Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doloy, A. Articles by Arlet, G. Search for Related Content PubMed PubMed Citation Articles by Doloy, A. Articles by Arlet, G.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, December 2006, p. 4177-4181, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00619-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Genetic Environment of Acquired bla_ACC-1 ß-Lactamase Gene in Enterobacteriaceae Isolates

A. Doloy,¹ C. Verdet,^1,2 V. Gautier,¹ D. Decré,¹ E. Ronco,³ A. Hammami,⁴ A. Philippon,⁵ and G. Arlet^1,2*

Université Paris VI, UPRES EA 2392, UFR de Médecine Pierre et Marie Curie, Paris, France,¹ AP-HP, Hôpital Tenon, Service de Bactériologie, Paris, France,² AP-HP, Hôpital Raymond Poincaré, Laboratoire de Microbiologie, Garches, France,³ Service de Microbiologie, CHU Habib Bourguiba, Sfax, Tunisia,⁴ AP-HP, Hôpital Cochin, Service de Bactériologie, Paris, France⁵

Received 19 May 2006/ Returned for modification 8 July 2006/ Accepted 6 September 2006

Top Abstract Text References

 
We studied the genetic organization of bla_ACC-1 in 14 isolates of Enterobacteriaceae from France, Tunisia, and Germany. In a common ancestor, ISEcp1 was likely involved in the mobilization of this gene from the Hafnia alvei chromosome to a plasmid. Other genetic events involving insertion sequences (particularly IS26), transposons (particularly Tn1696), or sulI-type integrons have occurred, leading to complex genetic environments.

Top Abstract Text References

 
The chromosomally encoded class C ß-lactamases confer a natural resistance to ß-lactams, which is typical of a number of gram-negative species. Since the 1980s, plasmid-encoded AmpC-type ß-lactamases have been found worldwide, most often in organisms lacking inducible chromosomal AmpC enzymes (13). Plasmid-carried ampC genes originate from the chromosomal ampC genes of organisms such as Citrobacter freundii (CMY type), Enterobacter spp. (MIR-1 and ACT-1), Morganella morganii (DHA type), and Hafnia alvei (ACC-1) (13).

ACC-1 was characterized in isolates of several species, such as Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli, from Germany, France, Tunisia, Spain, and The Netherlands (2, 3, 6-9, 11, 12, 15). In France, nosocomial outbreaks following the admission of a patient transferred from Tunisia, a country which seems to be a potential reservoir, were described (11, 12).

The ACC-1-producing rifampin-resistant K. pneumoniae SLK54 strain, isolated in Saint-Louis Hospital in Paris, France, was previously studied by cloning experiments, and the genetic organizations of both plasmid-mediated genes, bla_ACC-1 and arr-2, were established (1, 11). The presence of IS26 in both recombinant plasmids suggests that arr-2 and bla_ACC-1 could be linked in the K. pneumoniae SLK54 plasmid. In this study, we have tested this hypothesis, and we have analyzed the genetic environment of the bla_ACC-1 gene in a collection of ACC-1-producing isolates, including 11 K. pneumoniae, one P. mirabilis, and two S. enterica isolates from different settings.

Table 1 lists the 14 clinical isolates and their sources. Most isolates were previously studied (2, 8, 11, 12, 15).

View this table: [in this window] [in a new window]

TABLE 1. Origin, epidemiological features, and PCR mapping of clinical isolates

Genomic DNAs from K. pneumoniae isolates were prepared as described previously (5), digested with XbaI (New England Biolabs Inc., Saint Quentin en Yvelines, France), and subjected to pulsed-field gel electrophoresis in a CHEF DRIII device (Bio-Rad, Marnes-la-Coquette, France). DNA fragments were separated in a 1% (wt/vol) agarose gel in 0.5x Tris-borate-EDTA buffer at 200 V for 20 h, with pulse times ranging from 5 to 30 s. The pulsed-field gel electrophoresis profiles obtained for isolates C5900 (Tunisia) and KUS (Germany) (profiles C and D, respectively) were different from each other and from those of the nine other K. pneumoniae isolates (data not shown). According to the interpretation criteria of Tenover et al. (16), the nine French isolates could be divided into two possibly related clusters, profiles A and B (Table 1).

By mating experiments, E. coli K-12 J53-2 or E. coli K-12 HB101 transconjugants were obtained with ceftazidime (10 µg/ml) and either rifampin (250 µg/ml) or streptomycin (250 µg/ml) as selective agents. In case of failure, plasmid DNA was used to transform E. coli DH10B cells by electroporation (Bio-Rad). Transformants were selected with ceftazidime (10 µg/ml). Fingerprinting analysis was carried out with plasmid DNA purified from transformants or transconjugants by using a plasmid midi kit (QIAGEN, Courtaboeuf, France), digested with EcoRI, and subjected to electrophoresis in a 1.2% agarose gel at 80 V for 3 h. This analysis showed that plasmids present in the 10 French isolates were identical to those in the Tunisian K. pneumoniae isolate C5900 (profile A) (data not shown). Each of the other three isolates had unique plasmid profiles (Table 1). It seems that the spread of bla_ACC-1 in three different hospitals in France is linked to the spread of both a clonal strain and a plasmid that likely originated from Tunisia.

PCR experiments and DNA sequencing were carried out with the clinical isolates to detect bla_TEM, as previously described (17), and bla_ACC (Table 2 PCR A). The sequence analysis showed that, except for S. enterica serovar Mbandaka MMAS₄₀, all isolates were TEM-1 positive (data not shown), and it confirmed that all isolates and transformants or transconjugants were ACC-1-positive.

View this table: [in this window] [in a new window]

TABLE 2. Sequences of oligonucleotides used for PCR mapping

The genetic context of bla_ACC-1 was explored by PCR mapping using the bla_ACC-1-carrying plasmids from transconjugants or transformants. First, we tested whether bla_ACC-1 and arr-2 in K. pneumoniae SLK54 were linked on the same plasmid. Primers were designed to amplify the region between bla_ACC-1 and intlI (PCR F) (Table 2). As expected, the 2.3-kb segment (PCR F) overlapped with both sequences of SLK54 previously submitted to the sequence databases; the first comprises bla_ACC-1 (GenBank accession number AJ270942), and the second comprises arr-2 (GenBank accession number AJ277027) (Fig. 1). We then used several PCRs (PCRs B to H) to characterize the genetic context of bla_ACC-1 in the other 13 isolates (Fig. 1; Table 2). Eight isolates other than SLK54 (seven K. pneumoniae isolates and one P. mirabilis isolate) were positive in these PCRs (Table 1). Two aliquots of the PCR products were totally digested, one with TacI (New England Biolabs) and the other with HaeIII (New England Biolabs). The restriction profiles obtained from these PCR products were the same for each isolate (data not shown), suggesting that the genetic environments of bla_ACC-1 were at least similar, if not identical, in the plasmids from these nine isolates.

View larger version (25K): [in this window] [in a new window]

FIG. 1. Comparison of regions surrounding blaACC-1. ORF, open reading frame.

The genetic organization of bla_ACC-1 in five isolates (SLK55, C5900, KUS, MMAS₄₀, and B952) seemed to be different from that in SLK54. Therefore, we used cloning experiments to explore the region surrounding bla_ACC-1. The plasmid DNA was partially digested with SauIIIA, and the fragments were ligated into the BamHI site of pACYC184. Recombinant plasmids introduced into E. coli DH10B were then selected with ceftazidime (10 µg/ml) and chloramphenicol (50 µg/ml). Several clones with an insert encompassing bla_ACC-1 were obtained from each of the five isolates. The largest of these were selected, and their sequences were analyzed on both strands. Finally, overlapping inserts of recombinant plasmids together with PCR mapping segments (PCRs I and J) allowed us to map the unknown region surrounding bla_ACC-1 for these five isolates (Fig. 1). The genetic organizations of bla_ACC-1 from the 14 isolates of this study and from Salmonella enterica serovar Bareilly 60.50, isolated in The Netherlands (7), are shown in Fig. 1.

Common structures. The ACC-1-producing isolates all have the same genetic organization of plasmid-encoded bla_ACC-1 in the proximity of the ß-lactamase gene. In each isolate, a 1,318-bp region originating from the H. alvei chromosome, comprising ampC but lacking ampR (which is consistent with the high level of ß-lactamase production), was present. The total length of the sequence mobilized from H. alvei is unknown, as the previously sequenced region surrounding the ampC and ampR genes from H. alvei (accession number AF180952) is too short, with only the final 18-bp segment of gdha being present in this sequence. Nevertheless, gdha is present downstream from bla_ACC-1 in H. alvei (PCRs A and B) (Table 1) and in the six plasmid-carried bla_ACC-1 genetic organization profiles. Moreover, gdha, which encodes a glutamate dehydrogenase, is a very common housekeeping gene among proteobacteria, and its presence on the H. alvei genome would be expected. Thus, the gdha sequence present downstream from bla_ACC-1 is presumed to have been mobilized from the H. alvei chromosome together with bla_ACC-1.

The insertion sequence ISEcp1 upstream from bla_ACC-1 is always present in the same orientation but has variable deletions in the 5' part: a 1,433-bp deletion for SLK54 and SLK55; a 1,101-bp deletion for KUS, B952, and MMAS₄₀; and a 988-bp deletion for S. enterica serovar Bareilly 60.50. Nevertheless, the length between the end of ISEcp1 and the ATG codon of bla_ACC-1 is constant, which suggests that only one genetic recombination event has occurred. ISEcp1 is known to be involved in both the mobilization and expression of bla_CTX-M ß-lactamase genes (14). Although ISEcp1 is always truncated in the 5' part and can no longer function in transposition events, we cannot rule out the possibility that it was involved in the initial mobilization of bla_ACC-1 in an ancestor common to the six different profiles described here. Indeed, ISEcp1 transposase may recognize a wide range of DNA sequences as inverted right repeats and use them as ends in a mobilization process involving its adjacent sequences (14). The –35 and –10 regions corresponding to putative promoter sequences for acquired bla were different for the chromosomal bla_ACC type of H. alvei_. Indeed, whatever the deletion of ISEcp1, the promoter is likely provided by ISEcp1, as reported with bla_CTX-M (4).

Different structures. The genetic organization of plasmid-carried bla_ACC-1 differs beyond ISEcp1 and gdha, and it can be divided into three main patterns.

(i) the first pattern was found in SLK54 and in the related isolates SLK55 and C5900. In this pattern, ISEcp1 has a 1,433-bp deletion due to an insertion of an IS26 in the opposite orientation. A second copy of IS26 (IS26_L) is present 4,210 bp upstream from the first copy, IS26_R. The two copies of IS26 are in opposite orientations, with the 3' ends facing outwards (Fig. 1). Another copy of ISEcp1 (ISEcp1_L) is present downstream from IS26_L and is 3' truncated, with the deletion being exactly complementary to that of ISEcp1_R upstream from bla_ACC-1. This and the presence of two copies of IS26 suggest that a composite transposon with two directly repeated IS26s was inserted in ISEcp1 with the duplication of a 8-bp target site (characteristic of IS26 transposition) GACATTTT (10). However, we are still unsure as to how ISEcp1_L and IS26_L could have been secondarily inverted and moved elsewhere, downstream from bla_ACC-1.

(ii) The second pattern was found in KUS, B952, and MMAS₄₀. In this group, ISEcp1 has a 1,101-bp deletion due to the insertion of an IS26 in the same orientation. A second copy of IS26 (IS26_L) is present 2,742 bp downstream from bla_ACC-1 (Fig. 1). The two copies of IS26 are in opposite orientations, with the 3' ends facing inwards. The DNA sequence between the two copies of IS26 is 100% identical in KUS, B952, and MMAS₄₀. Beyond IS26, the DNA sequence was different in the three structures (Fig. 1). Thus, the structures from the KUS, B952, and MMAS40 isolates are likely derived from a common ancestor different from that of the first pattern, with a shorter deletion of ISEcp1 due to the insertion of IS26 in the opposite direction. After this event, several different genetic events seem to have occurred, mainly involving Tn1696 but also involving a class 1 integron carrying a dfrA21 gene cassette.

(iii) The third pattern was observed in S. enterica serovar Bareilly only (Fig. 1). This has an ISEcp1 deleted by 988 bp due to the insertion of the previously described orf7 (7). The 309-amino-acid protein encoded by orf7 is 99% identical to a transposase described for Yersinia enterocolitica strain 29979 (GenBank accession number CAA73750). Upstream from gdha, the three previously described open reading frames encode proteins, the first identical to a transposase from K. pneumoniae CG43 (accession number NP_943502), the second identical to a protein from the Y. enterocolitica plasmid p29930 (GenBank accession number CAD58550), and the third identical to a recombinase from the same plasmid (GenBank accession number CAE46772).

Studies of the molecular mechanisms of ampC gene transfer from chromosome to plasmids are in progress. It seems that several different DNA-mobilizing elements, such as common regions associated to integrons and insertion sequences, are involved in the movement of the ampC gene and the adjacent region (13, 17). Although IS26 has been found in the genetic organization of bla_ACC-1 in several clinical isolates, it is likely that this structure is not directly involved in the translocation of the ampC gene from the H. alvei chromosome to different plasmids. Nevertheless, its presence seems to be linked with recombination events occurring after the insertion of ISEcp1 upstream from bla_ACC-1 that lead to the deletion of the 5' end of ISEcp1 (Fig. 1). In all cases, ISEcp1 was never complete, and its deletion may have led to the stabilization of bla_ACC-1 on different plasmids.

Nucleotide sequence accession numbers. The nucleotide sequences in strains SLK54, SLK55, KUS, B952, and MMAS₄₀ have been submitted to GenBank and have been assigned accession numbers AJ870923, AJ870922, AJ870924, AJ870925, and AJ870926, respectively.

 
This work was supported by grants from Faculté de Médecine Saint-Antoine, Université Pierre et Marie Curie, and from the European Community (6th PCRD contract LSHM-CT 2003-503335). A. Doloy was supported by a grant from Fonds d'Etudes et de Recherche du Corps Médical des Hôpitaux de Paris, AP-HP.

 
* Corresponding author. Mailing address: AP-HP, Hôpital Tenon, Service de Bactériologie, Paris, France. Phone: 33 1 56 01 70 18. Fax: 33 1 56 01 61 08. E-mail: guillaume.arlet{at}tnn.ap-hop-paris.fr.

Published ahead of print on 18 September 2006.

Top Abstract Text References

 

1
1. Arlet, G., D. Nadjar, J. L. Herrmann, J. L. Donay, M. Rouveau, P. H. Lagrange, and A. Philippon. 2001. Plasmid-mediated rifampin resistance encoded by an arr-2-like gene cassette in Klebsiella pneumoniae producing an ACC-1 class C ß-lactamase. Antimicrob. Agents Chemother. 45:2971-2972.[Free Full Text]
2
2. Bauernfeind, A., I. Schneider, R. Jungwirth, H. Sahly, and U. Ullmann. 1999. A novel type of AmpC ß-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrob. Agents Chemother. 43:1924-1931.[Abstract/Free Full Text]
3
3. Bidet, P., B. Burghoffer, V. Gautier, N. Brahimi, P. Mariani-Kurkdjian, A. El-Ghoneimi, E. Bingen, and G. Arlet. 2005. In vivo transfer of plasmid-encoded ACC-1 AmpC from Klebsiella pneumoniae to Escherichia coli in an infant and selection of impermeability to imipenem in K. pneumoniae. Antimicrob. Agents Chemother. 49:3562-3565.[Abstract/Free Full Text]
4
4. Cao, V., T. Lambert, and P. Courvalin. 2002. ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum beta-lactamase CTX-M-17. Antimicrob. Agents Chemother. 46:1212-1217.[Abstract/Free Full Text]
5
5. Decré, D., B. Gachot, J. C. Lucet, G. Arlet, E. Bergogne-Bérézin, and B. Régnier. 1998. Clinical and bacteriological epidemiology of extended-spectrum ß-lactamase-producing strains of Klebsiella pneumoniae in a medical intensive care unit. Clin. Infect. Dis. 27:834-844.[Medline]
6
6. Girlich, D., A. Karim, C. Spicq, and P. Nordmann. 2000. Plasmid-mediated cephalosporinase ACC-1 in clinical isolates of Proteus mirabilis and Escherichia coli. Eur. J. Clin. Microbiol. Infect. Dis. 19:893-895.[CrossRef][Medline]
7
7. Hasman, H., D. Mevius, K. Veldman, I. Olesen, and F. M. Aarestrup. 2005. Beta-lactamases among extended-spectrum beta-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J. Antimicrob. Chemother. 56:115-121.[Abstract/Free Full Text]
8
8. Makanera, A., G. Arlet, V. Gautier, and M. Manai. 2003. Molecular epidemiology and characterization of plasmid-encoded ß-lactamases produced by Tunisian clinical isolates of Salmonella enterica serotype Mbandaka resistant to broad-spectrum cephalosporins. J. Clin. Microbiol. 41:2940-2945.[Abstract/Free Full Text]
9
9. Miró, E., B. Mirelis, F. Navarro, L. Matas, M. Gimenez, and C. Rabaza. 2005. Escherichia coli producing an ACC-1 class C beta-lactamase isolated in Barcelona, Spain. Antimicrob. Agents Chemother. 49:866-867.[Free Full Text]
10
10. Naas, T., Y. Mikami, T. Imai, L. Poirel, and P. Nordmann. 2001. Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes. J. Bacteriol. 183:235-249.[Abstract/Free Full Text]
11
11. Nadjar, D., M. Rouveau, C. Verdet, J. L. Donay, J. L. Herrmann, P. H. Lagrange, A. Philippon, and G. Arlet. 2000. Outbreak of Klebsiella pneumoniae producing transferable AmpC-type ß-lactamase (ACC-1) originating from Hafnia alvei. FEMS Microbiol. Lett. 187:35-40.[Medline]
12
12. Ohana, S., V. Leflon, E. Ronco, M. Rottman, D. Guillemot, S. Lortat-Jacob, P. Denys, G. Loubert, M. H. Nicolas-Chanoine, J. L. Gaillard, and C. Lawrence. 2005. Spread of a Klebsiella pneumoniae strain producing a plasmid-mediated ACC-1 AmpC ß-lactamase in a teaching hospital admitting disabled patients. Antimicrob. Agents Chemother. 49:2095-2097.[Abstract/Free Full Text]
13
13. Philippon, A., G. Arlet, and G. A. Jacoby. 2002. Plasmid-determined AmpC-type beta-lactamases. Antimicrob. Agents Chemother. 46:1-11.[Free Full Text]
14
14. Poirel, L., J. W. Decousser, and P. Nordmann. 2003. Insertion sequence ISEcp1B is involved in expression and mobilization of a bla_CTX-M ß-lactamase gene. Antimicrob. Agents Chemother. 47:2938-2945.[Abstract/Free Full Text]
15
15. Rhimi-Mahjoubi, F., M. Bernier, G. Arlet, Z. Ben Jemaa, P. Jouve, A. Hammami, and A. Philippon. 2002. Identification of plasmid-encoded cephalosporinase ACC-1 among several enterobacteria (Klebsiella pneumoniae, Proteus mirabilis, Salmonella) issued from a Tunisian hospital (Sfax, 1997-2000). Pathol. Biol. 50:7-11.
16
16. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulse-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
17
17. Verdet, C., Y. Benzerara, V. Gautier, O. Adam, Z. Ould-Hocine, and G. Arlet. 2006. Emergence of DHA-1-producing Klebsiella spp. in the Parisian region: genetic organization of the ampC and ampR genes originating from Morganella morganii. Antimicrob. Agents Chemother. 50:607-617.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, December 2006, p. 4177-4181, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00619-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Jacoby, G. A. (2009). AmpC {beta}-Lactamases. Clin. Microbiol. Rev. 22: 161-182 [Abstract] [Full Text]  
• Cattoir, V., Nordmann, P., Silva-Sanchez, J., Espinal, P., Poirel, L. (2008). ISEcp1-Mediated Transposition of qnrB-Like Gene in Escherichia coli. Antimicrob. Agents Chemother. 52: 2929-2932 [Abstract] [Full Text]  
• Papagiannitsis, C. C., Tzouvelekis, L. S., Tzelepi, E., Miriagou, V. (2007). Plasmid-Encoded ACC-4, an Extended-Spectrum Cephalosporinase Variant from Escherichia coli. Antimicrob. Agents Chemother. 51: 3763-3767 [Abstract] [Full Text]  
• Partridge, S. R., Arlet, G. (2007). Genetic Environment of ISEcp1 and blaACC-1. Antimicrob. Agents Chemother. 51: 2658-2659 [Full Text]  
• Papagiannitsis, C. C., Loli, A., Tzouvelekis, L. S., Tzelepi, E., Arlet, G., Miriagou, V. (2007). SCO-1, a Novel Plasmid-Mediated Class A {beta}-Lactamase with Carbenicillinase Characteristics from Escherichia coli. Antimicrob. Agents Chemother. 51: 2185-2188 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doloy, A. Articles by Arlet, G. Search for Related Content PubMed PubMed Citation Articles by Doloy, A. Articles by Arlet, G.