27.6 15.0 77.5 0 18.2 0 0 0 0

NA 0.0 NA 5.0 NA 0 NA 0 NA 0

0 0 0 0 0 0 0 0 0 0

0 0.6 0 12.5 0 0 25.0 75.0 0 0

0 0 0 0 0 0 50.0 25.0 0 0

a

CNS, category of nonsusceptibility; I-intermediate; R-resistant. AMP, Ampicillin; CIP, Ciprofloxacin; CLO, Chloramphenicol; ENR, Enrofloxacin; ERY, Erythromycin; STR, Streptomycin; FOS, Fosfomycin; GEN, Gentamicin; LEV, Levofloxacin; LZD, Linezolid; NIT, Nitrofurantoin; NOR; Norfloxacin; PEN, Penicillin; QID, Quinupristin/dalfopristin; RIF, Rifampicin; TEC, Teicoplanin; TET, Tetracycline; VAN, Vancomycin. c NA: Not applicable. b

while penicillin resistance was present in E. hirae and E. faecalis strains (7.5% and 5.0%, respectively). Streptomycin and gentamicin resistance were observed in two and one E. faecalis strains, respectively. A variety of antimicrobial resistance profiles (total of 107) were observed among the 231 enterococcal strains recovered from fecal samples of A. aestiva, seventy profiles being represented by a single strain. The resistance profile most frequently observed among E. faecalis (n = 9) strains comprised nonsusceptibility to enrofloxacin, erythromycin, quinupristin/dalfopristin and rifampicin (ENRI ERYI QIDR RIFR). The most common profiles among E. hirae strains were associated with nonsusceptibility to enrofloxacin and nitrofurantoin, and their different combinations: ENRI (21 strains), NITI (7 strains), ENRI NITI (6 strains), ENRI NITR (6 strains) and NITR (4 strains). According to the susceptibility data obtained, 111 (48.0%) out of 231 strains were found to be multidrug-resistant. E. hirae was the species most frequently associated with multiresistance, corresponding to 55.0% of the strains, followed by E. faecalis (34.2%), E. casseliflavus (8.1%) and E. gallinarum (2.7%). 3.3. Occurrence of antimicrobial resistance genes Among the 10 tetracycline-resistant strains observed, two harbored the tet(M) gene only, one harbored the tet(L) gene only, and seven harbored both tet(M) and tet(L) genes. None of them were positive for the tet(K) and tet(O) genes. Of the 37 erythromycin resistant strain, seven harbored the ermB gene only, three harbored the ermA gene only, two harbored the mefA gene only, four harbored both ermA and mefA genes, three harbored both ermA and ermB genes and two harbored both ermB and mefA genes. Sixteen other strains were negative for the presence of these three genes. The streptogramin A resistance gene vat(D) was found in 46 quinupristin/dalfopristin-resistant strains (thirty-five E. hirae, eight E. casseliflavus and three E. gallinarum). The vat(E) gene was not found. The two (0.8%) E. faecalis strains resistant to high-levels of streptomycin were negative for the presence of the genes ant(6′)-Ia, ant(3′)-Ia, ant(9′)-Ia and ant(9′)-Ib. Only one (0.4%) strain, identified as E. faecalis and carrying the aac(6′)-Ie-aph(2′)-Ia gene, was resistant to high levels of gentamicin. The vanC2/3 and vanC1 genes were detected in 11 E. casseliflavus strains and 4 E. gallinarum strains, respectively. The vanA, vanB, vanD, vanE and vanG genes were not found among the isolates investigated. 3.4. PFGE analysis Five groups or clusters of identical or closely related electrophoretic profiles as well as four nonrelated unique profiles were observed among the 74 E. hirae strains included in the analysis (Fig. 1). The five clonal groups were named by capital letters of the alphabet, as HA, HB, HC, HD and HE, and the four additional unique PFGE profiles (associated

to three strains from RIO-ZOO and one from CETAS-RJ, respectively) were denominated as HF, HG, HH and HI. The major clonal group, HA, consisted of 36 (48.6%) strains showing electrophoretic profiles indistinguishable from each other. Among these strains, 18 were isolated from birds from the CETAS-RJ and 18 birds from the RIO-ZOO. The other four clonal groups (HB, HC, HD, HE) were composed by strains recovered from of different birds, but belonging to the same institution. Strains included in these clonal groups showed indistinguishable PFGE profiles or profiles with 1 or 2 band differences and thus were considered closely related. An exception was the clonal group HE composed by seven strains, six isolated from the RIOZOO and one isolated from CETAS-RJ, that had profiles presenting a high number of band differences but still related. Four clonal groups and 7 distinct electrophoretic profiles were detected among the 18 strains of E. faecalis (Fig. 2). The dendrogram revealed that similarity among PFGE profiles of E. faecalis strains was relatively low, when adopting a coefficient of similarity of 90%. The clonal groups, named with letters of the alphabet (FA, FB, FC and FD) were composed by at least two strains. Each of these groups included strains recovered from birds restricted to one institution. 4. Discussion Studies on antimicrobial resistance, performed in different countries, have reported the presence of resistant bacteria among wild animal populations exposed to low levels of antimicrobial drugs or even never exposed (Radimersky et al., 2010; Radhouani et al., 2010; Radhouani et al., 2012; Silva et al., 2012; Oravcova et al., 2013; Oravcova et al., 2015; Roberts et al., 2016; Prichula et al., 2016). These findings raise at least two questions. One is related to the proximity to human activities influencing the antibiotic resistance profiles of the gut bacteria of wild animals (Radhouani et al., 2014). For this reason, the use of antimicrobials in agriculture and human and veterinary medicine as well as the procedures for disposal of domestic and hospital waste should be reviewed to promote environmental health. On the other hand, the number of potential resistance genes present in the natural resistome is still far from being correctly estimated, and studies in this field are needed in order to better understand the potential mechanisms of acquisition and dissemination of these genes by human and veterinary pathogens (Martinez, 2009). In this context, the enterococci may be considered as a key microbiota indicator to trace the spread and evolution of multidrug-resistant bacteria in environments and wildlife (Radhouani et al., 2014). In the present study, E. hirae was the most frequent enterococcal species identified in fecal samples obtained from A. aestiva. This observation is in agreement with an earlier study, conducted with healthy Psittaciformes birds, that reported E. hirae as a common microorganism of the intestinal microbiota of birds (Devriese et al., 1995). However,


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Fig. 1. A. Dendrogram showing genotypic relationships based on the analysis of SmaI PFGE profiles of Enterococcus hirae strains isolated from the fecal microbiota of captive Amazona aestiva in Rio de Janeiro, Brazil. B. PFGE profiles of SmaI-digested chromosomal DNA representative of the five clonal groups identified among E. hirae strains, as showing in panel A. Lanes 1 and 14, molecular size markers; lanes 2, 3, 5, 12 and 13, clonal group HA; lanes 7, 10 and 11, clonal group HB; lanes 8 and 9, clonal group HC; lane 6, clonal group HD; lane 4, clonal group HE.

in a more recent study by Allegretti et al. (2014) involving wild and captive A. aestiva, E. faecium and E. faecalis were the predominant enterococcal species, followed by E. hirae. Interestingly, in the study of Allegretti et al. (2014), both groups of birds living in different geographic areas of Brazil showed similarities in number of species and frequency of isolation, although a more diverse diet may be inferred for wild parrots than for captivity parrots (Xenoulis et al., 2010). Feeding regimes

and habitats may be important factors influencing the distribution of enterococcal species among A. aestiva. Whilst few generations separate A. aestiva kept as pet from those observed in their natural habitat, and, in many cases, captive A. aestiva are wild-caught, we still do not know the impact suffered in the intestinal microbiota during the transfer from the wildlife to captivity hosts (Xenoulis et al., 2010). Recent studies show that location and diet play greater roles in shaping the gut microbiota

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Fig. 2. A. Dendrogram showing genotypic relationships based on the analysis of SmaI PFGE profiles of Enterococcus faecalis strains isolated from the fecal microbiota of captive Amazona aestiva in Rio de Janeiro, Brazil. B. PFGE profiles of SmaI-digested chromosomal DNA representative of the four clonal groups identified among E. faecalis strains, as showing in panel A. Lanes 1 and 7, molecular size markers; lane 2, clonal group FC; lanes 3 and 5, clonal group FB; lane 4, clonal group FA; lane 6, clonal group FD.

of birds than the taxonomic identity (Stanley et al., 2013; Hird et al., 2014). In our study, the birds were kept in captivity in two different institutions distant from each other, although both located in Rio de Janeiro state. Limited attention has been given to species other than E. faecalis and E. faecium, as they show a relatively low prevalence and are more difficult to identify (Talarmin et al., 2011; Pãosinho et al., 2016). E. hirae is frequently associated with infections in different animals and it has been particularly associated with endocarditis in broilers (Chadfield et al., 2005), septicemia in Psittaciformes birds (Devriese et al., 1995), vertebral osteomyelitis in broilers (Braga et al., 2016), enteropathy in cats and piglets (Lapointe et al., 2000; Larsson et al., 2014) and mastitis in cows (Wu et al., 2016). Therefore, knowing the commensal microbiota of clinically healthy animals is an important step in the epidemiology of their infectious diseases. Atypical physiological profiles were observed in E. hirae, E. faecalis, E. casseliflavus and E. hermanniensis isolated from fecal samples of A. aestiva, in the present study. Atypical physiological profiles are observed in some enterococcal strains, especially among those isolated from nonhuman sources (Ramotar et al., 2000; Santestevan et al., 2015; Prichula et al., 2016) and can be explained, at least in part, by the large plasticity of the enterococcal genomes and by metabolic adaptation of these microorganisms to a variety of environments (Zhang et al., 2011; Arias and Murray, 2012). Another aspect that must be considered is that most of the phenotypic identification schemes that have been developed to identify enterococci, are based on testing large numbers of strains of human origin and none or few from other sources. Therefore, the diversity of phenotypic traits expressed by enterococcal strains from sources other than humans is still largely unknown and the use of a combination of methods is recommended in order to achieve an accurate identification (Teixeira et al., 2015). Difficulties in the precise identification of enterococci also represent a challenge for rarely found species with very little information available in the literature. E. hermanniensis is an example among the species identified in this study. To date, E. hermanniensis has been isolated from fresh meat broiler and canine tonsils (Koort et al., 2004), traditional fermented sausage type (fuet) in Spain (Martín et al., 2009) and a traditional Chinese fermented fish product in China (Dai et al., 2013). Resistance to drugs frequently used to treat different bacterial infections in human and veterinary medicine was frequent among the enterococcal strains identified in the present study, and included rifampicin (45.0%), enrofloxacin (69.3%), nitrofurantoin (37.7%),

ciprofloxacin (26.0%), and quinupristin/dalfopristin (24.6% in non-E. faecalis strains). Rifampicin has been used for decades to treat infections caused by Mycobacterium tuberculosis, and recently has aroused interest as an alternative treatment, in combination with other antimicrobials, for multidrug-resistant pathogens infections (Talarmin et al., 2011; Hu et al., 2016). Although rifampicin has not been used extensively to treat enterococcal infections, resistance is common in enterococci from different sources (Ghosh et al., 2012; Gawryszewska et al., 2016; Prichula et al., 2016), suggesting that other as-yet-unknown factors may contribute to the occurrence of resistant enterococcal strains (Kristich et al., 2014). Similarly, due to increasing multidrug resistance, the therapeutic choices are becoming fewer and old antibiotics such as nitrofurantoin have received attention. Interestingly, all the 87 strains resistant to nitrofurantoin in the present study were identified as E. hirae. Previous studies have revealed the occurrence of E. hirae resistant to nitrofurantoin (Santestevan et al., 2015; Wu et al., 2016). This study confirms the literature data that attribute good activity of nitrofurantoin to E. faecalis, a species that along with E. faecium, is responsible for the majority of enterococcal urinary tract infections in humans (Arias and Murray, 2012; Pãosinho et al., 2016). However, the lack of available data on the other species of enterococci should be pointed out. Enrofloxacin was introduced in a large scale in veterinary medicine, being used in both clinically impaired and farm animals, not only for urinary tract infections, but also for respiratory tract and gastrointestinal infections (Ruzauskas et al., 2009). In vivo, ciprofloxacin is the main metabolite formed from the hepatic metabolism of enrofloxacin and both active agents have cross-resistance (van den Bogaard et al., 2001). Therefore, the presence of strains not susceptible to both antimicrobials, enrofloxacin and ciprofloxacin, may be due to continued use of enrofloxacin in veterinary medicine. In the present study, most (70%) tetracycline-resistant enterococcal isolates harbored the tet(M) and tet(L) genes, concomitantly. Although the tet(M) gene is the most common determinant of resistance among tetracycline-resistant enterococci, the association between tet(M) and tet(L) has often been reported among isolates originating from wild birds (Radimersky et al., 2010; Radhouani et al., 2010; Radhouani et al., 2012; Oravcova et al., 2015). The erm(B) gene appears to be the most common among erythromycin-resistant enterococci isolated from humans and animals (Radimersky et al., 2010; Radhouani et al., 2010; Radhouani et al., 2012). However, in the present study, a high frequency of negative strains was observed for the three genes (ermB,


ermA and mefA), suggesting the involvement of other macrolide resistance determinants, such as msrC (Prichula et al., 2016) that was not tested by us. The vat(D) and vat(E) genes, coding for streptogramin A resistance in enterococci have been detected from wild animals in Europe (Radhouani et al., 2010; Radhouani et al., 2012; Oravcova et al., 2013). It is interesting to note the high proportion of vat(D) gene identified in our study. All the 47 quinupristin/dalfopristin-resistant non-E. faecalis strains carried the vat(D) gene. However, to our knowledge this is the first report on the detection of vat(D) among Enterococcus recovered from animals in Brazil. Another important point was the detection of the vat(D) gene among strains of different species (E. hirae and E. gallinarum) recovered from the same bird. This is an important finding that reflects that the intestinal tract of these animals could contain diverse vat(D)-positive strains, and the potential transfer of vat(D) genes among different bacteria in the intestinal tract cannot be discarded. Detection of quinupristin/dalfopristin resistant enterococci requires attention since this combination of antimicrobials is used to treat human infections caused by vancomycin-resistant enterococci (VRE) (Higuita and Huyck, 2014). Susceptibility to ampicillin among E. faecalis should not be used to predict susceptibility to penicillin, due to the emergence of penicillinresistant, ampicillin-susceptible E. faecalis (CLSI, 2016). Actually, there are few reports of this incongruous phenotype in the literature, but it has been reported in 31.4% of E. faecalis isolates from hospitalized patients in Greece (Metzidie et al., 2005). The same phenotype was found in E. faecalis isolated from the bloodstream in Denmark (Guardabassi et al., 2010) and also in Brazil (Conceição et al., 2014). Further studies involving sequencing of the pbp4 gene of our strains would help in clarifying whether a specific amino acid substitution in PBP4 found in E. faecalis isolates from Brazilian hospitals (Conceição et al., 2014; Infante et al., 2016) also occur in E. faecalis strains from the fecal microbiota of A. aestiva. In addition, mechanisms resistance to βlactam are usually investigated in E. faecalis and E. faecium, and studies with other species, such as E. hirae, are required. Although most E. faecalis remain susceptible to β-lactams, high-level resistance to aminoglycosides has been largely reported among strains from humans (Merlo et al., 2015; Gawryszewska et al., 2016) and animals (Ahmad et al., 2011; Silva et al., 2012), representing an important problem for the traditional combination therapy. Interestingly, resistance to high-levels of aminoglycosides was quite low among the strains isolated in the present study. One strain was resistant to highlevels of streptomycin and other was simultaneously resistant to highlevels of both streptomycin and gentamicin, representing a potential threat due to the possibility of dissemination to humans, as these compounds are important alternatives for treatment of E. faecalis in human medicine. Previous studies have revealed the spread of clones that colonize birds, to cause infections in humans (Poulsen et al., 2012; Dumke et al., 2015). Food chain and also direct contact with animals, as in the case of occupational exposure (veterinarians and animal breeders) or keeping birds as pet animals are possible sources of infection from birds to humans (Poulsen et al., 2012; Dumke et al., 2015). The results indicating absence of vancomycin-resistant enterococci (VRE) among enterococcal strains from A. aestiva are consistent with previous observations in wild animals in Brazil (Xavier et al., 2010; Silva et al., 2012; Santestevan et al., 2015; Prichula et al., 2016), suggesting that, in Brazil, this resistance characteristic remains restricted to the hospital environment (Almeida et al., 2014; Merlo et al., 2015; Infante et al., 2016). In contrast, VRE have been frequently isolated from animals in the United States (Marrow et al., 2009; Roberts et al., 2016; Oravcova et al., 2013) and in European countries (Radhouani et al., 2010; Oravcova et al., 2015). The 36 strains belonging to clonal group HA (isolated from 13 A. aestiva kept at the CETAS-RJ and from 11 A. aestiva kept at the RIOZOO) were distributed in most of all sectors of the two institutions and were isolated within a period of six months on subsequent visits

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to both institutions, suggesting that some conditions may be contributing to the spread and persistence of this predominant E. hirae clone. However, the 36 isolates belonging to clonal group HA displayed different antimicrobial susceptibility profiles. Such finding is agreement with observations by Poulsen et al. (2012), investigating E. faecalis isolated from urinary tract infections in humans and from poultry living in the same household in Vietnam. The characteristics that allow Enterococcus strains to survive for long periods in the environment, combined with the management of birds, may help in the spreading of these microorganisms among the different sectors of the two institutions. The food provided throughout the day on aviaries, composed of large-mesh screens, is attractive to insects and free-living birds that have no physical barriers to enter and exit the aviaries and can act as reservoirs or vectors, facilitating the spread between different aviaries and sectors of the institutions. The possibility of insects and free-living birds acting as carriers of enterococci harboring antibiotic resistance genes in farm animals has been well documented by Ahmad et al. (2011) and Garcia-Migura et al. (2005), respectively. The presence of a predominant E. hirae clone being carried by birds from the two distinct institutions (CETAS-RJ and RIO-ZOO), geographically distant, can also be related to transfers of animals between the two institutions. The conditions for management of birds are similar in both institutions and this clone would be easily adapted and disseminated among the different sectors. Our results indicated the presence of various strains having different PFGE profiles and carrying the vat(D) gene. Considering E. hirae strains from the two institutions investigated, at least one isolate per aviary contained the vat(D) gene. We were no able to determine the reason for the relatively high number of carriers of this gene among strains showing wide genetic diversity, but we can infer that this finding may be associated with the diet fed to birds, as this is a factor that they had in common. As the vat(D) gene encoding resistance to high-levels of streptogramin A is present in a plasmid, E. hirae isolated from the intestinal microbiota of A. aestiva can act as a reservoir of this gene. Virginiamycin, a streptogramin analogue of quinupristin/dalfopristin, has been used in animal production in Brazil (MAPA, 2015) and it is possible that quinupristin/dalfopristin-resistant enterococci have already emerged in farm animal populations, and then could be disseminated among enterococci in different ecological niches. Multidrug-resistant enterococcal populations are commonly isolated from humans (Almeida et al., 2014; Gawryszewska et al., 2016). Their presence in pets may represent a serious risk for dissemination of antibiotic resistance. In this work, E. hirae was the species most frequently associated with multiresistance. Our observations corroborate previous findings demonstrating that different species of enterococci can be associated with resistance to multiple drugs (Radhouani et al., 2010; Ahmad et al., 2011; Silva et al., 2012; Ghosh et al., 2012; Wu et al., 2016). 5. Conclusions In the present study, enterococcal strains recovered from the fecal microbiota of A. aestiva belonged to five different species, including species that are commonly identified among strains from different sources and others that are rarely found. The predominant species was E. hirae, representing 75.3% of the isolates and associated with a major clonal group, suggesting that this clone was well adapted to the host and environments investigated. E. faecalis, the second most frequent species, comprised 17.3% of the isolates and showed a tendency to a more diverse genetic background. A considerable proportion (48.0%) of the strains was multidrug-resistant (resistant to ≥3 antimicrobial classes) and diverse genetic determinants associated with antimicrobial resistance were identified. The occurrence of enterococcal strains exhibiting antimicrobial resistance traits and carrying genetic determinants that represent potential threats to the health of both humans and animals, in the intestinal microbiota of A. aestiva, highlights the need for

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additional monitoring studies to elucidate the bacterial population structure and the dynamics of transmission of these microorganisms among animals, humans and the environment.

Acknowledgements This work was supported in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES: 465718/2014-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: 486583/2012-0 and 309776/2015-5), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ: E-26/201.314/2014).The authors have no conflict of interest to declare. References Ahmad, A., Ghosh, A., Schal, C., Zurek, L., 2011. Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiol. 11, 1–13. Allegretti, L., Revolledo, L., Astolfi-Ferreira, C.S., Chacón, J.L., Martins, L.M., Seixas, G.H., Ferreira, A.J., 2014. Isolation and molecular identification of lactic acid bacteria and Bifidobacterium spp. from faeces of the blue-fronted Amazon parrot in Brazil. Benefic. Microbes 5, 497–503. 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