A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and β-lactam resistance genes in the gut microbiota
© Fouhy et al.; licensee BioMed Central Ltd. 2014
Received: 12 August 2013
Accepted: 3 February 2014
Published: 5 February 2014
The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes.
The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including bla OXA, bla TEM, bla SHV and bla CTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes.
This study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.
KeywordsAntibiotic resistance Aminoglycosides β-lactam Gut microbiota PCR
Almost as soon as the widespread therapeutic use of antibiotics occurred, bacteria displaying diverse and complex mechanisms of resistance became problematic [1, 2]. As the human gut is one of the most densely populated microbial environments, it has been postulated that it can act as a considerable reservoir for antibiotic resistance genes . Thus, gut microbes may disseminate antibiotic resistance genes to other commensals or to bacteria transiently colonising the gut . Given that antibiotics are known to exert significant and sustained negative effects on the gut microbiota [5, 6], possessing resistance genes can provide a significant selective advantage to a subpopulation of microorganisms in individuals undergoing antibiotic treatment . The aminoglycosides and β-lactams are two large families of antibiotics which are frequently employed in clinical settings. The aminoglycosides, which were first characterised in 1944,  function by binding to the 30S subunit of the prokaryotic ribosome resulting in disruption to protein synthesis. Resistance to aminoglycosides can be through reduced aminoglycoside uptake or enzymatic modification of the aminoglycoside through acetylation (AAC), adenylation (ANT) or phosphorylation (APH). β-lactam antibiotics include the penicillins and cephalosporins and inhibit bacteria through disruption of cell wall biosynthesis [9, 10]. Resistance to β-lactams can be due to alterations to penicillin binding proteins or to the porins in the outer membrane (in Gram negative targets) or alternatively through the production of β-lactamases, which hydrolyse the eponymous β-lactam ring rendering the antibiotic inactive [11, 12].
The question of the evolutionary origin of antibiotic resistance genes has been the subject of much attention [9, 13, 14]. For quite some time it was thought that resistance evolved following exposure of bacteria to new antibiotics . However, it is now apparent that repositories of antibiotic resistance genes exist such that, following the development and application of new antibiotics, bacteria possessing or acquiring such genes will gain a selective advantage and thus resistance will increase over time [16, 17]. Previous studies have employed PCR to detect resistance genes in specific pathogens [18, 19], though studies employing PCR to detect resistance genes in complex microbial environments have been limited. In one instance, a PCR-based approach was used to investigate the prevalence of gentamycin resistance genes in resistant isolates from sewage, faeces (from cattle and chickens), municipal and hospital sewage water and coastal water . The utilisation of a PCR approach in that instance resulted in the identification of diverse genes encoding gentamycin modifying enzymes from across a broad host range, thus demonstrating the suitability of a PCR-based approach to investigate resistance genes present in complex environments. However, the study did not investigate antibiotic resistance genes in human gut microbiota and, to our knowledge, to date no such PCR-based studies exist. Given these findings and other indications that there exist large natural pools of antibiotic resistance genes within complex microbial populations, it is likely that the human gut also contains many such genes. However, until now, PCR-based strategies to detect antibiotic resistance genes in the gut microbiota have involved an initial culture-based screen for resistant isolates, followed by subsequent PCR-based approaches to identify the associated resistance genes. This does not take into consideration the fact that the vast majority of gut microbes are not easily cultured , and thus antibiotic resistance genes from such microorganisms would typically be overlooked.
Here we utilise degenerate PCR primers to investigate the presence of β-lactam resistance genes and each of the three categories of aminoglycoside modifying enzymes within human metagenomic DNA and in doing so demonstrate that the human gut microbiota is a reservoir for antibiotic resistance genes. Additionally, we establish that a PCR-based approach allows the rapid detection of such genes in the complex gut microbiota environment, without the need for an initial isolation of strains.
Recruitment of volunteers
Forty adults were recruited and each provided written, informed consent for participation in this study. Approval for this trial was received from the Clinical Research Ethics Committee of the Cork Teaching Hospitals, Cork, Ireland. Volunteers were aged 28.8 ± 3.8 years, were free from gastrointestinal disorders and had not been treated with antibiotics in the 6 months prior to sample collection. Fresh faecal samples were collected and stored at −80°C until processed.
Stool samples were weighed, homogenised and due to the total volume provided by each individual, samples had to be pooled to achieve the required volume for our metagenomic DNA extraction protocol. To facilitate this, an equal volume (250 mg) from each individual was taken and pooled to form one sample, from which metagenomic DNA was extracted. The DNA extraction procedure used was optimised for total bacterial genomic DNA extraction from stool samples. The stool sample was homogenized in PBS and centrifuged at 1000 g × 5 mins and the supernatant was removed and retained. This was repeated 3 times. The supernatant then underwent Nycodenz (Axis Shield, UK) density gradient centrifugation separation, to separate out the bacterial cells from faecal matter. Following enzymatic lysis of bacterial cells using lysozyme and mutanolysin (Sigma Aldrich, Dublin, Ireland) protein precipitation using Proteinase K and ammonium acetate (Sigma Aldrich) was completed. Bacterial DNA was then precipitated and washed using standard chloroform and ethanol procedures. DNA was eluted in TE buffer.
PCR-based detection of β-lactam resistance gene homologues
Primers used for the detection of β-lactamase and aminoglycoside resistant genes
Amplicon Size (bp)
Bailey et al. (2011) 
Tenover et al. (1994) 
Tenover et al. (1994) 
Briñas et al. (2002) 
Briñas et al. (2002) 
Monstein et al. (2009) 
AG resistant genes
Heuer et al. (2002) 
aac (6′)-Ie-aph (2″)-Ia
De Fatίma Silva Lopes et al. (2003) 
Schmitz et al. (1999) 
PCR-based detection of aminoglycoside resistance gene homologues
For the detection of aminoglycoside resistant genes, degenerate primer sets were used which had previously been designed and shown to amplify all known genes encoding gentamycin-modifying enzymes and similar, but as yet undiscovered, sequences . PCRs were completed using primer sets (MWG Eurofins, Germany) for genes belonging to each group of aminoglycoside modifying enzymes namely, acetylation, adenylation and phosphorylation enzymes. DNA from positive controls (kindly gifted to us from the Smalla laboratory, JKI, Braunschweig) namely Escherichia coli S17-1 pAB2002 (aac (3)-Ia), Pseudomonas aeruginosa 88.341 F (aac (3)-Ib), Enterobacter aerogenes 17798 VDK (aac (3)-IIa), E. coli DH5α pSCH4203 (aac (3)-IIb), E. coli DH5α pSCH4101 (aac (3)-VIa), P. aeruginosa PST-1 (aac (3)-IIIa), Acinetobacter baumannii LBL.3 (aac (6′)-Ib), P. aeruginosa F-03 (aac (6′)-IIa), E. coli DH5α pSCH5102 (aac (6′)-IIb), E. coli CV600 pIE723 (ant (2″)-I), E. coli DH5α pAM6306 (aph (2″)-Ic) and E. coli NC95 (aph (2″)-Id) were used as positive controls for the PCR reactions. This ensured the specificity of the respective primer pairs. PCRs for the detection of acetylation genes aac (3)-I, aac (3)-II, aac (3)-III, aac (3)-VI and aac (6), adenylation genes ant (2″)-Ia and phosphorylation genes aph (2″)-Ic and aph (2″)-Id were completed as previously outlined  (Table 1). Additionally, PCRs using primers for the bifunctional gene aac (6″)-Ie-aph (2″) [26, 27] (which encodes enzymes responsible for high level gentamycin resistance, as well as concomitant resistance to tobramycin and kanamycin) [27–31] were completed as follows: heated lid 110°C, 94°C × 5 mins followed by 30 cycles of 94°C × 30s, 47°C × 30s, 72°C × 30s, with a final extension step of 72°C × 10 mins and held at 4°C. All PCRs contained 25 μl Biomix Red (Bioline, UK), 1 μl forward primer (10pmol concentration), 1 μl reverse primer (10pmol concentration), metagenomic DNA (64 ng) and PCR grade water (Bioline, UK), to a final volume of 50 μl. Negative controls were run for all primer sets. All PCRs were performed in triplicate and analysed using gel electrophoresis, as described above.
Cloning of PCR amplicons
Triplicate samples from successful PCR reactions were pooled and cleaned using AMPure magnetic bead-based PCR clean up kit (Beckman Coulter, UK). TOPO cloning reactions were performed on purified PCR products using the TOPO TA cloning kit (Invitrogen, Dublin, Ireland) to facilitate the sequencing of individual gene fragments. TOPO cloning reactions were then cloned into TOP10 E. coli (Invitrogen) as per the manufacturer’s instructions and plated onto LB (Difco) containing the appropriate antibiotic (either ampicillin 50 μg/ml or kanamycin 50 μg/ml; Sigma Aldrich, Dublin, Ireland) to select for the presence of the cloning vector. Transformants were selected from each TOPO cloning reaction and grown overnight in LB broth containing the suitable selective antibiotic (either ampicillin 50 μg/ml or kanamycin 50 μg/ml). Plasmids were extracted from overnight samples using QIAprep Spin Mini Prep kit (Qiagen, Sussex, UK) according to the manufacturer’s instructions and sent for Sanger sequencing (Source BioSciences, Dublin, Ireland).
Following Sanger sequencing, sequence reads were analysed using the NCBI protein database (BlastX; (http://blast.ncbi.nlm.nih.gov/)). In the event where multiple hits occurred, the BLAST hit which displayed greatest homology is reported.
Results and discussion
A PCR-based approach highlights the presence of β-lactamase gene homologues in the gut microbiota
Homologues of β-lactamase genes detected in the human gut microbiota via PCR techniques
S. enterica subsp. enterica
ES β-lactamase TEM-116
Uncultured soil bacterium
Sar 86 cluster bacterium
KPC-2 β lactamase
Hypothetical protein SH0313
Hypothetical protein HMPREF 9332
K. pneumoniae subsp. pneumoniae
Hypothetical protein HMPREF 1077
ES β-lactamase SHV120
ES β lactamase CTX-M-15
β-lactamase penicillin hydrolase
Using the bla SHV primers, multiple genes sharing homology with genes from members of the Enterobacteriaceae, and Klebsiella and E. coli in particular were detected. In addition, amplicons with low percentage identity to genes from Alloprevotella rava and Parabacteroides johnsonii, respectively, were also identified. This is again consistent with existing research which states that Enterobacteriaceae are the primary source of bla SHV genes [39–43]. Furthermore, the amplicons sequenced resembled various different types of ESBL-encoding SHV genes, including bla SHV-132, bla SHV-140 and bla SHV-48, thus again highlighting the genuine degeneracy of the primers used.
Additional PCRs were completed to identify other ESBLs, specifically CTX-M- and OXA-type β-lactamases (Table 2). A number of different CTX-M β-lactamases were detected, including CTX-M-1, CTX-M-15 and CTX-M-36. The fact that many of the β-lactamase genes detected using our approach share homology with resistance genes found in members of the phylum Proteobacteria is not surprising as, despite being typically less common than the Bacteroidetes or Firmicutes in the gut microbiota of healthy adults , members of this genus have been identified as sources of antibiotic resistance genes and have been frequently associated with nosocomial infections and outbreaks [36, 39, 44, 45]. In the 1990s, TEM- and SHV-type ESBLs were the β-lactamases most frequently observed among Enterobacteriaceae. However, more recently, CTX-M-type ESBLs have spread rapidly and are now the most prevalent ESBL in Enterobacteriaceae in several parts of the world . In a recent report on antibiotic resistance threats in the USA, the Centre for Disease Control stated that ESBL-producing Enterobacteriaceae were a serious public health threat . The report estimates that 26,000 infections and 1,700 deaths that occur each year in the United States are attributable to ESBLs and that upwards of 140,000 health-care related Enterobacteriaceae infections occur annually. Therefore the detection of homologues of ESBL-encoding genes in the gut microbiota of healthy individuals is significant and provides evidence of the ubiquitous nature of these resistance genes, even in the absence of recent antibiotic exposure. With respect to the CTX-M-type ESBLs, it is particularly notable that homologues of the bla CTX-M-15 gene were detected, as these have received significant attention due to their recent rapid spread and their association with multi-drug resistant E. coli responsible for outbreaks of antibiotic resistant infections [48, 49]. In such cases, these genes have been found on multi-drug resistance-encoding regions of plasmids, thus facilitating the rapid transfer of these genes. The presence of such genes within the gut microbiota raises concerns that horizontal gene transfer may occur between commensals or to bacteria passing through the gut. If the resistance genes detected in our study are, or were to become, mobile, it would enable the gut to act not only as a source of resistance genes, but also as a site of resistance gene transfer. Although outside the scope of this study, studies investigating whether these genes are located on or near mobile genetic elements would be pertinent to ascertain the risk of the gut acting as a site for horizontal gene transfer.
When the bla ROB primer set was employed to detect the presence of homologues of these ampicillin resistance-encoding genes, all amplicons sequenced were identical and shared 44% identity to Staphylococcus haemolyticus bla ROB gene. Finally, this study did not detect bla OXA gene homologues in our metagenomic sample. These findings are unexpected and may have occurred as a result of the particular affinity of the primer sets used.
A PCR-based approach highlights the presence of aminoglycoside resistance encoding gene homologues in the gut microbiota
Homologues of aminoglycoside resistance genes detected in the human gut microbiota via PCR techniques
Hypothetical protein Colaer00186
aac (6′)- Ie-aph (2″)
Bifunctional AAC/APH partial sequence
Bifunctional AG modifying enzyme
6′ AG acetyltransferase
Bifunctional AG 6′-N acetytransferase/2′-AG phosphotransferases
Gentamycin resistance protein
aph (2″) Id
Chain A crystal structure AG 2′ phosphotransferases
Aph2″ chain a
AG modifying enzyme
ant (2″) Ia
2′ AG nucleotidlytransferase
S. enterica subsp enterica
Homologues of aminoglycoside phosphorylation-encoding genes were also detected using a PCR-based approach, with both aph (2″)-Ic and aph (2″)-Id like genes being detected. These genes shared homology with genes from Enterococcus species, including E. faecium and E. casseliflavus. Aminoglycoside resistant E. faecium have received significant attention due to their role in nosocomial infections [58, 59]. Notably, the role of mobile genetic elements in the maintenance and dissemination of multi-drug resistance in Enterococcus faecalis and E. faecium has previously been highlighted [30, 60, 61]. While it is not certain that the genes identified in this study are also associated with mobile elements, the possibility that resistance genes could be transferred to commensals is a concern. Homologues of aminoglycoside adenylation genes, ant (2″)-Ia, were also successfully detected. These resembled genes from Pasteurella, Acinetobacter and E. coli (Table 3), and the findings are thus consistent with previous research showing that these genes are most frequently detected in Gram negative bacteria . Overall, the results demonstrate that the gut microbiota is a source of diverse aminoglycoside and β-lactam resistance genes, despite having had no recent antibiotic exposure. If these genes are expressed there is the potential that if antibiotic exposure occurred, bacteria containing these resistance genes would become the dominant component of the gut microbiota, as has been shown in previous studies [5, 63].
This study has highlighted the merits of applying a PCR-based approach to detect antibiotic resistance genes within the human gut microbiome. The results clearly demonstrate that the human gut microbiota is a considerable reservoir for resistance genes. Further studies are required to determine the exact sources of these genes and to determine if they have the potential to become mobile. Additionally, we have highlighted the successful application of a PCR-based screen of a complex environment without prior isolation of resistant isolates. The possibility exists to couple this approach with lower throughput next generation sequencing strategies, such as that provided by the Ion PGM 314 chip, in instances where great diversity is likely. Our approach could also be used in conjunction with functional screening of metagenomic libraries to enable the detection of genes present in a complex environment at a low threshold and that may have avoided capture in the metagenomic library, as shown in a recent study . Such a PCR-based approach is not being proposed as a substitute for ultra-deep high-throughput shotgun sequencing of metagenomic DNA, rather it is a lower cost, more targeted, alternative which facilitates the detection and in silico analysis of specific gene sets of interest. Finally, while this study demonstrates that the gut microbiota is a source of diverse resistance genes, further studies are required to investigate the exact sources of these genes, their expression and whether they have the potential to become mobile. As the scientific community continues to gain knowledge with respect to the genetic mechanisms involved in providing resistance to various antibiotics, the design of additional sets of degenerate primers will be possible and will provide further opportunities for the use of PCR to rapidly and efficiently detect antibiotic resistance genes in complex microbial environments, including the human gut microbiota.
Availability of supporting data
The data sets supporting results of this article are available in the LabArchives repository, [http://dx.doi.org/10.6070/H42V2D1V].
Extended spectrum β-lactamase
The authors wish to acknowledge the advice, assistance and protocols received from Dr. Brian Jones and Dr. Lesley Ogilvie regarding metagenomic sample preparation and analysis. Additionally the authors acknowledge the gift of control bacteria strains from the Smalla laboratory, JKI, Braunschweig. Fiona Fouhy is in receipt of an Irish Research Council EMBARK scholarship and is a Teagasc Walsh fellow. Research in the PDC laboratory is also supported by the Irish Government under the National Development Plan through the Science Foundation Ireland Investigator award 11/PI/1137.
- Davies J, Davies D: Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev. 2010, 74: 417-433. 10.1128/MMBR.00016-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Abraham E, Chain E: An enzyme from bacteria able to destroy penicillin. Nature. 1940, 146: 837-837.View ArticleGoogle Scholar
- Salyers AA, Gupta A, Wang Y: Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol. 2004, 12: 412-416. 10.1016/j.tim.2004.07.004.View ArticlePubMedGoogle Scholar
- Broaders E, Gahan CG, Marchesi JR: Mobile genetic elements of the human gastrointestinal tract: potential for spread of antibiotic resistance genes. Gut microbes. 2013, 4: 271-280. 10.4161/gmic.24627.PubMed CentralView ArticlePubMedGoogle Scholar
- Dethlefsen L, Huse S, Sogin ML, Relman DA: The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol. 2008, 6: e280-10.1371/journal.pbio.0060280. 210.137/journal.pbio.0060280PubMed CentralView ArticlePubMedGoogle Scholar
- Cotter P, Stanton C, Ross R, Hill C: The impact of antibiotics on the gut microbiota as revealed by high throughput DNA sequencing. Discov Med. 2012, 13: 193-199.PubMedGoogle Scholar
- Sommer MOA, Dantas G, Church GM: Functional characterization of the antibiotic resistance reservoir in the human microflora. Sci. 2009, 325: 1128-1131. 10.1126/science.1176950.View ArticleGoogle Scholar
- Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM: Aminoglycosides: activity and resistance. Antimicrob Agents Chemother. 1999, 43: 727-737.PubMed CentralPubMedGoogle Scholar
- Page MGP: Beta-Lactam Antibiotics. Antibiot Discov Dev. 2012, 1: 79-117.View ArticleGoogle Scholar
- Tipper DJ, Strominger JL: Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine. Proc Natl Acad Sci U S A. 1965, 54: 1133-1141. 10.1073/pnas.54.4.1133.PubMed CentralView ArticlePubMedGoogle Scholar
- Bush K: Characterization of beta-lactamases. Antimicrob Agents Chemother. 1989, 33: 259-263. 10.1128/AAC.33.3.259.PubMed CentralView ArticlePubMedGoogle Scholar
- Bush K: Alarming beta-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr Opin Microbiol. 2010, 13: 558-564. 10.1016/j.mib.2010.09.006.View ArticlePubMedGoogle Scholar
- Kotra LP, Mobashery S: β-Lactam antibiotics, β-lactamases and bacterial resistance. Bull Inst Pasteur. 1998, 96: 139-150. 10.1016/S0020-2452(98)80009-2.View ArticleGoogle Scholar
- Tipper D: Mode of action of β-lactam antibiotics. Rev Infect Dis. 1979, 1: 39-53. 10.1093/clinids/1.1.39.View ArticlePubMedGoogle Scholar
- Hughes VM, Datta N: Conjugative plasmids in bacteria of the ‘pre-antibiotic’ era. Nature. 1983, 302: 725-726. 10.1038/302725a0.View ArticlePubMedGoogle Scholar
- Bhullar K, Waglechner N, Pawlowski A, Koteva K, Banks ED, Johnston MD, Barton HA, Wright GD: Antibiotic resistance is prevalent in an isolated cave microbiome. PLoS ONE. 2012, 7: e34953-10.1371/journal.pone.0034953.PubMed CentralView ArticlePubMedGoogle Scholar
- D’Costa VM, King CE, Kalan L, Morar M, Sung WWL, Schwarz C, Froese D, Zazula G, Calmels F, Debruyne R: Antibiotic resistance is ancient. Nature. 2011, 477: 457-461. 10.1038/nature10388.View ArticlePubMedGoogle Scholar
- Dallenne C, Da Costa A, Decré D, Favier C, Arlet G: Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010, 65: 490-495. 10.1093/jac/dkp498.View ArticlePubMedGoogle Scholar
- Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala J-L: Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol. 1995, 33: 2864-2867.PubMed CentralPubMedGoogle Scholar
- Heuer H, Krögerrecklenfort E, Wellington E, Egan S, Elsas J, Overbeek L, Collard JM, Guillaume G, Karagouni A, Nikolakopoulou T: Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Immunol Med Microbiol. 2002, 42: 289-302.Google Scholar
- Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA: Diversity of the human intestinal microbial flora. Science. 2005, 308: 1635-1638. 10.1126/science.1110591.PubMed CentralView ArticlePubMedGoogle Scholar
- Bailey JK, Pinyon JL, Anantham S, Hall RM: Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli. J Antimicrob Chemother. 2011, 66: 745-751. 10.1093/jac/dkq529.View ArticlePubMedGoogle Scholar
- Tenover FC, Huang MB, Rasheed JK, Persing DH: Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae. Eur J Clin Microbiol. 1994, 32: 2729-2737.Google Scholar
- Briñas L, Zarazaga M, Sáenz Y, Ruiz-Larrea F, Torres C: β-Lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob Agents Chemother. 2002, 46: 3156-3163. 10.1128/AAC.46.10.3156-3163.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Monstein H-J, Tärnberg M, Nilsson LE: Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons. BMC Infect Dis. 2009, 9: 7-15. 10.1186/1471-2334-9-7.PubMed CentralView ArticlePubMedGoogle Scholar
- De Fátima Silva Lopes M, Ribeiro T, Abrantes M, Figueiredo Marques JJ, Tenreiro R, Crespo MTB: Antimicrobial resistance profiles of dairy and clinical isolates and type strains of enterococci. Int J Food Microbiol. 2005, 103: 191-198. 10.1016/j.ijfoodmicro.2004.12.025.View ArticlePubMedGoogle Scholar
- Schmitz F-J, Fluit AC, Gondolf M, Beyrau R, Lindenlauf E, Verhoef J, Heinz H-P, Jones ME: The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals. J Antimicrob Chemother. 1999, 43: 253-259. 10.1093/jac/43.2.253.View ArticlePubMedGoogle Scholar
- Matsumura M, Katakura Y, Imanaka T, Aiba S: Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110. J bacteriol. 1984, 160: 413-420.PubMed CentralPubMedGoogle Scholar
- Ubukata K, Yamashita N, Gotoh A, Konno M: Purification and characterization of aminoglycoside-modifying enzymes from Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother. 1984, 25: 754-759. 10.1128/AAC.25.6.754.PubMed CentralView ArticlePubMedGoogle Scholar
- Hegstad K, Mikalsen T, Coque T, Werner G, Sundsfjord A: Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium. Clin Microbiol Infect. 2010, 16: 541-554. 10.1111/j.1469-0691.2010.03226.x.View ArticlePubMedGoogle Scholar
- Ferretti JJ, Gilmore K, Courvalin P: Nucleotide sequence analysis of the gene specifying the bifunctional 6′-aminoglycoside acetyltransferase 2″-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. J bacteriol. 1986, 167: 631-638.PubMed CentralPubMedGoogle Scholar
- Fouhy F, Ross RP, Fitzgerald GF, Stanton C, Cotter PD: PCR sequencing data of aminoglycoside and beta-lactam resistance genes. BMC microbiology. 2013, http://dx.doi.org/10.6070/H42V2D1V; 2013Google Scholar
- Morris D, Whelan M, Corbett-Feeney G, Cormican M, Hawkey P, Li X, Doran G: First Report of Extended-Spectrum-β-Lactamase-Producing Salmonella enterica Isolates in Ireland. Antimicrob Agents Chemother. 2006, 50: 1608-1609. 10.1128/AAC.50.4.1608-1609.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Perilli M, Felici A, Franceschini N, De Santis A, Pagani L, Luzzaro F, Oratore A, Rossolini GM, Knox JR, Amicosante G: Characterization of a new TEM-derived beta-lactamase produced in a Serratia marcescens strain. Antimicrob Agents Chemother. 1997, 41: 2374-2382.PubMed CentralPubMedGoogle Scholar
- Zhao W-H, Hu Z-Q, Chen G, Matsushita K, Fukuchi K, Shimamura T: Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type beta-lactamases encoded on a single plasmid. Microbiol Res. 2007, 162: 46-52. 10.1016/j.micres.2006.06.005.View ArticlePubMedGoogle Scholar
- Morosini MI, Canton R, Martinez-Beltran J, Negri MC, Perez-Diaz JC, Baquero F, Blazquez J: New extended-spectrum TEM-type beta-lactamase from Salmonella enterica subsp. enterica isolated in a nosocomial outbreak. Antimicrob Agents Chemother. 1995, 39: 458-461. 10.1128/AAC.39.2.458.PubMed CentralView ArticlePubMedGoogle Scholar
- Wong MHY, Liu M, Wan HY, Chen S: Characterization of Extended-Spectrum-β-Lactamase-Producing Vibrio parahaemolyticus. Antimicrob Agents Chemother. 2012, 56: 4026-4028. 10.1128/AAC.00385-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Mokracka J, Koczura R, Kaznowski A: Multiresistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant. Water Res. 2012, 46: 3353-3363. 10.1016/j.watres.2012.03.037.View ArticlePubMedGoogle Scholar
- Coque TM, Oliver A, Pérez-Díaz JC, Baquero F, Cantón R: Genes Encoding TEM-4, SHV-2, and CTX-M-10 Extended-Spectrum β-Lactamases Are Carried by Multiple Klebsiella pneumoniae Clones in a Single Hospital (Madrid, 1989 to 2000). Antimicrob Agents Chemother. 2002, 46: 500-510. 10.1128/AAC.46.2.500-510.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Paterson DL, Hujer KM, Hujer AM, Yeiser B, Bonomo MD, Rice LB, Bonomo RA: Extended-spectrum β-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV-and CTX-M-type β-lactamases. Antimicrob Agents Chemother. 2003, 47: 3554-3560. 10.1128/AAC.47.11.3554-3560.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Heritage J, M’Zali FH, Gascoyne-Binzi D, Hawkey PM: Evolution and spread of SHV extended-spectrum β-lactamases in Gram-negative bacteria. Journal of antimicrobial chemotherapy. 1999, 44: 309-318. 10.1093/jac/44.3.309.View ArticlePubMedGoogle Scholar
- Babini GS, Livermore DM: Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997–1998. J Antimicrob Chemother. 2000, 45: 183-189. 10.1093/jac/45.2.183.View ArticlePubMedGoogle Scholar
- Pitout J, Sanders C, Sanders W: Antimicrobial resistance with focus on beta-lactam resistance in gram-negative bacilli. Am J Med. 1997, 103: 51-59. 10.1016/S0002-9343(97)00044-2.View ArticlePubMedGoogle Scholar
- Bonnet R: Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004, 48: 1-14. 10.1128/AAC.48.1.1-14.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Pitout JDD, Laupland KB: Extended-spectrum [beta]-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis. 2008, 8: 159-166. 10.1016/S1473-3099(08)70041-0.View ArticlePubMedGoogle Scholar
- Coque T, Baquero F, Canton R: Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveillance. 2008, 13: 19-29.Google Scholar
- CDC: Antibiotic resistance threats in the United States. 2013, Retrieved from http://www.cdc.gov/drugresistance/threat-report-2013/ Google Scholar
- Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey BM, Bryce E, Gardam M, Nordmann P, Mulvey MR: Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother. 2004, 48: 3758-3764. 10.1128/AAC.48.10.3758-3764.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Lavollay M, Mamlouk K, Frank T, Akpabie A, Burghoffer B, Redjeb SB, Bercion R, Gautier V, Arlet G: Clonal dissemination of a CTX-M-15 β-lactamase-producing Escherichia coli strain in the Paris area, Tunis, and Bangui. Antimicrob Agents Chemother. 2006, 50: 2433-2438. 10.1128/AAC.00150-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Cho YJ, Moon DC, Jin JS, Choi CH, Lee YC, Lee JC: Genetic basis of resistance to aminoglycosides in Acinetobacter spp. and spread of armA in Acinetobacter baumannii sequence group 1 in Korean hospitals. Diagn Microbiol Infect Dis. 2009, 64: 185-190. 10.1016/j.diagmicrobio.2009.02.010.View ArticlePubMedGoogle Scholar
- Lambert T, Gerbaud G, Courvalin P: Characterization of the chromosomal aac (6′)-Ij gene of Acinetobacter sp. 13 and the aac (6′)-Ih plasmid gene of Acinetobacter baumannii. Antimicrob Agents Chemother. 1994, 38: 1883-1889. 10.1128/AAC.38.9.1883.PubMed CentralView ArticlePubMedGoogle Scholar
- Shaw K, Cramer C, Rizzo M, Mierzwa R, Gewain K, Miller G, Hare R: Isolation, characterization, and DNA sequence analysis of an AAC (6′)-II gene from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1989, 33: 2052-2062. 10.1128/AAC.33.12.2052.PubMed CentralView ArticlePubMedGoogle Scholar
- Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC: Prevalence in the United States of aac (6′)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother. 2006, 50: 3953-3955. 10.1128/AAC.00915-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Dijkshoorn L, Nemec A, Seifert H: An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol. 2007, 5: 939-951. 10.1038/nrmicro1789.View ArticlePubMedGoogle Scholar
- Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA: Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother. 2007, 51: 3471-3484. 10.1128/AAC.01464-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Vakulenko SB, Donabedian SM, Voskresenskiy AM, Zervos MJ, Lerner SA, Chow JW: Multiplex PCR for detection of aminoglycoside resistance genes in enterococci. Antimicrob Agents Chemother. 2003, 47: 1423-1426. 10.1128/AAC.47.4.1423-1426.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Vanhoof R, Godard C, Content J, Nyssen H, Hannecart-Pokorni E: Detection by polymerase chain reaction of genes encoding aminoglycoside-modifying enzymes in methicillin-resistant Staphylococcus aureus isolates of epidemic phage types. J Med Microbiol. 1994, 41: 282-290. 10.1099/00222615-41-4-282.View ArticlePubMedGoogle Scholar
- Han D, Unno T, Jang J, Lim K, Lee S-N, Ko G, Sadowsky MJ, Hur H-G: The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea. Int J Food Microbiol. 2011, 144: 387-392. 10.1016/j.ijfoodmicro.2010.10.024.View ArticlePubMedGoogle Scholar
- Montecalvo MA, Horowitz H, Gedris C, Carbonaro C, Tenover FC, Issah A, Cook P, Wormser GP: Outbreak of vancomycin-, ampicillin-, and aminoglycoside-resistant Enterococcus faecium bacteremia in an adult oncology unit. Antimicrob Agents Chemother. 1994, 38: 1363-1367. 10.1128/AAC.38.6.1363.PubMed CentralView ArticlePubMedGoogle Scholar
- Leclercq R: Enterococci acquire new kinds of resistance. Clin Infect Dis. 1997, 24: S80-S84. 10.1093/clinids/24.Supplement_1.S80.View ArticlePubMedGoogle Scholar
- McKay G, Thompson P, Wright G: Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity. Biochemistry. 1994, 33: 6936-6944. 10.1021/bi00188a024.View ArticlePubMedGoogle Scholar
- Shaw K, Rather P, Hare R, Miller G: Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev. 1993, 57: 138-163.PubMed CentralPubMedGoogle Scholar
- Fouhy F, Guinane CM, Hussey S, Wall R, Ryan CA, Dempsey EM, Murphy B, Ross RP, Fitzgerald GF, Stanton C: High-throughput sequencing reveals the incomplete, short-term, recovery of the infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamycin. Antimicrob Agents Chemother. 2012, 56: 5811-5820. 10.1128/AAC.00789-12.PubMed CentralView ArticlePubMedGoogle Scholar
- de Vries LE, Vallès Y, Agersø Y, Vaishampayan PA, García-Montaner A, Kuehl JV, Christensen H, Barlow M, Francino MP: The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant. PLoS ONE. 2011, 6: e21644-10.1371/journal.pone.0021644.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.