Abstract
Resistance to minocycline has emerged in multidrug-resistant Acinetobacter baumannii isolates from Buenos Aires hospitals. Few reports about the description and dispersion of tet genes in this species have been published. We observed the presence of tet(B) in all minocycline-resistant isolates. This gene was found to be associated with the ISCR2 mobile element, which may, in part, explain its dispersion.
TEXT
Recently, misconceptions regarding Acinetobacter baumannii have been advanced, exposing the importance of this species as a significant nosocomial pathogen (1–3). One of the important features of A. baumannii, which may contribute to its success in surviving in the hospital environment, is the intrinsic ability to develop resistance to all available antibiotics to treat A. baumannii infections (1–3).
A review of the existing literature indicates that little is described regarding the dispersion of tetracycline resistance determinants (tet) among A. baumannii clinical isolates (4–8). The presence of resistance genes such as tet(A) and tet(B) for tetracyclines, most of them tetracycline efflux pumps, has been described (4–12). It is known that Tet(A) confers resistance to tetracycline (TET) while Tet(B) confers resistance to TET and minocycline (MIN). In Argentina, the emergence of minocycline resistance has been observed in the past few years, varying from 10 to 40% resistance among different centers (13).
As there are scarce data about the resistance determinants for tetracyclines in A. baumannii and the potential therapeutic benefit of using tetracyclines in combination treatment with colistin and carbapenems (14, 15), we decided to search for the presence of tetracycline resistance genes in 47 epidemiologically unrelated A. baumannii isolates. The studied isolates were selected from a collection of 250 A. baumannii clinical isolates. Criteria for selection included temporal representation (1983 to 2011), different hospitals (n = 11), and different countries (Argentina, Uruguay, and Chile). The selected isolates possessed different antibiotic resistance profiles, with all of them resistant to tetracycline and 12 also resistant to minocycline (Table 1). Using molecular typing techniques, we observed the presence of a prevalent clone among the minocycline-resistant isolates (Table 1).
Table 1.
Description of the isolates included in the study and PCR amplification results of tet(A) and tet(B)
| Isolate | Hospital/country | Yr | Clone | Tetracycline resistance | tet(A) | tet(B) |
|---|---|---|---|---|---|---|
| Ab102 | H7/Argentina | 1983 | II | TET | − | − |
| Ab155 | H7/Argentina | 1994 | IV | TET | + | − |
| Ab115 | H7/Argentina | 1994 | IV | TET | + | − |
| Ab1 | H2/Chile | 1997 | III | TET | − | − |
| Ab3 | H2/Chile | 1997 | III | TET | − | − |
| Ab6 | H2/Chile | 1998 | III | TET | − | − |
| Ab181 | H7/Argentina | 2001 | I | TET | − | − |
| Ab7 | H3/Chile | 2006 | III | TET | − | − |
| Ab287 | H4/Argentina | 2006 | XIV | TET | + | − |
| Ab374 | H4/Argentina | 2007 | IV | TET | − | − |
| Ab326 | H4/Argentina | 2007 | XIV | TET | − | − |
| Ab375 | H5/Argentina | 2007 | I | TET | − | − |
| Ab394 | H4/Argentina | 2007 | I | TET | − | − |
| Ab49631 | H6/Argentina | 2008 | III | TET | − | − |
| Abu1 | H1/Uruguay | 2008 | IV | TET | − | − |
| Abu2 | H1/Uruguay | 2008 | IV | TET | − | − |
| Ab66285 | H6/Argentina | 2009 | XIV | TET | + | − |
| Ab98 | H8/Argentina | 2009 | III | TET | − | − |
| Ab04 | H11/Argentina | 2010 | III | TET | − | − |
| Ab21 | H10/Argentina | 2010 | III | TET | − | − |
| Ab908 | H8/Argentina | 2011 | III | TET | − | − |
| Ab13205 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab13338 | H8/Argentina | 2011 | III | TET | − | − |
| Ab13494 | H8/Argentina | 2011 | III | TET | − | − |
| Ab14393 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab15111 | H8/Argentina | 2011 | III | TET | − | − |
| Ab47 | H8/Argentina | 2011 | III | TET | − | − |
| Ab15424 | H8/Argentina | 2011 | III | TET | + | − |
| Ab15975 | H8/Argentina | 2011 | III | TET | − | − |
| Ab15799 | H8/Argentina | 2011 | III | TET | + | − |
| Ab15897 | H8/Argentina | 2011 | III | TET | + | − |
| Ab15 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab20 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab42 | H8/Argentina | 2011 | III | TET | + | − |
| Ab53 | H8/Argentina | 2011 | III | TET | − | − |
| Ab103 | H8/Argentina | 2011 | III | TET | − | − |
| Ab105 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab106 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab107 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab112 | H8/Argentina | 2011 | III | TET | − | − |
| Ab120 | H8/Argentina | 2011 | III | TET | − | − |
| Ab129 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab133 | H8/Argentina | 2011 | V | TET, MIN | − | + |
| Ab741019 | H9/Argentina | 2011 | V | TET, MIN | − | + |
| Ab761457 | H9/Argentina | 2011 | V | TET, MIN | − | + |
| Ab767339 | H9/Argentina | 2011 | V | TET, MIN | − | + |
| Ab795672 | H9/Argentina | 2011 | VI | TET | − | + |
To determine the presence of tet genes, we extracted total DNA and used it to perform PCR amplification reactions according to the manufacturer's instructions (Promega, Madison, WI). Specific primers for tet(A), tet(B), tet(M), tet(39), and tet(H) were used (see Table S1 in the supplemental material). We obtained positive results for the tet(A) and tet(B) genes, and the rest of the amplification reactions were negative (Table 1). All PCR amplification products were sequenced on both DNA strands using an ABI Prism 3100 BioAnalyzer and Taq FS Terminator Chemistry (Taq FS; Perkin-Elmer), and sequence analysis was performed with the Sequencher 4.7 software (Gene Codes Corp.) and BLAST (version 2.0) software (http://www.ncbi.nlm.nih.gov/BLAST/).
The tet(B) gene was found in 13 extensively drug-resistant A. baumannii isolates of our collection, with 12 of them minocycline resistant (Table 1). All positive isolates were recovered in the last year, exposing the recent emergence of this determinant (Table 1). There is scant information about tet(B) gene distribution among A. baumannii clinical isolates. The few reports which investigated the presence of this gene in A. baumannii clinical isolates showed a high prevalence of tet(B) among multidrug-resistant A. baumannii isolates (6, 12). Lately, the description of an AbaR-type genomic island in strains belonging to the global clone 2 revealed the presence of a tet(B) determinant in this context (9–11).
To determine the genetic platform of tet(B) in our isolates, inverse PCR and PCRs with primers annealing in the described structures were performed. Sequence analysis of the positive inverse PCR products revealed the presence of the ISCR2 element downstream of tet(R) and a sequence derived from Tn10, which is preceded by the ΔISCR2 deletion, upstream of tet(B) (Fig. 1). The genetic platform surrounding tet(R) resembled the recently described genetic context found in the AbaR4-type islands (9–11). As Tn5393, containing the strA and strB genes, was found following the ISCR2 element in the recently described tet(B) genetic platform (9–11), PCRs were carried out to determine their presence. All tet(B)-positive isolates contained the strA and strB genes associated with the ISCR2 mobile element.
Fig 1.
Schematic representation of the tet(B) gene context found in this study and the recently described context found in the AbaR4 islands. Boxes and lines of different thicknesses and colors represent the different determinants and elements (antibiotic resistance genes and ISCR2). Vertical bars indicate the inverted repeats and the ori and ter sites of the ISCR2 element. ISCR2Δ is a partial version of the ISCR2 element, and tet(B)* is a truncated version of the tet(B) gene. Genes are shown by horizontal arrows with the gene name listed below. The tet(B) genetic context found in Ab13205 resembles the recently described tet(B) context within the AbaR island found in strain A91 (10). Phosphoglucosamine mutase (glmM) and the transcription regulator of ArsR family genes are represented as g and r, respectively. The structure found in the study is available from GenBank under accession number JX566450. Information in parentheses after each strain name indicates the GenBank accession number for that strain.
Concerning the structure upstream of the tet(B) gene, Nigro and Hall (10) recently found this gene within a novel AbaR4-type island, named Tn6167 and containing the sul2 gene and a partial copy of CR2, which is the same genetic context identified in all tet(B)-positive isolates from our study. To determine that tet(B) was within the AbaR-type genomic organization, specific PCRs showing that the comM gene was intact were done (16). This gene was recognized as a target for the insertion of AbaR-type resistance islands in multidrug-resistant strains enclosing different resistance determinants (10, 16–18). Therefore, the difference concerning the tet(B) genetic platform is that our isolates do not have the tet(B) gene linked to AbaR islands (Fig. 1).
Plasmidic DNA from Ab13205 was extracted using the QIAfilter midi kit (Qiagen) and used to transform Escherichia coli Top10 competent cells. Colonies were obtained in plates containing 3 μg/ml of TET and subjected to plasmid DNA extraction as described above (data not shown). To confirm tet(B) presence, plasmidic DNA was used as the template for PCR amplification, obtaining positive results. Moreover, the MICs to TET and MIN of the transformed E. coli Top10 cells were determined using the Etest strips as recommended (19). MIC results show an increase in both MICs, supporting the plasmid location (Table 2).
Table 2.
MICs to TET and MINa
| Strain | MIC (mg/liter) |
|
|---|---|---|
| TET | MIN | |
| A. baumannii Ab13205 | 32 | 12 |
| E. coli TOP10 | 1 | 0.5 |
| E. coli TOP10 p13205 | 24 | 3 |
The MICs to TET and MIN were determined by following CLSI recommendations.
On the other hand, we observed the presence of the tet(A) gene in 8 out of 47 isolates. These tet(A)-positive isolates were recovered in different years (1994, 2006, and 2011) and different hospitals (Table 1). This gene was reported to be associated with Tn1721-like transposon, which was reported in a clinical A. baumannii isolate (7). In order to determine if this genetic structure was present in our isolates, PCRs with specific primers for the Tn1721-like transposase (tnpA) and tet(A) gene were carried out (see Table S1 in the supplemental material). When we tested the primer combinations tetAbTnpA′R/tetAF and tetAbTnpAF/tetAR, negative results were obtained in all tet(A)-positive isolates, exposing the absence of this transposon in our isolates and suggesting a different genetic context for this gene.
Our results exposed a complex genetic background associated with tetracycline resistance in our A. baumannii population, including tet(A), tet(B), and yet-unknown elements. We also observed the presence of the tet(B) gene associated with the plasmid-mediated ISCR2 mobile element. Lastly, we noted that this element can be transferred and increased the MICs to tetracycline and minocycline in E. coli. This new genetic platform of the tet(B) gene, linked to the insertion sequence ISCR2, supports a mechanism by which spread among A. baumannii isolates is possible.
Nucleotide sequence accession number.
The sequence of the tet(B) context has been submitted to GenBank under accession no. JX566450.
Supplementary Material
ACKNOWLEDGMENTS
M.S.R. and D.C. are career investigators of CONICET, Argentina. E.V. and G.M.T. have doctoral fellowships from CONICET. This study was supported by grant PIP 11420100100152 to M.S.R. and grant UBACyTs 2011-2012 to M.S.R. and D.C., Buenos Aires, Argentina.
Footnotes
Published ahead of print 12 November 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01751-12.
REFERENCES
- 1. McConnell MJ, Actis L, Pachon J. 8 May 2012, posting date Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol. Rev. doi:10.1111/j.1574-6976.2012.00344.x [DOI] [PubMed] [Google Scholar]
- 2. Roca I, Espinal P, Vila-Farres X, Vila J. 2012. The Acinetobacter baumannii oxymoron: commensal hospital dweller turned pan-drug-resistant menace. Front. Microbiol. 3:148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Towner KJ. 2009. Acinetobacter: an old friend, but a new enemy. J. Hosp. Infect. 73:355–363 [DOI] [PubMed] [Google Scholar]
- 4. Agerso Y, Guardabassi L. 2005. Identification of Tet 39, a novel class of tetracycline resistance determinant in Acinetobacter spp. of environmental and clinical origin. J. Antimicrob. Chemother. 55:566–569 [DOI] [PubMed] [Google Scholar]
- 5. Guardabassi L, Dijkshoorn L, Collard JM, Olsen JE, Dalsgaard A. 2000. Distribution and in-vitro transfer of tetracycline resistance determinants in clinical and aquatic Acinetobacter strains. J. Med. Microbiol. 49:929–936 [DOI] [PubMed] [Google Scholar]
- 6. Marti S, Fernandez-Cuenca F, Pascual A, Ribera A, Rodriguez-Bano J, Bou G, Miguel Cisneros J, Pachon J, Martinez-Martinez L, Vila J. 2006. Prevalence of the tetA and tetB genes as mechanisms of resistance to tetracycline and minocycline in Acinetobacter baumannii clinical isolates. Enferm. Infecc. Microbiol. Clin. 24:77–80 (In Spanish.) [DOI] [PubMed] [Google Scholar]
- 7. Ribera A, Roca I, Ruiz J, Gibert I, Vila J. 2003. Partial characterization of a transposon containing the tet(A) determinant in a clinical isolate of Acinetobacter baumannii. J. Antimicrob. Chemother. 52:477–480 [DOI] [PubMed] [Google Scholar]
- 8. Ribera A, Ruiz J, Vila J. 2003. Presence of the Tet M determinant in a clinical isolate of Acinetobacter baumannii. Antimicrob. Agents Chemother. 47:2310–2312 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Nigro SJ, Hall RM. 2012. Antibiotic resistance islands in A320 (RUH134), the reference strain for Acinetobacter baumannii global clone 2. J. Antimicrob. Chemother. 67:335–338 [DOI] [PubMed] [Google Scholar]
- 10. Nigro SJ, Hall RM. 2012. Tn6167, an antibiotic resistance island in an Australian carbapenem-resistant Acinetobacter baumannii GC2, ST92 isolate. J. Antimicrob. Chemother. 67:1342–1346 [DOI] [PubMed] [Google Scholar]
- 11. Seputiene V, Povilonis J, Suziedeliene E. 2012. Novel variants of AbaR resistance islands with a common backbone in Acinetobacter baumannii isolates of European clone II. Antimicrob. Agents Chemother. 56:1969–1973 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Srinivasan VB, Rajamohan G, Pancholi P, Stevenson K, Tadesse D, Patchanee P, Marcon M, Gebreyes WA. 2009. Genetic relatedness and molecular characterization of multidrug resistant Acinetobacter baumannii isolated in central Ohio, USA. Ann. Clin. Microbiol. Antimicrob. 8:21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Fiorilli G, Vay NMC, Famiglietti A, Rodriguez CH. 2012. Caracterización Feno y Genotípica de la resistencia a Tetraciclinas en Acinetobacter baumannii. RAM 44(Suppl 1):59 [Google Scholar]
- 14. Liang W, Liu XF, Huang J, Zhu DM, Li J, Zhang J. 2011. Activities of colistin- and minocycline-based combinations against extensive drug resistant Acinetobacter baumannii isolates from intensive care unit patients. BMC Infect. Dis. 11:109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Ozbek B, Senturk A. 2010. Postantibiotic effects of tigecycline, colistin sulfate, and levofloxacin alone or tigecycline-colistin sulfate and tigecycline-levofloxacin combinations against Acinetobacter baumannii. Chemotherapy 56:466–471 [DOI] [PubMed] [Google Scholar]
- 16. Post V, White PA, Hall RM. 2010. Evolution of AbaR-type genomic resistance islands in multiply antibiotic-resistant Acinetobacter baumannii. J. Antimicrob. Chemother. 65:1162–1170 [DOI] [PubMed] [Google Scholar]
- 17. Krizova L, Dijkshoorn L, Nemec A. 2011. Diversity and evolution of AbaR genomic resistance islands in Acinetobacter baumannii strains of European clone I. Antimicrob. Agents Chemother. 55:3201–3206 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Shaikh F, Spence RP, Levi K, Ou HY, Deng Z, Towner KJ, Rajakumar K. 2009. ATPase genes of diverse multidrug-resistant Acinetobacter baumannii isolates frequently harbour integrated DNA. J. Antimicrob. Chemother. 63:260–264 [DOI] [PubMed] [Google Scholar]
- 19. CLSI 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.