High Level Aminoglycoside Resistance in Enterococcus, Pediococcus and Lactobacillus Species from Farm Animals and Commercial Meat Products

Ann Microbiol (2016) 66:101–110 DOI 10.1007/s13213-015-1086-1

ORIGINAL ARTICLE

High level aminoglycoside resistance in Enterococcus, Pediococcus and Lactobacillus species from farm animals and commercial meat products

George Jaimee1 & Prakash M. Halami1

Received: 10 September 2014 /Accepted: 8 April 2015 /Published online: 2 May 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract Inappropriate use of aminoglycosides in animal I40a suggesting its involvement in antibiotic resistant gene husbandry has led to the selection and emergence of high- transfer. Besides, strains of L. plantarum, a species used as level aminoglycoside resistance (HLAR) in lactic acid bacte- probiotic, isolated in this study showed the occurrence of ria (LAB). The objective of this study was to assess the pres- aph(3′)IIIa as well as aac (6′)Ie-aph(2″)Ia genes that could ence of aminoglycoside resistant LAB in farm animals and be of concern in human health. The findings of the study meat products. Gentamicin resistant LAB (n=138) were se- highlight the spread and emergence of multi-resistance genes lectively isolated from 50 different meat and farm animal for aminoglycoside antibiotics among beneficial LAB. sources. These native isolates of LAB were subsequently characterized for their minimum inhibitory concentration to Keywords Lactic acid bacteria . Minimum inhibitory seven different aminoglycoside antibiotics. HLAR to genta- concentration . Aminoglycoside . Random amplified micin, kanamycin and streptomycin was found to be 38 %, polymorphic DNA . Integrase 45 % and 15 %, respectively. Selected cultures of LAB were identified by random amplified polymorphic DNA (RAPD)-PCR and 16S rDNA gene sequencing. Subsequent Introduction detection for the presence of nine aminoglycoside modifying genes [aac(6′)Ie-aph(2″)Ia, aph(3′)IIIa, aad6, ant(6)Ia, The spread of resistance to antibiotics among bacteria is ant(9)Ia, ant(9)Ib, aph(2″)Ib, aph(2″)Ic and aph(2″)Id] was alarming. In the global scenario, the development and carried out by PCR. The Enterococcus spp. (n=64) and spread of resistance to clinically significant drugs in com- Lactobacillus plantarum (n=6) isolated from farm animals mensal bacteria is associated with their improper use in and chicken sausages, respectively, were positive for the animal husbandry (Mathur and Singh 2005). The situation bifunctional gene, aac(6′)Ie-aph(2″)Ia in PCR. In addition, is of serious concern when it comes to lactic acid bacteria Enterococcus sp. (n=17), Lactobacillus plantarum (n=3), (LAB), which are beneficial microflora that can develop and Pediococcus lolii (n=2) possessed the kanamycin resis- resistance to life-saving aminoglycoside drugs (Jackson tance gene, aph(3′)IIIa. Other LAB viz. Enterococcus faecalis et al. 2010). Aminoglycosides are regarded as vital drugs (n=2), E. faecium (n=2) and L. plantarum (n=1) harbored for the treatment of life-threatening infections (United the streptomycin resistance gene, aad6. The integrase (int) States Pharmacopeial Convention 2008). High level resis- gene, characteristic to Tn 916-Tn 1545 was detected in tance to aminoglycosides in bacteria may lead to ineffec- Enterococcus faecalis CS11+ and Enterococcus cecorum tive therapeutic crisis. Many countries have banned the administration of certain antibiotics in animal husbandry due to their preferred usage in human medicine (Shwarz * Prakash M. Halami et al. 2000). However, aminoglycosides are recommended [email protected] for therapy and prophylaxis in farm animals owing to their efficient bactericidal mode of action against Gram- 1 Microbiology and Fermentation Technology, CSIR-Central Food negative and Gram-positive bacteria (United States Technological Research Institute, Mysore 570020, India Pharmacopeial Convention 2008). 102 Ann Microbiol (2016) 66:101–110

Bacterial resistance to aminoglycosides occurs due to mu- LAB are in demand industrially, because of their beneficial tations, impaired transport and acquired resistance (Ramirez aspects in fermented food and pharma-based therapeutics and Tolmasky 2010). The most common mode of aminogly- (Popova et al. 2012). The quality presumption safety (QPS) coside resistance in Gram-positive bacteria is the acquisition status, which ensures the absence of acquired resistance, has of aminoglycoside-modifying genes (Bismuth and Courvalin to be attained in order to use LAB in food or probiotic product 2010). Clinically, the bifunctional gene aac(6′)Ie-aph(2″)Ia development (EFSA 2012). However, this norm becomes di- confers resistance to almost all aminoglycosides except strep- luted in the case of food products, in view of the scale of tomycin. It has been associated with high-level gentamicin operation, wherein such stringent tests to avoid the inclusion resistance (HLGR) and high level kanamycin resistance of resistant LAB are ignored. Most countries in Asia and (HLKR) with minimum inhibitory concentration (MIC) Africa lack proper guidelines on the judicious use of antibi- values >500 μg/mL (Murray 1990; Del Campo et al. 2000; otics and there is much use of low cost antibiotics in animal Donabedian et al. 2003; Jackson et al. 2004; Bismuth and husbandry that is not being monitored. The persistent use of Courvalin 2010). This bifunctional gene is highly prevalent aminoglycoside antibiotics such as apramycin, neomycin, among clinical strains of Staphylococcus and Enterococcus streptomycin in farms may co-select for antibiotic resistance spp. (Culebras and Martinez 1999), and its frequent spread in LAB and pathogenic species via the food chain among Gram-positive organisms has been attributed to its (Guardabassi et al. 2004). In such instances, antibiotics deliver lower G+C content (Byrne et al. 1989). Of late, the selective a competitive advantage, thereby triggering the acquisition advantage of this fused gene has been related to the structure and rapid spread of resistance genes via horizontal gene trans- and rigidity of the two encoded functional enzymes (Boehr fer (Nielsen et al. 2014). The hazards of acquired resistance et al. 2004). The monofunctional genes [aph(2″)Ib, aph(2″)Ic and its rampant spread in nosocomial enterococci is of major and aph(2″)Id] depicted lower MIC values (≤500 μg/mL) for concern (Kuhn et al. 2005; Hammerum et al. 2010). Such gentamicin, while aph(2″)Ie was observed with HLGR findings illustrate that there is a high risk of acquisition and (≥1024 μg/mL) in enterococci (Chow et al. 1997;Tsaietal. transfer of aminoglycoside resistance by LAB via the food 1998; Kao et al. 2000;Chenetal.2006). The aminoglycoside chain. Thus, extreme care should be taken to avoid the emer- resistance genes mentioned above are known to modify gence of aminoglycoside-resistant LAB, as they can be a po- 4,6 deoxystreptamines, especially clinically significant antibi- tent source of antibiotic resistance genes (Mathur and Singh otics such as amikacin, gentamicin, kanamycin and tobramycin 2005). As a result, protocols to monitor the development of (Chen et al. 2006; Bismuth and Courvalin 2010). Other aminoglycoside resistance among LAB from farm animals are resistance-conferring genes found in Gram-positive organisms required. are kanamycin modifying aph(3′)IIIa, streptomycin modifying From the above understanding of the risk related to amino- aad6, ant(6)Ia and spectinomycin modifying ant(9′)Ia, glycoside resistance and its spread via the food chain, an at- ant(9′)Ib (Bismuth and Courvalin 2010). A high-level strepto- tempt was made in the present study to evaluate the presence mycin resistance (HLSR) in enterococci, despite the absence of of aminoglycoside resistance in native LAB from farm and aad6orant(6)Ia genes has been explained due to a mutation in meat origin in the Mysore region of Karnataka, India. We the 30S ribosome (Simjee and Gill 1997). Therefore, high MIC report the widespread presence of the aminoglycoside resis- levels may not necessarily correlate with the presence of the tant bifunctional gene along with the kanamycin and strepto- above-mentioned resistance genes. mycin modifying genes in different native isolates of In the past decade, the rise and spread of aminoglycoside Enterococcus sp., Lactobacillus plantarum,andPediococcus resistance in Enterococcus sp. from domestic and other farm lolii. animals has been documented (Donabedian et al. 2003; Jackson et al. 2004, 2009, 2010; Ramos et al. 2012; Klibi et al. 2014). Enterococci from clinical settings and farm ani- Material and methods mals from the same region were positive for aminoglycoside resistance, and the genetic relatedness signified their rapid Media, antibiotics, chemicals and control strains spread among the human population (Donabedian et al. 2003). Earlier, Tenorio et al. (2001) described the emergence The Iso-sensitest broth was purchased from Thermo Fisher of aac(6′)Ie-aph(2″)Ia in Lactobacillus acidophilus, Scientific (Basingstoke, UK). de Mann, Rogosa and Sharpe Lactobacillus salivarus and Pediococcus acidilactici from fe- (MRS) medium and antibiotics such as amikacin, apramycin, cal samples of healthy pigs and pets, which is the only report gentamicin, kanamycin, neomycin, streptomycin and specti- of its kind to date to have described the gene in Lactobacillus nomycin were procured from Hi-Media Laboratories and Pediococcus sp. Documented studies on the occurrence of (Mumbai, India). Antibiotic powder was weighed as required aminoglycoside resistance in LAB from food origin have been and dissolved in water. The dNTPs mix, 25 mM MgCl2,ly- very limited (Hammerum et al. 2010). sozyme and 10 Kb DNA ladder were procured from GeNei Ann Microbiol (2016) 66:101–110 103

(Merck, Bangalore, India). Proteinase K, Trizma base, Taq addition, the viable count of LAB was recorded as CFU/g. DNA polymerase, primer M13, aminoglycoside resistance The isolates were confirmed to be LAB by Gram-staining gene specific primers, 16S rDNA primer and PCR purification and catalase tests. Purified colonies were stored at −40 °C in kit were purchased from Sigma-Aldrich (St. Louis, MO). The MRS broth containing 20 % glycerol. Further, LAB isolates primers used in this study are detailed in Table 1. Sodium were tested for carbohydrate fermentation, growth at different hydroxide, sodium chloride, bromophenol blue, sodium do- temperatures (15, 45 and 50 °C), pH (4.5 and 5.5) and 6.5 % decyl sulphate, EDTA disodium salt and phenol were obtained NaCl concentration and tentatively identified to genus level as from Sisco Research Laboratories (Mumbai, India). The con- described in Bergey’s manual of Determinative Bacteriology trol strains Enterococcus faecalis JH2-2 and E. faecalis RE-25 (Holt et al. 1994). were obtained from C.M.A.P Franz, Federal Research Centre for Nutrition and Food, Institute of Hygiene and Toxicology, Determination of minimum inhibitory concentration Karlsruhe, Germany. The minimum inhibitory concentration (MIC) of individual Selective screening of gentamicin-resistant LAB antibiotics like amikacin, apramycin, gentamicin, kanamycin, neomycin, streptomycin and spectinomycin was determined Aminoglycoside resistant LAB from the intestines of by the broth microdilution method using 96-well microtiter slaughtered animals [chicken (n=25), sheep (n=7), beef (n= plates in LAB susceptibility test medium (LSM-90 % Iso- 3), pigs (n=5)] and meat products [raw meat (n=4) and chick- sensitest broth supplemented with 10 % MRS broth) accord- en sausages (n=3), hams (n=2), salami (n=1)] were isolated ing to the CLSI (2012) standards. Quantification was per- by selective screening on MRS agar containing gentamicin formed using an ELISA reader (Model No:1510, (64 μg/mL). Briefly, to describe the isolation of LAB, homog- Thermofischer Scientific, Vantaa, Finland) after incubation enized samples in 10 g quantities were emulsified in 90 mL for 24 h at 37 °C. The lowest concentration of antibiotic at 0.1 % peptone water and aliquots of appropriate serial dilu- which no growth was observed was taken as the MIC values tions were plated on gentamicin-containing MRS agar. Plates of different aminoglycosides (CLSI 2012). The strain were incubated for 48 h at 37 °C and typical gentamicin resis- Enterococcus faecalis JH2-2 was used a negative control in tant colonies were selected at random and isolated. In MIC tests. E. faecalis RE-25 was used as a positive control for kanamycin and streptomycin resistance.

Table 1 Primersusedinthisstudy Random amplified polymorphic DNA polymerase chain Target Primer sequences 5′-3′ Annealing PCR reaction (RAPD-PCR) gene temperature product Tm (°C) (bp) The total DNA extracted (Mora et al. 2000) from individual LAB isolates was employed as a template for initial grouping aph(2″)Ib F- GCAAATGGCACAGTAT 56 958 AATATGC of LAB by RAPD-PCR. The PCR reactions were carried out R- GCTTGTGTTTGTAGCA according to the method described by Schillinger et al. (2003). ATTCAG The 25 μL reaction mixture for all the PCR amplification aph(3′)IIIa F- GATACGGAAGGAATGT 56 663 carried out in this study consisted of 0.4 μM M13 primer, CTCC 25 ng/μL total DNA, 1.5 mM MgCl2, 0.5 mM dNTPs, 1X R- GCTTGATCCCCAGTAA GTC PCR buffer, 0.3 U Taq DNA polymerase and DNase-free ster- aad6/ ant6 F - CTTTAGCAGAACAGGA 52 844 ile water. The amplified products were examined on a 1.8 % TG agarose gel in TAE buffer at a voltage of 65 V for 4–5h.The R- CCGGCCTCTCTTCTAT RAPD profiles were evaluated using the Quantity one® soft- ATC ware (Bio-Rad, Milan, Italy). ant(6)Ia F- CGCCTCTCTTCTATATCA 50 844 R- CTTTAGCAGAACAGGA 16S rDNA amplification TG ant(9)Ia F-CCAAATCAAGCGATTC 54 690 AAAC To identify the LAB isolates, 16S rDNA gene amplification R- TCCTTCCCACTTATCA were performed according to Kimprasit et al. (2013). TCC Representative cultures from each group with similar RAPD ant(9)Ib F- GATCAGGAGTTGAGAG 46 543 banding patterns as well as tentatively identified Enterococcus TG and Lactobacillus isolates that showed varied MIC levels for R- GAGAAGATTCAGCCAC TG gentamicin, kanamycin and streptomycin were also considered for 16S rDNA gene sequencing. The sequences obtained 104 Ann Microbiol (2016) 66:101–110 were further subjected to BLAST search (Altschul et al. 1997) lactobacilli (n=32) and pediococci (n=5) species. The LAB analysis for comparison with known sequences from the pub- isolates were assessed by determination of MIC to seven dif- lic database. ferent aminoglycosides. The MIC values obtained for gentamicin, kanamycin and streptomycin were compared with Detection of aminoglycoside resistance genes established breakpoint levels (EFSA 2012); As stated by EFSA (2008), LAB tolerant to these three antibiotics lowers The presence of aminoglycoside resistance genes in gentami- the risk of acquired resistance to other aminoglycoside. In this cin resistant LAB was evaluated by PCR employing total study, LAB were subjected to MIC determination to other DNA as a template. The primer sequences for aac(6′)Ie- aminoglycosides to investigate the level of resistance. aph(2″)Ia, aph(2″)Ic, aph(2″)Id and int were taken from liter- Likewise, the percentage of native LAB isolates resistant to ature (Doherty et al. 2000;Donabedianetal.2003). As de- apramycin, streptomycin, neomycin, amikacin, spectinomy- tailed in Table 1 the primers were designed based on the cin, kanamycin and gentamicin were evaluated. A MIC value GenBank accession numbers of aph(3′)IIIa (X92945.2), of ≤64 μg/mL for apramycin, streptomycin, neomycin, aad6 (X92945.2), ant(6)Ia (GQ900487.1), ant(9)Ia amikacin, spectinomycin, kanamycin and gentamicin was ob- (AY764268.1), ant(9)Ib (M69221.1), aph(2″)Ib served in 25 %, 22 %, 37 %, 42 %, 18 %, 31 % and 41 % of (AF207840.1). The isolates positive for the presence of ami- LAB isolates, respectively. Similarly, 68 %, 63 %, 48 %, noglycoside resistance genes were further assessed for trans- 39 %, 77 %, 24 % and 21 %, respectively, of LAB isolates poson integrase (int)genepertainingtoTn916-Tn 1545fam- displayed MIC values of ≥128–≥512 μg/mL and 7 %, 15 %, ily. The amplified product was checked on 1–1.2 % agarose 15 %, 19 %, 5 %, 45 % and 38 %, respectively, showed MIC gel in 1X TAE buffer as described previously. in the range ≥1024–≥4096 μg/mL. The MIC results inferred that LAB obtained in this study exhibited low (≤64 μg/mL), Gene sequencing, nucleotide accession numbers, moderate (≥128–≥512 μg/mL) and high (≥1024–≥2048 μg/ and phylogenetic tree construction mL) level resistance to aminoglycosides, as also documented in earlier studies (Murray 1990; Simjee and Gill 1997; The amplified products of the aminoglycoside resistance Donabedian et al. 2003; Bismuth and Courvalin 2010). In genes and 16S rDNA were purified using a PCR purification comparison to the MIC levels, the majority of isolates showed kit and sequenced at Amnion Biosciences (Bangalore, India). resistance in the range of ≥128–≥512 μg/mL to all the amino- The GenBank accession numbers obtained for 16S rDNA glycosides, except amikacin, kanamycin and gentamicin. nucleotide sequences of the LAB cultures [I40a, CS31+, S9, However, a few LAB cultures exhibited high level resistance chl, chl3, C11(5)], [J12G1, B20G3, P1, CS32+, 39-1, CHB1], to all the aminoglycoside antibiotics. The low level resistance [33-1, CS12+, CS13+, C26a, CSG-8, L32] and J10G3 are among a few of the LAB isolates observed in our study could [KJ420396–401], [KM016947–952], [KM016954–959] and be attributed to its anaerobic nature (Lopes et al. 2003). KJ995522, respectively. Likewise, accession numbers for the However, these isolates were susceptible to higher doses and sequences of the bifunctional gene in isolates I40a, CS31+, 5, do not exhibit acquired resistance. Enterococci from our study S9 and C11(5) are KJ420402–406. The accession numbers for showed HLGR (38 %), HLKR (45 %) and HLSR (15 %), kanamycin and streptomycin resistance genes aph(3′)IIIa and which was consistent with earlier studies (Lopes et al. 2003; aad6 in cultures CS11+ and 33-1 are KJ995521 and Del Campo et al. 2005; Ramos et al. 2012). KM016953, respectively. The int gene detected in the culture The resistance pattern to clinically relevant aminoglyco- I40a was also deposited with GenBank under the accession sides of enterococci, lactobacilli and pediococci is shown in number KJ420407. To construct the phylogenetic tree, DNA Table 2.Enterococci(n=101) was the most prevalent, follow- fingerprinting by RAPD-PCR was compared and a dendro- ed by lactobacilli (n=32) and pediococci (n=5) isolates. A gram created by the cluster analysis method using dice coef- significant number of LAB isolates from poultry were resis- ficient values by the NTYSIS software [Version 2.02e] tant to gentamicin (n=67),kanamycin(n=55) and streptomy- (Jamshidi and Jamshidi 2011). Patterns were grouped with cin (n=69), followed by sheep, pork and beef. Earlier studies unweighted pair group algorithm averages (UPGMA). indicated a higher prevalence of HLAR in enterococci from pig, cattle and sheep (Ramos et al. 2012; Klibi et al. 2014). The higher frequency of aminoglycoside resistance in entero- Results and discussion cocci from poultry indicates the significant use of these antibiotics in poultry farms. Pediococci isolated from poultry and A total of 138 LAB cultures were isolated by selective screen- pork were resistant to gentamicin (n=4), kanamycin (n=4) ing against gentamicin from the samples analyzed. The LAB and streptomycin (n=3). In contrast, meat products such as isolates were tentatively identified by initial biochemical char- chicken sausages were found to be contaminated with acterization and classified into enterococci (n =101), aminoglycoside-resistant enterococci and lactobacilli. Ann Microbiol (2016) 66:101–110 105

Lactobacilli derived from meat products only were resistant to gentamicin (13 %) and kanamycin (19 %). Eleven lactobacilli were observed with MIC values of ≥128 μg/mL for =0) n Meat products ( streptomycin. RAPD-PCR was carried out to group the aminoglycoside-

=3) resistant LAB that were selected based on the breakpoint MIC n Pork ( values as indicated in Fig. 1. Based on the RAPD profiles, a dendrogram was constructed and species-specific clusters of =0) n

Beef ( E. faecalis, E. faecium, E. avium, E. hirae, E. durans, E. cecorum, L. plantarum and Pediococcus lolii were b observed using the dice coefficient parameter and UPGMA. =0) sp. n Sheep ( The banding patterns revealed that the majority of gentamicin-

Number of isolates and kanamycin-resistant isolates from chicken intestine, n followed by sheep and chicken sausages, matched those of =2) n 2 0010 Poultry ( Pediococcus E. faecalis. Representative LAB isolates with similar RAPD profiles from each cluster were subjected to taxonomic identification by 16S rDNA gene sequencing. Enterococcus, Lactobacillus and Pediococcus isolates from different sources with low, moderate and higher MIC values for gentamicin, =32) c g/mL, respectively n Meat products ( μ kanamycin and streptomycin, as well as the presence of an aminoglycoside-resistant gene, was the criteria set to select

=0) them for 16S rDNA identification. Additionally, isolates of n Pork ( Minimal inhibitory concentration.

=16 and 64 Enterococcus and Lactobacillus derived from chicken meat,

MIC chicken intestine and chicken sausage with similar banding =0)

n patterns were also sequenced since they exhibited varying Beef ( MIC levels. In brief, E. faecalis CS12+ showed HLGR, a HLSR and a moderate MIC (512 μg/mL) for kanamycin, sp. =0) n Sheep ( while two isolates, E. faecalis CHL3 and E. faecalis CHL showed moderate MIC values (≥128–≥512 μg/mL) to the Lactobacillus plantarum above mentioned three aminoglycosides. E. faecalis 33-1 =0) n Poultry ( Lactobacillus and E. faecalis CS13+ with comparable RAPD profiles were g/mL, respectively

μ sequenced as they exhibited high level resistance to all seven aminoglycosides (≥2048–≥4096 µg/mL). Similarly, in the case of Lactobacillus species, CSG-8, CSG-21, L32, C11(5) and S9 showed different MIC levels (range 4–128 μg/mL) for =13) n Meat products ( gentamicin and for kanamycin (16–128 μg/mL), while

sp. =16, 64 and 64 L. plantarum Lb6 was sensitive to gentamicin and exhibited

=3) kanamycin resistance (≥128 μg/mL). Since it is important to n Pork ( of gentamicin, and kanamycin for identify contamination as well as the risk associated with the presence of aminoglycoside-resistant LAB in meat processing Pediococcus =2)

n units, it was thought appropriate to identify the above- Beef ( mentioned isolates. The phenotypically resistant LAB that were identified in this study include: E. faecalis (n=52), sp. =11)

n E. faecium (n =2), E. durans (n =4), E. hirae (n =6), Sheep ( E. avium (n=3), E. cecorum (n=1), P. lolii (n=2) and L. plantarum (n=32). Thus, E. faecalis species was prevalent =72)

n in all food animals and chicken sausage, which is in line with 9 103697 0 000nr Poultry ( Enterococcus earlier studies (Silva et al. 2012; Ramos et al. 2012).

2012 ), microbiological cut off values However, it was noted that E. faecalis was frequently iden-

Prevalence of aminoglycoside-resistant lactic acid bacteria (LAB) obtained from different sources. tified from poultry sources. Aminoglycoside-resistant E. faecium, E. durans, and E. cecorum were isolated from poultry, while E. hirae was identified from chicken, beef (128) g/mL) Gentamicin, kanamycin and streptomycin for As per EFSA ( Not required Gentamicin (32)Kanamycin (512) 67Streptomycin 55 10 6 1 0 0 2 4 4 0 0 0 0 0 0 0 0 4 6 2 2 0 0 0 0 2 2 0 0 Antibiotic (MIC μ Table 2 a b c and sheep. In similar studies conducted by Kuhn et al. 106 Ann Microbiol (2016) 66:101–110

Isolates Source MIC (µg/mL)

Fig. 1 Dendrogram showing the coefficient of similarity of the were subjected to 16S rDNA sequencing. The MIC values indicated in aminoglycoside-resistant lactic acid bacteria (LAB). The representative bold represent the isolates harboring the respective aminoglycoside cultures shown in bold carried the aminoglycoside resistance genes and resistance genes. Gm gentamicin, Km kanamycin, Str streptomycin

(2003), the association of E. faecalis with poultry and From our studies, it was evident that the food chain com- E. hirae with pigs and cattle was described. prises diverse groups of enterococcal populations. Similar Ann Microbiol (2016) 66:101–110 107 observations have been reported with Enterococcus species E. faecalis and E. faecium, while several isolates displayed found in food animals (Kuhn et al. 2003; Poeta et al. 2005; HLSR. Silva et al. 2012). Aminoglycoside resistant enterococcal spe- In the case of L. plantarum (n=6), varying MIC levels cies associated with nosocomial infections (Murray 1990)like (4–128 μg/mL) were observed against gentamicin; this E. avium, E. durans, E. faecalis, E. faecium and E. hirae from LAB was also found to possess the widespread food sources were also observed. The 16S rDNA sequence aac(6′)Ie-aph(2″)Ia gene. The aph(3′)IIIa kanamycin data of the Pediococcus and Enterococcus isolates, namely resistance gene and aad6 streptomycin modifying P1 and I40a, exhibited 100 % homology to P. lolii (acces- gene were found in three and one isolate, respectively. sion no. NR041640) and 98 % to E. cecorum (accession Isolates of L. plantarum revealed the presence of no. NR024905). It was interesting to note HLAR in lesser- aac(6′)Ie-aph(2″)Ia, aph(3′)IIIa and aad6 genes in earlier known species like P. lolii and E. cecorum fromfarman- investigations (Ouoba et al. 2008;Shaoetal.2015). The imals because E. cecorum was regarded as a part of the high MIC values and presence of gentamicin, kanamycin avian gut microflora until it was found to be an emerging and streptomycin resistance genes in L. plantarum isolat- pathogen in unhealthy chickens and infected patients ed from chicken sausages point out the unanticipated risks (Hseueh et al. 2000; Stalker et al. 2010). The occurrence relating to its generally regarded as safe (GRAS) status. of intestinal microbiota such as P. lolii and L. plantarum in Pediococcus lolii (n=2) harbored the aph(3′)IIIa gene. pork intestine and chicken sausage, respectively, are worth That no prior existence of HLKR in P. lolii was found in the mentioning. The occurrence of HLAR in rare microbiota literature signifies the transmission of kanamycin resistance relates significantly to selective pressure induced by the despite strict prohibition in its usage in animal husbandry improper use of antibiotics in farms. Additionally, (United States Pharmacopeial Convention 2008). L. plantarum, part of the characteristic microbiota of the Furthermore, acquired resistance and display of HLKR from human gut and also considered as a potential strain for food animals call into question the prophylactic use of amino- probiotic applications, can also be regarded as a possible glycosides in farm animals as they are used in human therapy reservoir of aminoglycoside resistance. (Guardabassi et al. 2008). All the isolates with MIC above the The phenotypically resistant LAB isolates were subjected breakpoint values were tested for gentamicin resistance genes to PCR for the presence of gentamicin, kanamycin and strep- such as aac(6′)Ie-aph(2″)Ia, aph(2″)Ib, aph(2″)Ic and tomycin resistance genes previously found in different entero- aph(2″)Id. Contrary to an earlier investigation (Donabedian coccal species of food origin. Isolates of E. faecalis (n=44), et al. 2003), the bifunctional gene was the only gentamicin- three each of E. durans and E. avium, as well as one each of modifying gene predominant in this study. However, one iso- E. hirae and E. cecorum showed MIC values associated to late of E. faecalis from chicken sausage showed gentamicin HLAR and were found to carry the aac(6′)Ie - aph(2″)Ia gene MICvaluesof1024μg/mL, which failed to generate ampli- (Table 3). Interestingly, the bifunctional gene was also identification for any of the above genes and therefore may contain fied in E. faecalis (n=5),E.hirae(n=5), E. avium (n=1) and a novel resistance mechanism or even a gene that needs to be E. durans (n=1), with moderate gentamicin MIC values in the investigated. Nine percent (n=12) of the LAB isolated from range of 128–256 μg/mL, similar to those found in an earlier food origin were observed with both the aac(6′)Ie-aph(2″)Ia investigation (Donabedian et al. 2003). To the best of our and aph(3′)IIIa genes conferring resistance to clinically signif- knowledge, the presence of this gene in E. cecorum and icant gentamicin and kanamycin. One isolate each of E. avium, which are otherwise considered to be the normal E. faecalis and L. plantarum was observed to harbor all three microbiota of the chicken intestine, has not been documented resistance genes [aac(6′)Ie-aph(2″)Ia, aph(3′)IIIa and aad6] previously. This study highlights the emergence of HLAR in conferring resistance to gentamicin, kanamycin and strepto- normal microbiota and its selection in the farm environment, mycin, respectively, which was also found to correlate with and the results portray an increasing risk of aminoglycoside higher MIC values. gene transfer from farm animals to the human population via The transmission of the bifunctional and other genes could the food chain. The PCR detection of the kanamycin resis- be related to transposons and horizontal gene transfer among tance gene, aph(3′)IIIa revealed its prevalence in E. faecalis the normal microflora. Studies have revealed the involvement (n=11), E. faecium (n=2) and E. avium (n=4). The kanamy- of a transpositional genetic element (Tn 5281)inE. faecalis cin modifying gene was found frequently in enterococci, similar to the staphylococcal transposons Tn 4001 and Tn along with aac(2″)Ie-aph(2″)Ia, which was previously associ- 4031 (Hodel-Christian and Murray 1991). The presence of ated with HLKR with MIC values of ≥1024 μg/mL (Del transposons in clinical strains of E. faecalis (Ferretti et al. Campo et al. 2000; Lopes et al. 2003; Ramos et al. 2012). 1986)andE. faecium (Eliopoulos et al. 1988), indicates the However, in our study it was detected in one strain of possibility of a direct gene exchange between species and a E. faecalis with MIC of 500 μg/mL. Also, a streptomycin common ancestral origin of gentamicin resistance. resistance gene (aad6) was found in two isolates each of Conjugative elements of the Tn 916-Tn 1545 family are 108 Ann Microbiol (2016) 66:101–110

Table 3 PCR detection of aminoglycoside resistance genes Antibiotic Species Resistance gene (no. of isolates) among native LAB isolates Gentamicin aac(6′)Ie - aph(2″)Ia MIC = ≥1024–≥4096μg/mL Enterococcus faecalis (n=43) E. hirae (n=1) E. durans (n=3) E. avium (n=3) MIC = ≥128–≥512μg/mL Lactobacillus plantarum (n=1) E. hirae (n=6) E. avium (n=1) E. durans (n=1) E. faecalis (n=5) E. cecorum (n=1) aac(6′)Ie - aph(2″)Ia MIC = ≥16–≥32μg/mL L. plantarum (n=2) MIC = ≥4–≥8μg/mL L. plantarum (n=3) Kanamycin aac(6′)Ie - aph(2″)Ia MIC = ≥1024–≥4096μg/mL E. faecalis (n=48) E. hirae (n=1) E. avium (n=4) E. cecorum (n=1) MIC = ≥128–≥512μg/mL E. hirae (n=5) E. durans (n=4) aph(3′)IIIa MIC = ≥1024–≥4096μg/mL E. faecalis (n=11) E. faecium (n=2) E. avium (n=4) Pediococcus (n=2) lolii MIC = ≥64–≥128μg/mL L. plantarum (n=3) Streptomycin aad6 MIC = ≥1024μg/mL E. faecalis ()n=2 E. faecium (n=2) MIC = 64–≥128μg/mL L. plantarum (n=1) Tn 916-1545 int Integrase E. faecalis (n=2) E. cecorum (n=1)

responsible for the dissemination of many antimicrobial resis- Overall in this study, we have observed aminoglycoside tance genes, usually related to tetracyclines and macrolides resistance genes in LAB isolated from chicken, sheep and (Thumu and Halami 2012, 2013). In the present study, the beef, while gentamicin resistance was not found in LAB de- integrase (int) gene inherent to the Tn 916-Tn 1545 family rived from pork. Spectinomycin-modifying genes ant(9)Ia was detected in E. faecalis (n=2) and E. cecorum (n=1) iso- and ant(9)Ib were also not found. Kanamycin has not been lated from chicken sausage and chicken intestine, respectively recommended for veterinary medicine; however, aph(3′)IIIa (Table 3). Since kanamycin resistance has been found only gene was the second most detected gene, in combination with rarely in these transposons, it was interesting to note that only the bifunctional gene. Combinations of aminoglycoside- one isolate E. faecalis CS11+ contained both the int and kana- modifying genes have been described in different species of mycin resistance [aph(3′)IIIa] genes that can be found in the enterococci(Jacksonetal.2004, 2009). Likewise, our study Tn 916-Tn 1545 family. Further studies are required to inves- revealed the presence of multiple aminoglycoside resistance tigate the localization and expression of aac(6′)Ie-aph(2″)Ia, genes in enterococci and lactobacilli. The presence of multi- aph(3′)IIIa and aad6 genes among LAB isolates. resistance genes to aminoglycoside in known nosocomial Ann Microbiol (2016) 66:101–110 109 pathogenic cultures like E. faecalis, E. faecium, E. durans and References E. hirae could be a great threat in developing nations. In addition, its emergence in the normal microflora (E. cecorum Altschul SF, Maddan TL, Schaffer AA, Zhang J, Zhang Z, Miller W, and E. avium) of farm animals can be compounded by the Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new genera- lack of proper monitoring of antibiotic use. Since enterococci tion of protein database search programs. Nucleic Acids Res 25: 3389–3402 are capable of acquiring and transferring genes horizontally, a Bismuth R, Courvalin P (2010) Aminoglycosides and Gram-positive high risk lies in the rapid emergence of the bifunctional gene bacteria: antibiogram, 3rd edn. ESKA, Portland among other genera of LAB as well as pathogenic strains in Boehr DD, Daigle DM, Wright GD (2004) Domain–domain interactions ′ – the human gut (Mathur and Singh 2005; Devirgiliis et al. in the aminoglycoside antibiotic resistance enzyme AAC (6 ) APH (2″). Biochemistry 43:9846–9855 2011). Acquired resistance in L. plantarum from meat prod- Byrne ME, Rouch DA, Skurray RA (1989) Nucleotide sequence analysis ucts indicates the contamination of food by resistant strains of IS256 from the Staphylococcus aureus gentamicin-tobramycin- that are otherwise regarded as harmless. The use of an amino- kanamycin-resistance transposon Tn4001. Gene 81:361–367 glycoside in farms may enhance the risk of acquiring resis- Chen YG, Qu TT, Yu YS, Zhou JY, Li LJ (2006) Insertion sequence ISEcp1- like element connected with a novel aph(2″)allele tance to other antibiotics of the same class (Guardabassi et al. [aph(2″)-Ie] conferring high level gentamicin resistance and a novel 2008). For instance, inappropriate use of apramycin was re- streptomycin adenylyltransferase gene in Enterococcus. J Med ported to trigger cross-resistance to gentamicin among bene- Microbiol 55:1521–1525 ficial microflora (Yates et al. 2004). In a recent study, Chow JW, Zervos MJ, Lerner SA, Thal LA, Donabedian SM, Jaworski DD, Tsai S, Shaw KJ, Clewell DB (1997) A novel gentamicin re- L. plantarum induced with streptomycin for a period of sistance gene in Enterococcus. Antimicrob Agents Chemother 41: 30 days was described to show higher MIC values for genta- 511–514 micin, kanamycin and neomycin to which it was previously CLSI (2012) Performance standards for antimicrobial susceptibility test- susceptible (Shao et al. 2015). Likewise, use of gentamicin in ing; twenty second informational supplement. In: CLSI document M100-S22. Clinical and Laboratory Standards Institute, Wayne animal husbandry may have propagated the selection and ac- Culebras E, Martinez JL (1999) Aminoglycoside resistance mediated by quisition of the bifunctional gene in LAB that exhibits cross- the bifunctional enzyme 6′ N aminoglycoside acetyltransferase - 2″- resistance to other aminoglycosides (except streptomycin). O-aminoglycoside phosphotransferase. Front Biosci 4:1–8 Del Campo R, Tenorio C, Rubio C, Castillo J, Torres C, Gomez-Lus R (2000) Aminoglycoside-modifying enzymes in high level streptomycin and gentamicin resistant Enterococcus spp. in Spain. Int J Conclusion Antimicrob Agents 15:221–226 Del Campo R, Galán JC, Tenorio C, Ruiz-Garbajosa P, Zarazaga M, Torres C, Baquero F (2005) New aac(6')-I genes in Enterococcus The overuse or underuse of antibiotics in livestock, as well as hirae and Enterococcus durans: effect on {beta}-lactam/aminogly- improper filtration of waste water from clinical settings and coside synergy. J Antimicrob Chemother 55(6):1053–1055 drug manufacturers could be the major reasons underlying the Devirgiliis C, Barile S, Perozzi G (2011) Antibiotic resistance determi- plague of antibiotic resistance in developing economies like nants in the interplay between food and gut microbiota. Genes Nutr 6:275–284 India. In addition, a lack of initiative to investigate and pro- Doherty N, Trzcinski K, Pickerill P, Zawadzki P, Dowson CG (2000) duce consistent data on the prevalence of developing resis- Genetic diversity of the tet(M) gene in tetracycline resistant clonal tance in these nations is a major drawback. There have been lineages of Streptococcus pneumoniae. Antimicrob Agents only few reports on aminoglycoside resistant LAB from dif- Chemother 44:2979–2984 ferent parts of the world, while these antibiotics are used rou- Donabedian SM, Thal LA, Hershberger E, Perri MB, Chow JW, Bartlett P, Jones R, Joyce K, Rositer S, Gay K, Johnson J, Mackinson C, tinely for prophylaxis and treatment in both humans and ani- Debess E, Madden J, Angulo F, Zervos MJ (2003) Molecular char- mals. The sudden emergence of resistance genes against broad acterization of gentamicin-resistant enterococci in the United States: spectrum aminoglycosides among the LAB via the food chain evidence of spread from animals to humans through food. J Clin – cannot be ignored. If the situation prevails, it could threaten Microbiol 41:1109 1113 EFSA (2008) Update of the criteria used in the assessment of bacterial the existing efficacy of broad spectrum activity aminoglyco- resistance to antibiotics of human or veterinary importance. EFSA J sides, thus increasing the demand for newer antibiotics. 732:1–15 EFSA (2012) Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J 10:1– 10 Acknowledgments The authors would like to thank Prof. Ram Eliopoulos GM, Wennersten C, Zighelboim-Daum S, Reiszner E, Rajasekharan, Director, CSIR-CFTRI, Mysore, for use of facilities. J.G. Goldmann D, Moellering RC Jr (1988) High level resistance to acknowledges the UGC for the grant of MAN fellowship. We also thank gentamicin in clinical isolates of Enterococcus faecium. Indian Council of Medical Research, New Delhi for financial assistance Antimicrob Agents Chemother 32:1528–1532 under the scheme of antimicrobial drug resistance. The authors wish to Ferretti JJ, Gilmore KS, Courvalin P (1986) Nucleotide sequence analysis sincerely acknowledge Dr M.C. Varadaraj for critical comments on the of the gene specifying the bifunctional 6′- aminoglycoside acetyl- manuscript. transferase 2″ aminoglycoside phosphtransferase enzyme in Streptococcus faecalis and identification and cloning of gene re- Conflict of interest The authors have no conflict of interest. gions specifying the two activities. J Bacteriol 167:631–638 110 Ann Microbiol (2016) 66:101–110

Guardabassi L, Schwarz S, Lloyd DH (2004) Pet animals as reservoirs of between pediocin AcH/PA-1 producing and non-producing strains. antimicrobial-resistant bacteria. J Antimicrob Chemother 54:321– Microbiology 46:2027–2038 332 Murray BE (1990) The life and times of the Enterococcus.ClinMicrobiol Guardabassi L, Jensen LB, Kruse H (2008) Guide to antimicrobial use in Rev 3:46–65 animals. Blackwell, UK Nielsen KM, Bohn T, Townsend JP (2014) Detecting rare gene transfer Hammerum AM, Lester CH, Heuer OE (2010) Antimicrobial-resistant events in bacterial populations. Front Microbiol 4:1–12 enterococci in animals and meat: a human health hazard? Foodborne Ouoba LII, Lei V, Jensen LB (2008) Resistance of potential probiotic Pathog Dis 7:1137–1146 lactic acid bacteria and bifidobacteria of African and European ori- Hodel-Christian SL, Murray BE (1991) Characterization of the gentami- gin to antimicrobials: determination and transferability of the resis- cin resistance transposon Tn 5281 from Enterococcus faecalis and tance genes to other bacteria. Int J Food Microbiol 121:217–224 comparison to staphylococcal transposons Tn 4001 and Tn 4031. Poeta P, Costa D, Saenz Y, Klibi N, Ruiz-Larrea F, Rodrigues J, Torres C Antimicrob Agents Chemother 35:1147–1152 (2005) Characterization of antibiotic resistance genes and virulence Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergey’s factors in faecal enterococci of wild animals in Portugal. J Vet Med Manual of Determinative Bacteriology, 9th edn. Williams & B Infect Dis Vet Public Health 52:396–402 Wilkins, Baltimore Popova M, Molimard P, Courau S, Crociani J, Dufour C, Le Vacon F, Hseueh PR, Teng LJ, Chen YC, Yang PC, Ho SW, Luh KT (2000) Carton T (2012) Beneficial effects of probiotics in upper respiratory Recurrent bacteremic peritonitis caused by Enterococcus cecorum tract infections and their mechanical actions to antagonize patho- – in a patient with liver cirrhosis. J Clin Microbiol 38:2450 2452 gens. J Appl Microbiol 113:1305–1318 Jackson CR, Fedorka-Cray PJ, Barrett JB, Ladely SR (2004) Genetic Ramirez MS, Tolmasky ME (2010) Aminoglycoside modifying en- relatedness of high level aminoglycoside resistant enterococci iso- zymes. Drug Resist Updat 13:151–171 – lated from poultry carcasses. Avian Dis 48:100 107 Ramos S, Igrejas G, Capelo-Martinez JL, Poeta P (2012) Antibiotic re- Jackson CR, Fedorka-Cray PJ, Davies JA, Barrett JB, Frye JG (2009) sistance and mechanisms implicated in fecal enterococci recovered Prevalence, species distribution and antimicrobial resistance of en- from pigs, cattle and sheep in a Portuguese slaughterhouse. Ann terococci isolated from dogs and cats in the United States. J Appl Microbiol 62:1485–1494 Microbiol 107:1269–1278 Schillinger U, Yousif NMK, Sesar L, Franz CMAP (2003) Use of group Jackson CR, Lombard JE, Dargatz DA, Fedorka-Cray PJ (2010) specific and RAPD PCR analyses for rapid differentiation of Prevalence, species distribution and antimicrobial resistance of en- – Lactobacillus strains from probiotic yoghurts. Curr Microbiol 47: terococci isolated from US dairy cattle. Lett Appl Microbiol 52:41 453–456 48 Shao Y, Zhang W, Guo H, Pan L, Zhang H, Sun T (2015) Comparitive Jamshidi S, Jamshidi S (2011) NTSYSpc 2.02e implementation in mo- studies on antibiotic resistance in Lactobacillus casei and lecular biodata analysis (clustering, screening, and individual selec- Lactobacillus plantarum. Food Control 50:250–258 tion). 2011 International Conference on Environmental and Shwarz S, Kehrenberg C, Walsh TR (2000) Use of antimicrobial agents in Computer Science. IPCBEE, vol.19. IACSIT, Singapore, pp 165– veterinary medicine and food animal production. Int J Antimicrob 169 Agents 17:431–437 Kao SJ, You IL, Clewell DB, Donabedian SM, Zervos MJ, Petrin J, Shaw KJ, Chow JW (2000) Detection of the high-level aminoglycoside Silva N, Igrejas G, Gonçalves A, Poeta P (2012) Commensal gut bacteria: resistance gene aph(2″)-Ib in Enterococcus faecium. Antimicrob distribution of Enterococcus species and prevalence of E. coli phy- – logenetic groups in animals and humans in Portugal. Ann Microbiol Agents Chemother 44:2876 2879 – Kimprasit T, Sukontasing S, Amavisit P (2013) In vitro selection of po- 62:449 459 tential lactic acid bacteria isolated from ducks and geese in Thailand. Simjee S, Gill MJ (1997) Gene transfer, gentamicin resistance and en- – Kasetsart J (Nat Sci) 47:261–270 terococci. J Hosp Infect 36:249 259 Klibi N, Aouini R, Borgo F, Said LB, Ferrario C, Dziri R, Boudabous A, Stalker MJ, Brash ML, Weisz A, Ouckama RM, Slavic D (2010) Arthritis Torres C, Slama KB (2014) Antibiotic resistance and virulence of and osteomyelitis associated with Enterococcus cecorum infection faecal enterococci isolated from food-producing animals in Tunisia. in broiler and broiler breeder chickens in Ontario, Canada. J Vet – Ann Microbiol. doi:10.1007/s13213-014-0908-x Diagn Investig 22:643 645 ′ Kuhn I, Iverson A, Burman LG, Ollsson-Liljequist B, Franklin A, Finn Tenorio C, Zarazaga M, Martinez C, Torres C (2001) Bifunctional 6 -N- ″ M, Aarestrup F, Seyfarth AM, Blanch AR, Vilanova X, Taylor H, aminoglycoside acetyltransferase-2 -O-aminoglycoside phospho- Caplin J, Moreno MA, Dominguez L, Herrero IA, Möllby R (2003) transferase in Lactobacillus and Pedicoccus isolates of animal ori- – Comparison of enterococcal populations in animals, humans, and gin. J Clin Microbiol 39:824 825 the environment—a European study. Int J Food Microbiol 88:133– The United States Pharmacopeial Convention, Inc (2008) The United 145 States pharmacopeia 31/the national formulary 26, Supp. 1, 8-1- Kuhn I, Iversen A, Finn M, Greko C, Burman LG, Blanch AR, Vilanova 08, online. United State Pharmacopeial Convention, Rockville X, Manero A, Taylor H, Caplin J, Dominguez L, Herrero IA, Thumu SCR, Halami PM (2012) Presence of erythromycin and tetracy- Moreno MA, Mollby R (2005) Occurrence and relatedness of cline resistance genes in lactic acid bacteria from fermented foods of vancomycin-resistant enterococci in animals, humans, and the envi- Indian origin. Antonie Van Leeuwenhoek 102:541–551 ronment in different European regions. Appl Environ Microbiol 71: Thumu SCR, Halami PM (2013) Phenotypic expression, molecular char- 5383–5390 acterization and transferability of erythromycin resistance genes in Lopes MFS, Ribeiro T, Martins MP, Tenreiro R, Crespo MTB (2003) Enterococcus spp. isolated from naturally fermented food. J Appl Gentamicin resistance in dairy and clinical enterococcal isolates Microbiol 116:689–699 and in reference strains. J Antimicrob Chemother 52:214–219 Tsai SF, Zervos MJ, Clewell DB, Donabedian SM, Sahm DF, Chow JW Mathur S, Singh R (2005) Antibiotic resistance in food lactic acid bacte- (1998) A new high-level gentamicin resistance gene, aph(2″)-Id, in ria—a review. Int J Food Microbiol 105:281–295 Enterococcus spp. Antimicrob Agents Chemother 42:1229–1232 Mora D, Fortina MG, Parini C, Daffonchio D, Manachini PL (2000) Yates CM, Pearce MC, Woolhouse ME, Amyes SG (2004) High frequen- Genomic subpopulations within the species Pediococcus cy transfer and horizontal spread of apramycin resistance in calf acidilactici detected by multilocus typing analysis: relationships faecal E. coli. J Antimicrob Chemother 54:534–537