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Bifunctional Enzyme 6′-N-Aminoglycoside Acetyltransferase-2"-O- Aminoglycoside Phosphotransferase in Lactobacillus and Pediococcus Isolates of Animal Origin

Lactic acid bacteria (LAB) are saprophytic bacteria in human and animal habitats. However, they are also involved in human infections as opportunistic pathogens (1). There are few reports on the susceptibility of these bacteria to antibiotics, and data on aminoglycoside resistance are scarce (9). The bifunctional aminoglycoside-modifying enzyme 6′-N-aminoglycoside acetyltransferase-2"-O-aminoglycoside phosphotransferase [AAC(6′)-APH(2")] has been previously reported in Enterococcus (4), Staphylococcus (7), and Streptococcus agalactiae isolates (5). The expression of the aac(6′)Ie-aph(2")Ia gene confers high-level gentamicin resistance in these genera in addition to resistance to other aminoglycosides, such as tobramycin, kanamycin, and netilmicin. Recently, two new 2"-O-aminoglycoside phosphotransferases that confer moderate or high-level gentamicin resistance have been described in enterococci, APH(2")-Ic (2) and APH(2")-Id (11). There are no previous reports of gentamicin resistance or aminoglycoside-inactivating enzymes in LAB. 
 
Thirty-nine LAB isolates (36 Lactobacillus and 3 Pediococcus isolates; the species are given in Table ​Table1)1) were obtained from fecal samples of 26 healthy pigs and 15 pets. Each fecal sample was diluted in sterile saline solution, and 25 μl was seeded in nonsupplemented MRS agar plates and incubated for 48 h at 30°C in 5% CO2. One colony per sample was selected and identified by the API 50 CHL system (BioMerieux). The MIC of gentamicin was determined by the agar dilution method (6) using brain heart infusion agar and an atmosphere of 5% CO2. Specific PCR amplifications were performed to determine the presence of the aac(6′)Ie-aph(2")Ia (12), aph(2")Id (11), and aph(2")Ic (2) genes, using primers and conditions previously described. In each PCR, DNA from one positive control was included (Enterococcus faecalis E-114, Enterococcus gallinarum SF9117, and Enterococcus casseliflavus UC73). 
 
 
 
TABLE 1 
 
MICs of gentamicin and mechanisms of resistance involved in 36 Lactobacillus and 3 Pediococcus isolates included in this study 
 
 
 
A gentamicin MIC of ≥64 μg/ml was found for 9 of 39 LAB isolates analyzed. Seven of these isolates (five L. salivarius, one L. acidophilus, and one P. acidilactici) gave positive aac(6′)Ie-aph(2")Ia PCR results and negative ones for aph(2")-Id and aph(2")-Ic genes. The other two gentamicin-resistant isolates (Lactobacillus salivarius) gave negative PCR results for the aac(6′)Ie-aph(2")Ia, aph(2")-Id, and aph(2")-Ic genes (Table ​(Table1).1). A gentamicin MIC of <8 μg/ml was found for the additional 30 LAB isolates, and these isolates gave negative results for all three genes analyzed by PCR (Table ​(Table1).1). The seven aac(6′)Ie-aph(2")Ia amplicons were purified and sequenced and showed 100% identity with the amino acid sequence of the AAC(6′)-APH(2") aminoglycoside-modifying enzyme of E. faecalis (accession no. {"type":"entrez-nucleotide","attrs":{"text":"A26048","term_id":"904820"}}A26048) and Staphylococcus aureus (accession no. S26353). 
 
This is the first report of the AAC(6′)-APH(2") aminoglycoside-modifying enzyme in Lactobacillus and Pediococcus isolates. The high conservation of this gene in Enterococcus and Staphylococcus indicates that it has been recently transferred from one of them to the other (3). The bifunctional gene has been described in different Enterococcus species (5). Transference of the bifunctional enzyme and other genes involved in aminoglycoside resistance between genera has been demonstrated under natural conditions (8, 10). Lactobacilli, pediococci, and enterococci share the same intestinal habitat in humans and animals, and the transference in the intestinal gut of the aac(6′)Ie-aph(2")Ia gene from Enterococcus to Lactobacillus and Pediococcus is a possibility cannot be excluded. Antibiotic resistance genes could be picked up by LAB from other bacteria when they survive in an antibiotic-containing habitat, such as the intestine (9).