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16S rRNA Gene Mutations Associated with Decreased Susceptibility to
Tetracycline in Mycoplasma bovis

E. Amram,a,b I. Mikula,a C. Schnee,c R. D. Ayling,d R. A. J. Nicholas,d R. S. Rosales,d S. Harrus,b I. Lysnyanskya
Division of Avian and Aquatic Diseases, Kimron Veterinary Institute, Bet Dagan, Israela; Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israelb; Friedrich-Loeffler-Institute, Jena, Germanyc; Animal and Plant Health Agency, Addlestone, United Kingdomd Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired
molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis
. Sequence analysis
of the two 16S rRNA-encoding genes (rrs3
and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site)
was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16
g/ml. Mutations at positions A965T, A967T/C
(Escherichia coli
numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetra-
cycline (MICs,
>2 g/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and
G1058) of the two rrs
alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis iso-
lates. The data presented correlate (P
< 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with de-
creased susceptibility to tetracycline.

The bacterial pathogen Mycoplasma bovis causes a variety of (rrl3 [MBOPG45 0957] and rrl4 [MBOPG45 0959]), while in
clinical manifestations in cattle, including respiratory disease, strain Hubei-1, only one rrn was identified No putative mastitis, arthritis, and otitis, which result in substantial economic tet(M) determinant was found in the annotated genomes of M. losses The tetracyclines are among the few important antimi- crobial agents that may be used to treat M. bovis infections The aim of this study was to investigate the mechanisms asso- Tetracyclines are broad-spectrum antimicrobials that have ciated with acquired decreased susceptibilities to tetracyclines in been widely used in human and veterinary medicine They M. bovis isolates.
inhibit protein synthesis by binding to the 30S ribosomal subunitand blocking an attachment of aminoacyl-tRNA to the A site MATERIALS AND METHODS
Resistance to tetracyclines is common in many bacterial speciesand may be achieved by (i) an energy-dependent efflux of the drug Mycoplasma bovis isolates and growth conditions. A total of 70 M. bovis
field isolates from Israel (n ⫽ 33; 1995 to 2011), the United Kingdom (n
across the cell membrane mediated by efflux pumps, (ii) the pres- 11; 2000 to 2009), Germany (n ⫽ 11; 1978 to 1991), Spain (n ⫽ 2; 1993), ence of ribosomal protection proteins that confer tetracycline re- Australia (n ⫽ 3; 2006), Hungary (n ⫽ 6; 2006 to 2010), Lithuania (n ⫽ 3; sistance, either by a reduction of the affinity of ribosomes to tet- 2007 to 2010), and Cuba (n ⫽ 1; 1980 were tested in this study, and racyclines or by releasing the bound antimicrobial from the their details are given in Each isolate originated from different ribosome, (iii) the enzymatic inactivation of the drug, or (iv) the farms, was selected at random, and had no epidemiological link to other mutations in the 16S rRNA genes that affect the binding sites of isolates unless indicated otherwise. The reference type strain M. bovis PG45 was obtained from the National Collection of Type Cultures, In Mollicutes, two mechanisms of resistance to tetracyclines United Kingdom (strain NCTC10131, corresponding to ATCC 25523).
have been identified so far, both of which are in Mollicutes species All the isolates were propagated at 37°C in standard M. bovis broth that infect humans. These include ribosomal protection by tet(M) medium supplemented with 0.5% (wt/vol) sodium pyruvate and determinants, described in naturally tetracycline-resistant strains 0.005% (wt/vol) phenol red (pH 7.8) Isolates of M. bovis were iden- of Mycoplasma hominis and Ureaplasma spp. as well as tified by immunofluorescence of colonies using species-specific conju- target modification with point mutation(s) in the 16S rRNA genes gated antiserum. Aliquots of cultures were stored at ⫺80°C until used. An of in vitro obtained mutants of Mycoplasma pneumoniae (posi- aliquot was then thawed, and the number of CFU per ml was determined tions 968 and 1193) and M. hominis (positions 346, 965 to 967, by performing serial 10-fold dilutions in broth, plating each dilution on Even though high MICs to tetracyclines have been identified in many Mycoplasma spp. of veterinary importance (reviewed in ref- Received 15 July 2014 Returned for modification 31 July 2014Accepted 5 November 2014 erence including M. bovis the genetic background for Accepted manuscript posted online 17 November 2014 decreased susceptibility has not been elucidated in either field iso- Citation Amram E, Mikula I, Schnee C, Ayling RD, Nicholas RAJ, Rosales RS, lates or mutants selected in vitro. M. bovis contains one or two Harrus S, Lysnyansky I. 2015. 16S rRNA gene mutations associated with decreased rRNA operons (rrn). Indeed, analysis of genome sequences of M. susceptibility to tetracycline in Mycoplasma bovis. Antimicrob Agents Chemother bovis type strain PG45 and two field strains, isolated in China, revealed that two out of the three annotated genomes (type strain Address correspondence to I. Lysnyansky,
PG45 and HB0801) contained two tandem rrn alleles Copyright 2015, American Society for Microbiology. All Rights Reserved.
(rrn3 and rrn4), both of which consisted of 16S rRNA (rrs3 [gene ID MBOPG45 0956] and rrs4 [MBOPG45 0958]) and 23S rRNA Antimicrobial Agents and Chemotherapy February 2015 Volume 59 Number 2 16S rRNA Mutations and Susceptibility in M. bovis TABLE 1 Molecular characterization of the primary binding pocket for tetracycline in rrs genes of M. bovis field isolates with different
susceptibilities to tetracycline
Mutation in the Tet-1 site of: (Continued on following page) February 2015 Volume 59 Number 2 Antimicrobial Agents and Chemotherapy Amram et al.
TABLE 1 (Continued)
Mutation in the Tet-1 site of: a The same superscript uppercase letter (A to F) indicates that M. bovis strains were isolated from the same shipment or on the same farm.
b NR, not relevant, M. bovis isolate with one rrn.
c M. bovis isolates isolated in Israel from imported calves in quarantine stations.
agar, incubating for 96 h, and then counting the colonies as described well 16 apparatus (Promega) according to the manufacturer's instruc- tions. The 16S rRNA-encoding gene (rrs3 and rrs4 alleles) containing the Tetracycline susceptibility. The susceptibilities of M. bovis isolates to
Tet-1 site, formed by residues 1054 to 1056 and 1196 to 1200 of helix 34 tetracycline hydrochloride (ⱖ95% active substance; Sigma, Rehovot, Is- and residues 964 to 967 of helix 31 (numbers corresponding to Escherichia rael) were tested by the agar dilution method as described previously coli 16S rRNA), was amplified in M. bovis isolates using the primers listed using M. bovis agar plates Briefly, 2-fold dilutions of tetracycline in The primer sets MB-282-F–MB-tet3/4-R and MB-rrs-3F–MB- from 0.03 to 32 ␮g/ml were incorporated onto the agar plates. Five mi- 287-R allowed the amplification of rrs3 and rrs4, respectively croliters of each isolate, containing 1 ⫻ 105 to 1 ⫻ 106 CFU/ml, was The primers were developed and commercially synthesized (Sigma, Re- spotted onto the agar plates. Plates were incubated at 37°C with 5% CO hovot, Israel) based on the nucleotide sequence of M. bovis type strain for 4 days. The NCTC M. bovis type strain PG45, which is regularly tested PG45 (NCBI reference sequence and strain HB0801 in the laboratory, was used as a control to ensure that the results were consistent with results obtained previously The procedure was re- PCRs were carried out in 50-␮l volumes containing 250 ng of template peated independently three times for the reference strain PG45 and for DNA, 1 ␮l of Phire Hot Start II DNA polymerase (Thermo Scientific, 50% of the isolates, selected at random, with the same results obtained Waltham, MA, USA), 10 ␮l of 5⫻ Phire reaction buffer, 1 ␮l of 10 mM within a single 2-fold dilution (data not shown).
deoxynucleoside triphosphate (dNTP), and 1 ␮l of 20 ␮M each primer.
Amplification of the M. bovis primary tetracycline binding site. Se-
PCR amplifications were carried out in a C1000 series thermocycler (Bio- quence analysis of the primary binding pocket for tetracycline (the Tet-1 Rad, Hercules, CA, USA). The conditions for the PCRs are specified in site) was performed for all 70 M. bovis field isolates tested in this study.
Genomic DNA was extracted from 400 ␮l of logarithmic-phase broth The amplicons were then extracted and purified from the gel using the culture using the Maxwell DNA isolation kit for cells/tissues and the Max- MEGAquick-spin PCR and agarose gel DNA extraction system (iNtRON TABLE 2 Primers and PCR amplification programs used in this study
Size of the amplified Primer sequence (5= to 3=) Amplification protocol product (bp)a 98°C for 30 s; 40 cycles of 98°C for 5 s, (3= end), rrs3 60°C for 5 s, 72°C for 1 min; a finalstage at 72°C for 3 min rrs3 and rrs4 rrs3 and rrs4 rrs4 (3= end), 98°C for 30 s; 40 cycles at 98°C for 5 s, 64°C for 5 s, 72°C for 2 min; a final stage at 72°C for 3 min a The length of the PCR products is according to M. bovis type strain PG45.
Antimicrobial Agents and Chemotherapy February 2015 Volume 59 Number 2

16S rRNA Mutations and Susceptibility in M. bovis the MICs was identified, indicating decreased susceptibilities inisolates in the higher range of MICs (ⱖ2 ␮g/ml) Indeed,44/70 (63%) isolates had MICs of 2 to 16 ␮g/ml. At least twoisolates per defined MIC were identified for further analyses, ex-cept at the MIC of 16 ␮g/ml, as only one isolate was found The sequence analysis of the Tet-1 site of 16S rRNA-encoding genes (rrs3 and rrs4 alleles) showed that 5/70 M. bovis isolates hadone rrs allele (two isolates with an MIC of 0.25 ␮g/ml, two with anMIC of 0.5 ␮g/ml, and one with an MIC of 8 ␮g/ml). The distri-bution of mutations in the Tet-1 site relative to the tetracyclineMICs is shown in Two cohorts of isolates were identified.
The first cohort contained 26 M. bovis isolates with MICs of ⱕ1 FIG 1 Distribution of Mycoplasma bovis isolate MICs to tetracycline. MICs of
␮g/ml. In 13/26 isolates, no mutations were identified at positions tetracycline tested in this study are shown on the x axis; the number of M. bovis 964 to 967 of helix 31 or at positions 1054 to 1056 and 1196 to 1200 isolates corresponding to each MIC is shown on the y axis.
of helix 34 in either rrs allele. However, a single mutation at posi-tion A965T or A967T of the rrs3 allele was found in seven isolates,two isolates contained the A965T and A967T mutations in rrs4, Biotechnology, South Korea). Sequencing was performed at the DNA and four isolates had mutations at position A967T/C in rrs3 and Sequencing Unit at the Weizmann Institute (Rehovot, Israel). Sequence rrs4 alleles isolate numbers 1 to 26).
editing, consensus, and alignment construction were performed usingDNASTAR software, version 5.06/5.51 (Lasergene, Inc., Madison, WI, The second cohort contained 44 M. bovis isolates with MICs of USA) and BioEdit (Ibis Biosciences The numbering of each nucle- ⱖ2 ␮g/ml. Twenty-two out of 44 isolates possessed the double otide was based on the respective 16S rRNA gene of E. coli unless indicated mutations AGA 965 to 967 TGT in the two rrs alleles, 18 isolates contained triple mutations (AGA 965 to 967 TGT/C and G1058A) Screening of M. bovis isolates for the presence of tetracycline resis-
in the two rrs alleles, and 3 isolates contained the double muta- tance determinants. To check for the presence of tet(M), tet(O), and
tions AGA 965 to 967 TGT in the two rrs alleles and a G1058C tet(L) determinants, all 70 M. bovis isolates and the PG45 type strain were mutation in rrs3 (one isolate) or a G1058A mutation in rrs4 (two subjected to DNA dot blot analysis. Briefly, for each isolate, approximately isolates). In addition, one isolate (with only one rrn) contained the 1 ␮g of M. bovis genomic DNA was spotted onto a positively charged double mutations AGA 965 to 967 TGT isolate numbers nylon membrane. The tet(M)-, tet(O)-, and tet(L)-related probes were amplified using genomic DNA of beta-hemolytic streptococci group G(GGS) containing these determinants (obtained from a collection of the In addition, three isolates with MICs of 2 to 4 ␮g/ml tetracy- Department of Clinical Microbiology and Infectious Diseases, Hadassah- cline had a U1199C mutation in rrs3, and 20 M. bovis isolates with Hebrew University Medical Center, Jerusalem, Israel). The primers and MICs of 2 to 8 ␮g/ml tetracycline contained the C1192A mutation PCR conditions used for tet(M), tet(O), and tet(L) amplifications were as in one rrs (12 isolates) or in both rrs alleles (8 isolates). These previously described The obtained tet(M), tet(O), and tet(L) mutations were not identified in M. bovis isolates with MICs of PCR amplicons were purified from the gel as described above and se- 0.125 to 1 ␮g/ml (data not shown). It should be noted that other quenced, and their nucleotide sequences were compared to the data pres- mutations/nucleotide substitutions were also identified within the ent in the NCBI Nucleotide Database rrs3 allele of some isolates (at positions 1005 [2 strains, with MICs The tet(M), tet(O), and tet(L) amplicons were subsequently labeled by of 0.125 and 0.5 ␮g/ml], 1013 [24, with MICs of 0.125 to 8 ␮g/ml], digoxigenin (DIG) following the manufacturer's protocol (Roche Diag- 1281 [6, with MICs of 0.125 to 8 ␮g/ml], and 1331 [1, with an MIC nostics GmbH, Mannheim, Germany). Spotted DNA was then hybridizedwith DIG-labeled probes, washed, incubated with alkaline phosphatase of 0.5 ␮g/ml]) and/or within the rrs4 allele (at positions 1005 [2, (AP)-tagged anti-DIG antibody, and detected as previously described by with MICs of 0.125 and 0.5 ␮g/ml), 1013 [37, with MICs of 0.25 to Lysnyansky et al. Chemiluminescence detection and imaging were 16 ␮g/ml], 1153 [61, with MICs of 0.125 to 16 ␮g/ml], 1184 [4, performed using a G:BOX Chemi XR5 scanner (Syngene, Cambridge, with MICs of 0.25 to 0.5 ␮g/ml], 1189 [1, with an MIC of 0.125 United Kingdom). Genomic DNAs of the GGS isolates containing tet(M), ␮g/ml], 1268 [59, with MICs of 0.125 to 16 ␮g/ml], and 1331 [1, tet(O), and tet(L) determinants were used as positive controls.
with an MIC of 4 ␮g/ml]). None of these positions is located In addition, PCR amplification of tet(M) was performed on genomic within or close to the Tet-1 site, and nucleotide substitution at DNA of all 70 M. bovis isolates using the primers (tetMF and tetMR) and position 1013 (A to G in rrs3 and G to A in rrs4) appeared to PCR conditions previously described by Blanchard et al. represent intra-rrs variability.
Statistical analysis. The significance of observed associations between
Screening of the M. bovis genomic DNA for the presence of
the presence of mutations within the 16S rRNA gene(s) and decreasedsusceptibility to Tet was analyzed using Fisher's exact test. Two-tailed P tet(M), tet(O), and tet(L) determinants. Genomic DNA samples
values were calculated using the GraphPad QuickCalc website of 70 M. bovis isolates with tetracycline MICs of 0.25 to 16 ␮g/ml P ⬍ 0.05 was considered sta- were screened by dot blot analysis for the presence of tet(M), tet(O), and tet(L) genes as described in Materials and Methods. Nopositive signals were identified by any of the DIG-labeled probes (data not shown). In contrast, strong signals were detected for DNA sequence analysis of the 16S rRNA-encoding genes of M.
GGS-tet(M), tet(O), and tet(L) determinants used as positive con- bovis isolates with different susceptibilities to tetracycline. The
trols. In addition, no PCR product was amplified using genomic MICs of the 70 M. bovis field isolates ranged from 0.125 to 16 DNA of the 70 M. bovis isolates and the PCR system developed by ␮g/ml, as shown in and A bimodal distribution of Blanchard et al. February 2015 Volume 59 Number 2 Antimicrobial Agents and Chemotherapy Amram et al.
pionibacterium acnes, Brachyspira hyodysenteriae, and Brachyspira Tetracyclines are often used for the treatment of M. bovis-related intermedia It has been suggested that a mutation at this infections, and thus tetracycline resistance in this organism is of position can influence the base pairing G1058 with U1199 and increasing concern. In this study, the molecular mechanisms as- might lead to a conformational change and closing of the Tet-1 sociated with M. bovis-decreased susceptibility to tetracycline binding pocket In our study, two additional mutated posi- were investigated. Our results showed that an increase of MICs to tions, C1192A and U1199C, were identified in groups of isolates tetracycline (ⱖ2 ␮g/ml) is correlated with the number of mutated with MICs of 2 to 8 and 2 to 4 ␮g/ml, respectively (data not nucleotide positions within the Tet-1 site of M. bovis field isolates.
shown). While the mutation U1199C itself may influence the Indeed, 43/44 M. bovis isolates with MICs of ⱖ2 ␮g/ml contained binding of tetracycline to the Tet-1 site, mutation at position 1192 two (A965T and A967T/C) or three (A965T, A967T/C, and was shown to be associated with resistance to spectinomycin in E. G1058A/C) mutations in both the rrs3 and rrs4 alleles (from 4 to 6 mutated sites in total; P ⬍ 0.0001). The additional isolate had only It is well documented that tetracycline resistance in M. hominis one rrs allele, which contained A965T and A967T mutations and Ureaplasma spp. with high MICs (ⱖ32 ␮g/ml) is associated number 61). In contrast, only single- or double-base-pair with the tet(M) determinant However, tetracycline- mutations (maximum of two mutated positions) were identified susceptible Ureaplasma urealyticum and two M. hominis isolates in 13/26 M. bovis isolates with MICs of ⱕ1 ␮g/ml tetracycline harboring the tet(M) gene were recently identified Whileno mutations in the coding region of the tet(M) gene or in the numbers 1 to 26). It is likely that the impact of single- or promoter region were found in the tetracycline-susceptible U. double-base-pair mutations on the susceptibility to tetracycline of urealyticum isolate, one of two M. hominis isolates had an inser- M. bovis isolates containing two rrs alleles is minor. This can be tion of insertion-like sequence ISMhom1 (IS30 gene family) up- explained by the fact that a nonmutated rrs copy may cover the stream to tet(M), which possibly caused the lack of tet(M) tran- function of the mutant copy. Indeed, from 12 out of 13 isolates scription in this isolate In this study, no tet(M), tet(O), or with two rrs alleles, 10 were heterozygotes with MICs of ⱕ0.5 tet(L) determinants were found in any of the 70 M. bovis isolates.
g/ml. Two additional isolates (116/93 and 145/93) were ho- Several assumptions can explain such results: (i) it may be that mozygous for the single mutation A967C and had an MIC of 1 tetracycline resistance in M. bovis is not acquired via tet determi- g/ml tetracycline. More homozygotes for a single mutation nants, and (ii) no M. bovis isolates with high MICs were identified should be tested to clarify whether one mutated position present and tested in this study, a fact that may have negatively influenced in both of these rrs alleles may account for a slight increase in MIC the chance to detect those genes. The existence of M. bovis isolates (1 ␮g/ml). In addition, it will be of interest to test the correlation with high MICs (ⱖ64 ␮g/ml) to different tetracyclines has been between the presence of single- or double-base-pair mutations shown (summarized in reference however, no isolates with and MICs of tetracycline in a cohort of M. bovis isolates with one such high MICs were found in our study, and only 1/70 isolates rrs allele (unfortunately, only 5 isolates with one rrs allele were had an MIC of 16 ␮g/ml The possible explanation for identified in our study numbers 5, 8, 16, 18, and 61]).
this discrepancy is that there are many difficulties in comparing The Tet-1 site consists of two domains in 16S rRNA: helix 34 results obtained from the different studies due to the lack of stan- (residues 1054 to 1056 and 1196 to 1200) and the loop next to dardization in the performance of the susceptibility assays, which helix 31 (residues 964 to 967). It lies in a clamp-like pocket at the includes use of different methods, different control isolates, and A site for binding of tRNA, as was previously shown by the crystal different tetracyclines. More isolates with MICs of ⱖ16 ␮g/ml structures of Thermus thermophilus 30S ribosome-tetracycline should be tested to confirm the results obtained in this study.
complexes Mutations detected in this study were located In summary, we have identified mutations in the primary within this site or close to it (position 1058). It has been shown binding pocket for tetracycline of clinical isolates of M. bovis, that low-level resistance to tetracycline in Helicobacter pylori re- which correlated with decreased susceptibilities to tetracycline.
sulted from single- and double-base-pair mutations at positions Moreover, the data presented here demonstrate that the increase 965 to 967 while high resistance to tetracycline was in the MICs for tetracycline (ⱖ2 ␮g/ml) in M. bovis field isolates achieved by triple-base-pair mutation AGA 965 to 967 TTC correlated with the number of nucleotide positions affected In another study, three isolates with a single mutation (at within the Tet-1 site of the rrn3 and rrn4 alleles.
positions 966, 967, or 1054), one isolate with a double mutation(at positions 346 and 965), and one isolate with a triple mutation (at positions 346, 965, and 966) were identified in mutants of M. We gratefully acknowledge the receipt of Streptococcus isolates from R.
hominis type strain PG21, selected in vitro using doxycycline Nir-Paz, Department of Clinical Microbiology and Infectious Diseases, In the same study, an in vitro-obtained mutant of M. pneumoniae Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
type strain FH harboring the single-base-pair mutation G1193A This research was supported by a research grant from the Emerging and another mutant with the double-base-pair mutations and Major Infectious Diseases of Livestock (EMIDA) Consortium from G1193A and T968C were also selected. It has been acknowledged the Chief Scientist of the Ministry of Agriculture, Israel (847-0369), and that compared to the parent M. hominis and M. pneumoniae the Israel Dairy Board (847-0366).
strains, the selected mutants showed decreased susceptibilities to We declare no conflicts of interest.
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Antimicrobial Agents and Chemotherapy February 2015 Volume 59 Number 2


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