on November 21, 2018 by guest

Transcription

1 AEM Accepts, published online ahead of print on 30 November 2012 Appl. Environ. Microbiol. doi: /aem Copyright 2012, American Society for Microbiology. All Rights Reserved Aeromonas and Pseudomonas spp. carriers of ampc FOX gene in the aquatic environment Veiko Voolaid, Tanel Tenson and Veljo Kisand Institute of Technology, University of Tartu Running title: ampc FOX genes in Aquatic Bacteria Resistance to beta-lactam antibiotics is evolutionarily ancient, and resistance to new drugs develops and quickly spreads. Most studies on beta-lactam resistance concentrate on pathogens in medical and veterinary settings. We show that ampc FOX genes can be found in natural environmental isolates. Address correspondence to Veljo Kisand, kisand@ut.ee Downloaded from on November 21, 2018 by guest

2 Beta-lactams are common antibiotics (AB) used to treat human and animal infections and for disease prevention (4). Their use has always been followed by the emergence of resistance, which is most frequently due to beta-lactamases, such as ampc (2, 5, 12). Both beta-lactam antibiotics and the resistance genes are evolutionarily ancient (12), and the natural origin of beta-lactamase genes might be manifested by their wider dissemination in the environment. Therefore we chose to investigate whether resistance genes from the class ampc, found among pathogenic Enterobacteria and are important resistance genes in medicine, could be found in fresh water (environmental) bacteria. We used a collection of antibiotic resistant (AR) isolates originating from freshwater ecosystems in Estonia (10), which was carried out on selective media with single AB (ampicillin, chloramphenicol, kanamycin, norfloxacin, and tetracycline) or double AB plates (ampicillin and kanamycin; tetracycline and chloramphenicol) as previously described (10). To detect ampc genes, DNA in the isolates was extracted from pure cultures (n=974) using the GuSCN silica method (1), which was amplified with multiplex PCR (9) for all major families of plasmid-encoded ampc genes (e.g. CMY, DHA, MOX, FOX, and ACC). Of our study population, ampc genes (all belonging to FOX family) were detected from 48 isolates (5% from all isolates). Forty-five FOX-positive isolates were isolated from ampicillin containing media, these isolates made up 31% of the total number of isolates that were isolated from ampicillin containing media. Other 3 FOX- positive isolates were isolated from tetracycline containing media but conformed as ampicillin resistant (10). To determine the preliminary phylogenetic affiliation of FOX-positive isolates, the 16S rrna gene was PCR amplified using the universal primers: pcrf (5 -AGAGTTTGATCATGGCTCAG-3, positions 6-26 in E. coli rdna) and pcrr (5 -TACGGYTACCTTGTTACGACTT-3, positions in E. coli rdna), the pcrf primer and RupA (5 -CGTATTACCGCGGCTGCT-3, position in E. coli rdna) (7) were used for sequencing nearly full 16S rrna gene. Phylogenetic affiliation was assessed using the Ribosome Database Project (11). Of the 48 FOX-positive isolates, 30 (63%) were Aeromonas spp. and 12 (25%) Pseudomonas spp. (Figure 1A). These genera were abundant in the whole population which contained 224 Pseudomonas spp (23%) and 71 Aeromonas spp. isolates (7%). The frequent presence of FOX genes in Aeromonas spp. and Pseudomonas spp. cannot be explained by an abundance of these genera in our strain collection, as we did not find proportionally as many ampc genes in isolates from other abundant genera, e.g. Stenotrophomonas (n = 87, 9%) and none from Chryseobacterium (n=95, 10%). We conclude that ampc FOX genes could be

3 found mostly from Aeromonas and Pseudomonas spp. culturable on media used (10) and isolated from the aquatic environment. We compared the FOX genes we had detected with previously described FOX gene variants (n = 10) from GenBank: accession numbers CP000644, AF462690, AJ277535, AJ703795, AJ703796, AY007369, AY034848, DQ478715, HM565917, JF896803; and (n=3) from EMBL- EBI: accession numbers X77455, Y10282, and Y Amplification products from multiplex PCR were purified, re-amplified and sequenced. All sequences were aligned and clustering of gene variants was obtained by analysis with Bayesian Inference of Phylogeny (MrBayes; Figure 1B). FOX genes from aquatic isolates were clustered into 4 groups (at intra-group similarity of 100%), which were phylogenetically in a separate node from previously characterized, mostly enterobacterial, FOX genes. The least similarity between the environmental FOX (or chromosomal putative beta-lactam in Aeromonas punctata AF462690, (3)) sequences and sequences from the databases was 87%. To see how environmental FOX variants are shared between various isolates, we mapped the 4 variants onto Figure 1A, from which we can see that one of the variants (VAR1) predominates. Aeromonas spp. had all the 4 variants, Pseudomonas spp. having only 2 variants. Other genera had too few bacterial isolates to reach any conclusion about the representation or distribution of the FOX variants. Since several FOX gene variants were found in AR isolates collected from a geographically small region of water bodies, the relationship between population structure and distribution of FOX gene variants was studied. We chose Aeromonas isolates because those that were FOX-positive were most numerous, and all 4 FOX variants were present. Multilocus sequence typing (MLST) was used to assess the population structure, using 6 housekeeping genes: gyrb, grol, glta, metg, ppsa, and reca (9). All the Aeromonas isolates were used in the analysis (n=71). Published MLST primers did not work perfectly with our environmental isolates, e.g. gyrb did not give a positive amplicon for 55 Aeromonas isolates (78%). The other 5 primer pairs worked more frequently, but there was no housekeeping gene for which the primers would have worked for all the isolates of the Aeromonas spp. according to the 16S rdna. All the isolates were unique, i.e. had novel sequence types bearing at least one nucleotide difference compared to the Aeromonas pubmlst database. Therefore, 3 genes were used for the population structure analysis (glta, metg, and reca, Genbank accession numbers JX JX899520, JX JX899645, and JX JX899477, respectively), with the highest number of sequences obtained (n = 19 of FOX containing isolates and n = 23 for isolates without FOX genes). Concatenated sequences of glta, metg and reca were aligned using ClustalW, and a maximum parsimony tree was constructed (Figure 2). Most of the FOX-positive isolates clustered together, separately form

4 the FOX-negative isolates. There was no association between the FOX gene variant and the location of origin of the isolate (chi-square test, p>0.05). However, the distribution of FOX gene variants was related to sampling season (p=0.02). Isolates carrying a FOX gene group together, as mentioned above, but separately from noncarriers (Figure 2). Thus we conclude that there has been no horizontal transfer of the FOX genes in recent evolutionary times since the clonally more similar Aeromonas isolates carry a specific FOX gene variant. In conclusion, ampc FOX genes from several environmental species have not previously been described. Four variants of FOX genes were detected, one variant predominating. The most numerous species bearing the FOX gene were of the Aeromonas genus, with 30 FOXpositive isolates from the total of 48. Thus the ampc gene might be more widely dispersed than previously suspected, including normal environmental bacteria. Therefore, gene exchange between medical settings and the natural environment might be a potential source of novel resistance genes in clinical isolates, although our study did not suggest such exchange as having happened recently because the environmental FOX gene variants differed from the FOX genes from medical strains, suggesting a separate evolution in the natural environment. Acknowledgements This work was supported by the European Regional Development Fund through the Center of Excellence in Chemical Biology. We appreciate valuable suggestions of 2 anonymous reviewers on an earlier version of this manuscript.

5 Figure legends Figure 1. (A) Maximum parsimony tree for the FOX-positive isolates based on 16S rdna sequences, bootstrap values of 100 replicates are shown (Genbank Accession numbers: JX JX899645) and (B) MrBayes phylogenetic tree of the FOX gene sequences from isolates from the study population and database sequences. The 4 FOX gene variants are also mapped on the 16S rdna phylogenetic tree in panel (A). Figure 2. Maximum parsimony tree for Aeromonas spp. from the study population, based on 3 concatenated gene sequences (glta-metg-reca), bootstrap values of 100 replicates are shown. Concatenated genes of glta (gi : ), metg (gi :c ), and reca (gi : ) sequences from Pseudomonas aeruginosa was used as root. Sampling time and the FOX variants identified by phylogenetic analysis (Figure 1B) are indicated. Downloaded from on November 21, 2018 by guest

6 Downloaded from on November 21, 2018 by guest

7

8 References 1. Alekshun MN, Levy SB Molecular mechanisms of antibacterial multidrug resistance. Cell 128: Bradford PA Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical microbiology reviews 14:933 51, table of contents. 3. Fosse T, Giraud-Morin C, Madinier I, Labia R Sequence analysis and biochemical characterisation of chromosomal CAV-1 ( Aeromonas caviae ), the parental cephalosporinase of plasmid-mediated AmpC FOX cluster. FEMS Microbiology Letters 222: Hawkey PM, Jones AM The changing epidemiology of resistance. The Journal of antimicrobial chemotherapy 64 Suppl 1:i Jacoby GA, Munoz-price LS The New beta-lactamases. Europe Jolley KA, Maiden MCJ BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC bioinformatics 11: Lane D S/23S rrna sequencing, p In Stackebrandt, E, Goodfellow, M (eds.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons. 8. Martino ME, Fasolato L, Montemurro F, Rosteghin M, Manfrin A, Patarnello T, Novelli E, Cardazzo B Determination of Microbial Diversity of Aeromonas Strains on the Basis of Multilocus Sequence Typing, Phenotype, and Presence of Putative Virulence Genes. Appl. Environ. Microbiol. 77: Pérez-Pérez FJ, Hanson ND Detection of plasmid-mediated AmpC betalactamase genes in clinical isolates by using multiplex PCR. Journal of clinical microbiology 40: Voolaid V, Jõers A, Kisand V, Tenson T Co-occurrence of resistance to different antibiotics among aquatic bacteria. BMC microbiology 12: Wang Q, Garrity GM, Tiedje JM, Cole JR Naive Bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy. Applied and environmental microbiology 73: van Hoek AHAM, Mevius D, Guerra B, Mullany P, Roberts AP, Aarts HJM Acquired Antibiotic Resistance Genes: An Overview. Frontiers in Microbiology 2:203.