- Email: [email protected]
Chromosomal ampC genes in Enterobacter species other than Enterobacter cloacae, and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to Enterobacter asburiae
FEMS Microbiology Letters 210 (2002) 87^92
www.fems-microbiology.org
Chromosomal ampC genes in Enterobacter species other than Enterobacter cloacae, and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to Enterobacter asburiae Martin Rottman
a;
a b
, Yahia Benzerara a , Be¤atrice Hanau-Berc ot b , Chantal Bizet c , Alain Philippon d , Guillaume Arlet a Service de Bacte¤riologie, Ho“pital Tenon, UFR Saint Antoine, Paris, France Service de Bacte¤riologie, Ho“pital Lariboisie're, Institut Pasteur, Paris, France c Collection de l’Institut Pasteur, Institut Pasteur, Paris, France d Service de Bacte¤riologie, Ho“pital Cochin, Paris, France First published online 12 April 2002
Abstract The amplification and sequence of ampC genes in Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei and Enterobacter intermedius bring the number of known cephalosporinase sequences from the genus Enterobacter to seven. Expression in Escherichia coli of the ampC genes from E. asburiae, E. hormaechei and E. intermedius established the functional nature of these genes. ampC from E. asburiae shows 96.5% identity to blaACT 1 encoding a plasmid-borne cephalosporinase previously believed to derive from Enterobacter cloacae. The reassignment of ACT-1 ancestry to E. asburiae is confirmed by the 95.5% identity between ampR upstream of blaACT 1 and ampR from E. asburiae. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : AmpC L-lactamase, chromosomal; AmpC L-lactamase, plasmid-encoded; AmpR transcriptional regulator; Enterobacter; Enterobacter asburiae
1. Introduction Enterobacter cloacae is a major nosocomial pathogen, the third most prevalent bacterium isolated in intensive care settings [1]. E. cloacae naturally exhibits resistance to ¢rst and second generation cephalosporins and to aminopenicillins, a mechanism mediated by the production of AmpC, a chromosomal Ambler class C L-lactamase [2]. In E. cloacae, ampC is divergently transcribed from an intercistronic promoter region shared with ampR. The transcription of ampC is under the control of the transcriptional regulator encoded by ampR [3]. The ampR^ampC genetic arrangement found in E. cloacae also exists in other bacteria with chromosomal inducible L-lactamases, in particular in Enterobacter aerogenes [4]. The constitutive hyperproduction of AmpC is of major concern since it confers resistance to most L-lactam antibiotics, sparing * Corresponding author. Present address: Service de Bacte¤riologie, Ho“pital Raymond Poincare¤, 104 Bd Raymond Poincare¤, 92380 Garches, France. Tel.: +33 (1) 47-10-79-46; Fax: +33 (1) 47-10-79-49. E-mail address : [email protected] (M. Rottman).
only carbapenems and ‘fourth generation’ cephalosporins (i.e. cefepime). This phenotype commonly occurs under antibiotic treatment, and Enterobacter isolates resistant to third generation cephalosporins account for over 30% of the isolate from intensive care units [5]. Still, this chromosomal resistance mechanism typically does not spread from strain to strain. These last decades, many authors reported outbreaks of infections due to Klebsiella pneumoniae harboring plasmidencoded cephalosporinases and the spread of this resistance mechanism to bacterial species naturally susceptible to cephamycins. While some of the plasmid-encoded class C L-lactamases are still not linked to known bacterial lineages, plasmid-encoded cephalosporinases derived from Morganella morganii, Citrobacter freundii and Hafnia alvei have been reported. These chromosomal ancestors have a nucleotide identity of over 95% with their plasmid-encoded descendants. MIR-1 [6] and ACT-1 [7] are plasmid-encoded cephalosporinases admitted to derive from E. cloacae but blaMIR 1 and blaACT 1 only share 85% identity with the ampC gene of E. cloacae. To this day the only plasmid-borne blaAmpC gene reported to be under the con-
0378-1097 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 1 3 - 4
FEMSLE 10445 14-5-02
88
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
trol of an ampR regulatory gene is derived from M. morganii [8]. The genetic variability of the ampC gene within wild-type E. cloacae strains has been studied [9], and current sequence analysis shows more than 95% nucleotide sequence identity with the exception of a single strain from Japan [10]. Very little is known about the genetic variability of the ampC gene among di¡erent species of a same genus, and E. cloacae and E. aerogenes [4] are the only examples of known ampC sequences from di¡erent species of a given enterobacterial genus. There are currently 13 recognized species in the genus Enterobacter and E. cloacae and E. aerogenes are the only species routinely isolated from human clinical specimens, the other species being mostly isolated from environmental or vegetal sources [11]. The putative L-lactamases from Enterobacter other than E. cloacae or E. aerogenes have so far only been studied in terms of the modal minimal inhibitory concentrations (MICs) of strains to L-lactam compounds, and their basic biochemical characteristics [12,13]. We have undertaken the study of ampC genes from di¡erent species belonging to the Enterobacter genus.
2. Materials and methods 2.1. Strains and sequence data The Enterobacter type strains studied were kindly provided by the Collection de l’Institut Pasteur (Institut Pasteur, Paris, France). The MIR-1 plasmid (pMG230) conjugated to Escherichia coli strain C600 was a gift from G.A. Jacoby [6]. Phenotypic identi¢cation was performed
using the commercial API 50 CHE, API 20 E and ID 32 E biochemical gallery systems from Biome¤rieux (Marcy l’Etoile, France). The pCR-Blunt (KanR) vector (Invitrogen, Cergy-Pontoise, France) was used for cloning and expression of the ampli¢ed ampC genes in TOP10 E. coli strain (Invitrogen). All nucleotide sequences are listed in Table 1. 2.2. L-Lactams susceptibility MICs of L-lactam antibiotics, alone or in association with clavulanate (2 Wg ml31 ) or tazobactam (4 Wg ml31 ), were determined by the agar dilution method with Mueller^Hinton agar (Bio-Rad, Marnes-la-Coquette, France). Inocula of 104 colony forming units per spot were delivered with a multipoint inoculator. The resistance and susceptibility patterns were determined by the disk di¡usion method on Mueller^Hinton agar. Plates were examined after overnight incubation at 37‡C. 2.3. Inducibility The inducibility of the L-lactamase was tested by a double disk agar di¡usion assay with cefotaxime (30 Wg antibiotic disk load) and ticarcillin (75 Wg) versus cefoxitin (30 Wg) and imipenem (10 Wg) as inducing compounds [14]. Antibiotic susceptibility assay disks and Mueller^Hinton agar were purchased from Bio-Rad. The inducibility of the L-lactamase assayed showed as an antagonistic image between inducing and non-inducing L-lactams, with a reduced inhibitory diameter of the non-inducing L-lactam facing the inducing L-lactam.
Table 1 Sequences used for phylogenetic analysis and identity matrices calculations Label
Gene
EMBL accession number
Origin
Reference
Ec P99 GN7471 MIR-1a ACT-1a Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1a Y.ent S.mar H.alv P.stu FOX-1a E.asb ACT-1
ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampR ampR
X07274 AB016611 M37839 U58495 AJ311172 AJ311363 AJ416709 AJ311364 AJ311365 AF211348 X03866 AF124205 AF055067 Y16410 X63149 AJ271368 AJ270942 Y17315 X77455 AJ421143 AF362955
E. cloacae P99 E. cloacae GN7471 K. pneumoniae plasmid pMG230 K. pneumoniae MCQ-95 E. asburiae type strain E. dissolvens type strain E. cancerogenus type strain E. hormaechei type strain E. intermedius type strain E. aerogenes C. freundii strain ‘OS60’ E. coli strain ‘E102’ M. morganii strain ‘GUI-1’ Salmonella enteritidis strain ‘KF92’ Yersinia enterocolitica strain ‘IP97’ Serratia marcescens strain ‘SLS73’ H. alvei Providentia stuartii strain ‘VDG96’ K. pneumoniae strain ‘BA32’ E. asburiae type strain upstream blaACT 1
[9] [10] [29] [7] This This This This This [4] [30] NR [8] [8] [31] NR [14] NR [23] This NR
NR : not currently referenced. Plasmid-encoded gene.
a
FEMSLE 10445 14-5-02
study study study study study
study
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
89
Table 2 Primers used for Enterobacter spp. gene ampli¢cation and sequence Reference sequence labelsa
Name
Sense
Sequenceb;c
Ec P99 (ampC) MIR-1, ACT-1 (ampC) Eaero (ampC) GN7471 (ampC) GN7471, Ec P99 (ampC) Ec P99 (ampC) MIR-1, ACT-1 (ampC) Eaero (ampC) GN7471 (ampC)
EBAC1 MirAct1 EaeroU JapU ampCU EBAC2 MirAct2 EaeroL JapL
5PC3P 5PC3P 5PC3P 5PC3P 5PC3P 3PC5P 3PC5P 3PC5P 3PC5P
(327) -TTG ACT CGC TAT TAC GGA AGA T(327) -TTG AAC TGC TAT TAC GGA AGA T(1) -ATG ATG AAA AAA TCC CTT ACA G(379) -GCC ACA GTC AAA TCC AAC AG(33) -MYK ATG ATG ANW AAA TCC CT(1153) -GCA ATG TTT TAC TGT AGC GC(1153) -GCA ATG TTT TAC TRC AGC GC(1159) -TTA CTG CAG CGC CTC AAG AA(1145) -CTG CAG AGC GCT CAG AAT AC-
ACT-1 (ampR) ACT-1 (ampR)
ampRFWD ampRREV
5PC3P 3PC5P
(1) -ATG ACG CGC AGC TAT CTC CCT(20) (881) -TGG TGG GGA AGA TGC AGA AAT AA- (860)
(36) (36) (21) (359) (16) (1133) (1133) (1141) (1127)
a
Gene sequence basing the design of the primer. Accession numbers and references are listed in Table 1. Numbers in parentheses refer to the position relative to the ¢rst nucleotide of the start codon of the reference genes. c M = A+C, Y = C+T, K = G+T, W = A+T, R = A+G. b
2.4. Enzymes Restriction endonucleases, T4 DNA polymerase (New England Biolabs, Ozyme, St Quentin en Yvelines, France) and T4 DNA ligase (Amersham Pharmacia Biotech, Saclay, France) were used according to manufacturers’ recommendations. 2.5. PCR Genomic DNA was obtained from exponential cultures in Mueller^Hinton medium, pelleted and washed in sterile water before being applied to a QIAamp column (Qiagen, Courtaboeuf, France) following the manufacturer’s protocol for bacterial DNA. Primers were speci¢cally designed to amplify the entire ampC gene from several Enterobacter-related cephalosporinases listed in Table 3. Additional primers were designed based on the ampR sequence found upstream of the blaACT 1 gene. Primers (listed in Table 2) were synthetized by OligoExpress (Montreuil, France). Ampli¢cations were performed with Taq DNA polymerase (Roche Molecular Biochemicals, Meylan, France) and Dynazyme EXT (Finnzymes, Ozyme) following the manufacturers’ recommendations. The PE 480 thermocycler (Perkin Elmer, France) was programmed as follows: initial denaturation at 94‡C for 6 min, followed by 35 cycles of ampli¢cation (94‡C for 60 s, 50‡C for 90 s and 72‡C for 120 s), with a ¢nal extension step of 600 s at 72‡C with mineral oil overlay of the samples. 2.6. Cloning of PCR products The PCR products were modi¢ed with T4 DNA polymerase according to the manufacturer’s protocol for blunt-ending restriction fragments, and ligated in the pCR-Blunt vector. Recombinant plasmids were transformed into the chemocompetent E. coli TOP10 by heat shock. Transformants were selected on the basis of resistance to amoxicillin (50 Wg ml31 ) and kanamycin (50 Wg
ml31 ) and were further characterized by analysis of their antibiotic susceptibility pattern. The size of the insert in the recombinant plasmid was estimated by PCR, EcoRI restriction enzyme digestion and electrophoresis in 1% agarose gels. 2.7. Sequencing and sequence analysis Double-stranded DNA sequencing was performed by Genome-Express (Montreuil, France). Sequence accession numbers are listed in Table 1. The BLASTN [15] program of the NCBI was used for database searches and the ClustalW [16] program was used to align multiple DNA and translated protein sequences. Phylogenetic analysis was performed using the PHYLogeny Inference Package [17]. All these programs where run at http://bioweb.pasteur.fr using the PISE interface [18]. Trees were displayed with Treeview32 [19]. Bioedit v5.0.9 [20] was employed for general purpose sequence editing and analysis.
3. Results and discussion 3.1. L-Lactams susceptibility MICs for L-lactam antibiotics of the Enterobacter type strains are listed in Table 3. The inducible phenotype (antagonism between cefotaxime and cefoxitin or imipenem) displayed by E. cloacae and E. aerogenes was also found in Enterobacter asburiae, Enterobacter cancerogenus and Enterobacter dissolvens. A lack of inducibility was observed with Enterobacter intermedius. An isolated resistance to cephalothin was found in Enterobacter amnigenus, Enterobacter gergoviae, Enterobacter nimipressuralis and Enterobacter sakazakii. Other Enterobacter species tested did not express resistance to cephalothin nor to amoxicillin, in concordance with previously reported data [12,13]. Transformant E. coli TOP10 expressing the cloned ampC genes from E. aerogenes, E. asburiae, Enterobacter hormae-
FEMSLE 10445 14-5-02
90
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
Table 3 MICs of 13 L-lactam antibiotics (in Wg ml31 ) alone or with clavulanate (2 Wg ml31 ) or tazobactam (4 Wg ml31 ) for Enterobacter sp. Strains E. E. E. E. E. E. E. E. E. E.
a
asburiae amnigenus cancerogenusa dissolvensa gergoviae hormaechei intermedius nimipressuralis pyrinus sakazakii
AMX
AMC
TIC
TCC
PIP
TZP
CEF
FOX
CTX
CAZ
FEP
ATM
IPM
s 128 8 64 128 4 4 128 4 4 8
s 128 8 128 s 128 4 4 64 4 4 4
4 4 4 4 16 4 4 4 4 4
4 2 4 4 8 4 4 4 4 4
8 1 1 4 8 1 1 1 2 1
4 1 1 2 8 1 1 1 1 1
s 128 128 s 128 s 128 128 8 64 16 16 64
128 16 s 128 128 8 4 16 2 16 4
0.25 0.12 0.06 0.12 1 0.06 0.25 0.06 0.25 0.06
0.25 0.25 0.25 0.25 1 0.25 0.25 0.25 0.25 0.25
0.03 0.03 0.06 0.03 0.25 0.03 0.06 0.03 0.06 0.03
0.125 0.03 0.03 0.06 0.25 0.03 0.06 0.03 0.03 0.03
1 0.25 0.06 0.06 0.125 0.06 0.06 0.03 0.03 0.03
AMX : amoxicillin ; AMC : amoxicillin clavulanate ; TIC: ticarcillin ; TCC: ticarcillin clavulanate; PIP : piperacillin ; TZP: piperacillin tazobactam; CEF : cephalothin; FOX: cefoxitin; CAZ: ceftazidime; FEP: cefepime; ATM: aztreonam; IPM: imipenem. a Inducible AmpC (double disk agar di¡usion assay).
chei and E. intermedius displayed identical antibiotic susceptibility patterns (constitutive resistance to amoxicillin and amoxicillin clavulanate, cephalothin and cefoxitin). Reported cases of E. hormaechei infections [21,22] describe antibiotic susceptibility patterns similar to E. cloacae. The lack of AmpC expression in the E. hormaechei type strain is not due to a deletion or a mutation of the ampC gene since it was functional in E. coli when plasmid-borne and under the control of the pCR-Blunt promoter. 3.2. ampC and ampR ampli¢cation Previously unknown ampC genes were successfully ampli¢ed from ¢ve of the Enterobacter type strains, notably all the strains resistant to amoxicillin, cephalothin and cefoxitin as well as the one from E. hormaechei. No detectable PCR product could be obtained from the six re-
maining species using our strategy (E. amnigenus, E. gergoviae, E. nimipressuralis, E. sakazaki, E. pyrinus and Enterobacter kobei), some of which have never been shown to possess a L-lactamase [12,13]. The strains for which no results have been obtained either lack the ampC gene altogether or display excessive variability to be ampli¢ed with E. cloacae-derived primers. The sequence of the ampR gene from E. asburiae was obtained from the sequencing of the PCR product obtained by ampli¢cation of E. asburiae chromosomal DNA with the ampRFWD and ampRREV primers based on the ampR sequence upstream of blaACT 1 submitted to the sequence databases. 3.3. Sequence analysis The search for homologous sequences for each of the obtained sequences in the public databases showed that
Table 4 Identity matrix for ampC nucleotide sequencesa from enterobacteria Ec P99 GN7471 MIR-1 ACT-1 Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1 Y.ent S.mar H.alv P.stu FOX-1 Ec P99 GN7471 MIR-1 ACT-1 Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1 Y.ent S.mar H.alv P.stu FOX-1 a
1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.816 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.850 0.859 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.849 0.850 0.902 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.848 0.850 0.909 0.965 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.781 0.786 0.814 0.799 0.803 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.784 0.772 0.788 0.787 0.794 0.777 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.937 0.819 0.849 0.847 0.850 0.780 0.775 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.795 0.792 0.801 0.806 0.811 0.773 0.772 0.789 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.727 0.720 0.729 0.730 0.739 0.694 0.697 0.732 0.727 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^
0.707 0.712 0.721 0.718 0.722 0.681 0.698 0.705 0.712 0.722 1.000 ^ ^ ^ ^ ^ ^ ^ ^
Sequences are cropped to a length of 1118 bp. Matrix computed using Bioedit.
FEMSLE 10445 14-5-02
0.655 0.674 0.672 0.681 0.682 0.647 0.645 0.653 0.679 0.681 0.701 1.000 ^ ^ ^ ^ ^ ^ ^
0.602 0.594 0.605 0.606 0.604 0.587 0.587 0.608 0.590 0.594 0.584 0.581 1.000 ^ ^ ^ ^ ^ ^
0.604 0.595 0.605 0.607 0.606 0.586 0.586 0.609 0.591 0.596 0.585 0.580 0.990 1.000 ^ ^ ^ ^ ^
0.574 0.586 0.583 0.583 0.585 0.564 0.557 0.581 0.573 0.575 0.585 0.564 0.555 0.557 1.000 ^ ^ ^ ^
0.532 0.515 0.523 0.523 0.531 0.490 0.504 0.528 0.525 0.537 0.496 0.493 0.506 0.507 0.478 1.000 ^ ^ ^
0.480 0.490 0.476 0.481 0.481 0.467 0.464 0.484 0.496 0.490 0.495 0.482 0.485 0.488 0.503 0.570 1.000 ^ ^
0.455 0.460 0.456 0.455 0.452 0.432 0.430 0.462 0.453 0.441 0.449 0.458 0.464 0.464 0.467 0.552 0.531 1.000 ^
0.529 0.525 0.515 0.509 0.512 0.500 0.503 0.519 0.505 0.516 0.496 0.477 0.517 0.516 0.489 0.553 0.507 0.449 1.000
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
91
Fig. 1. Unrooted radial tree of the ClustalW alignment of AmpC putative sequences of enterobacterial origin, computed with the Neighbor program applied to a matrix calculated with Protdist (PHYLIP package). The scale bar represents a distance of 0.1 amino acid substitutions per site.
although all sequences were previously unreported, they were close matches to other ampC entries. The AmpC from E. asburiae, E. cancerogenus, E. dissolvens, E. hormaechei and E. intermedius were respectively named Eas-1, Eca-1, Edi-1, Eho-1 and Ein-1 and their nucleotide sequences submitted to the EMBL nucleotide sequence database. The identity matrix for ampC nucleotide sequences from enterobacteria is displayed Table 4. Some of the sequences obtained in this study were partial since primers MirAct2 and EBAC2 overlapped the last nine 3P bases from the E. cloacae P99 sequence, and primer EaeroU matched the ¢rst 22 5P bases of the previously published E. aerogenes ampC sequence. The neighbor-joining analysis of amino acid sequences from enterobacterial chromosomal AmpC yielded a tree shown radially Fig. 1. The plasmid-encoded cephalosporinase FOX-1 [23] of unknown origin was included as an outside reference. E. aerogenes was close to C. freundii and E. coli, while all other Enterobacter-related sequences clustered together. Within this major Enterobacter cluster, all known E. cloacae ampC sequences were within 98% nucleotide identity (only AmpC from E. cloacae P99 is shown) except for E. cloacae GN7471. AmpC from this strain belonged to a cluster gathering Eas-1 and the plasmid-encoded ACT-1 and MIR-1. The homogeneity of the E. cloacae species has been questioned from phenotypic as well as DNA hybridization standpoints [24], and the breaking-up of E. cloacae into di¡erent species (most recently E. hormaechei [25], E. kobei [26], soon Enterobacter cowanii [27]) is in
progress. AmpC from strain GN7471 was ¢rst reported in 1980 [28], a time at which the species boundaries within the Enterobacter genus were very di¡erent from what they currently are. The analysis of strain GN7471 with current molecular methods would be of interest to investigate ampC sequence polymorphism between E. cloacae strains or biogroups, or to ascertain its belonging to another species. The blaACT 1 gene sequence and the chromosomal ampC gene from E. asburiae have a 96.5% nucleotide sequence identity translating into a 98% amino acid sequence identity. To con¢rm that ACT-1 is derived from Eas-1, the sequence of the ampR gene from E. asburiae was obtained and ampR from E. asburiae was found to be 95.5% identical to ampR upstream of blaACT 1 , the closest deposited sequence. The blaACT 1 gene has been shown to be capable of mobilization from plasmid to chromosome in epidemic strains [7], we hypothesize that the original mobilization event from the chromosome of E. asburiae most likely occurred in the environment rather than in patient care settings where E. asburiae is unlikely to be present [11].
Acknowledgements This work was supported in part by a grant from the French Ministe're de la Recherche (Re¤seau be¤ta-lactamase).
FEMSLE 10445 14-5-02
92
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
References [1] Sanders Jr., W. and Sanders, C. (1997) Enterobacter spp. : pathogens poised to £ourish at the turn of the century. Clin. Microbiol. Rev. 10, 220^241. [2] Ambler, R.P. (1980) The structure of L-lactamases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 289, 321^331. [3] Honore, N., Nicolas, M.H. and Cole, S.T. (1986) Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO J. 5, 3709^3714. [4] Preston, K.E., Radomski, C.C. and Venezia, R.A. (2000) Nucleotide sequence of the chromosomal ampC gene of Enterobacter aerogenes. Antimicrob. Agents Chemother. 44, 3158^3162. [5] National Nosocomial Infections Surveillance System (2000) Semi-annual Report. Aggregated Data from the National Nosocomial Infections Surveillance (NNIS) System (http://www.cdc.gov/ncidod/hip/ NNIS/DEC2000sar.PDF). [6] Papanicolaou, G.A., Medeiros, A.A. and Jacoby, G.A. (1990) Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34, 2200^ 2209. [7] Bradford, P.A., Urban, C., Mariano, N., Projan, S.J., Rahal, J.J. and Bush, K. (1997) Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41, 563^569. [8] Barnaud, G., Arlet, G., Verdet, C., Gaillot, O., Lagrange, P.H. and Philippon, A. (1998) Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrob. Agents Chemother. 42, 2352^2358. [9] Galleni, M., Lindberg, F., Normark, S., Cole, S., Honore, N., Joris, B. and Frere, J.M. (1988) Sequence and comparative analysis of three Enterobacter cloacae ampC beta-lactamase genes and their products. J. Biochem. 250, 753^760. [10] Kuga, A., Okamoto, R. and Inoue, M. (2000) ampR gene mutations that greatly increase class C beta-lactamase activity in Enterobacter cloacae. Antimicrob. Agents Chemother. 44, 561^567. [11] Abbot, S. (1999) Klebsiella, Enterobacter, Citrobacter and Serratia. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 475^480. American Society for Microbiology, Washington, DC. [12] Muytjens, H.L. and van der Ros-van de Repe, J. (1986) Comparative in vitro susceptibilities of eight Enterobacter species, with special reference to Enterobacter sakazakii. Antimicrob. Agents Chemother. 29, 367^370. [13] Pitout, J.D., Moland, E.S., Sanders, C.C., Thomson, K.S. and Fitzsimmons, S.R. (1997) Beta-lactamases and detection of beta-lactam resistance in Enterobacter spp.. Antimicrob. Agents Chemother. 41, 35^39. [14] Nadjar, D., Rouveau, M., Verdet, C., Donay, L., Herrmann, J., Lagrange, P.H., Philippon, A. and Arlet, G. (2000) Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. FEMS Microbiol. Lett. 187, 35^40.
[15] Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [16] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [17] Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) version 3.5c, Distributed by the author. Department of Genetics, University of Washington, Seattle, WA. [18] Letondal, C. (2001) A Web interface generator for molecular biology programs in Unix. Bioinformatics 17, 73^82. [19] Page, R.D. (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357^358. [20] Hall, T. (1999) BioEdit : a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95^98. [21] Wenger, P.N. et al. (1997) An outbreak of Enterobacter hormaechei infection and colonization in an intensive care nursery. Clin. Infect. Dis. 24, 1243^1244. [22] Davin-Regli, A., Bosi, C., Charrel, R., Ageron, E., Papazian, L., Grimont, P.A., Cremieux, A. and Bollet, C. (1997) A nosocomial outbreak due to Enterobacter cloacae strains with the E. hormaechei genotype in patients treated with £uoroquinolones. J. Clin. Microbiol. 35, 1008^1010. [23] Gonzalez Leiza, M., Perez-Diaz, J.C., Ayala, J., Casellas, J.M., Martinez-Beltran, J., Bush, K. and Baquero, F. (1994) Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants. Antimicrob. Agents Chemother. 38, 2150^2157. [24] Lindh, E. and Ursing, J. (1991) Genomic groups and biochemical pro¢les of clinical isolates of Enterobacter cloacae. APMIS 99, 507^ 514. [25] O’Hara, C.M., Steigerwalt, A.G., Hill, B.C., Farmer III, J.J., Fanning, G.R. and Brenner, D.J. (1989) Enterobacter hormaechei, a new species of the family Enterobacteriaceae formerly known as enteric group 75. J. Clin. Microbiol. 27, 2046^2049. [26] Kosako, Y., Tamura, K., Sakazaki, R. and Miki, K. (1996) Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae. Curr. Microbiol. 33, 261^265. [27] Inoue, K., Sugiyama, K., Kosako, Y., Sakazaki, R. and Yamai, S. (2000) Enterobacter cowanii sp. nov., a new species of the family Enterobacteriaceae. Curr. Microbiol. 41, 417^420. [28] Minami, S., Inoue, M. and Mitsuhashi, S. (1980) Puri¢cation and properties of a cephalosporinase from Enterobacter cloacae. Antimicrob. Agents Chemother. 18, 853^857. [29] Jacoby, G.A. and Tran, J. (1999) Sequence of the MIR-1 beta-lactamase gene. Antimicrob. Agents Chemother. 43, 1759^1760. [30] Lindberg, F. and Normark, S. (1986) Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene. Eur. J. Biochem. 156, 441^445. [31] Seoane, A., Francia, M.V. and Garcia Lobo, J.M. (1992) Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica. Antimicrob. Agents Chemother. 36, 1049^1052.
FEMSLE 10445 14-5-02
www.fems-microbiology.org
Chromosomal ampC genes in Enterobacter species other than Enterobacter cloacae, and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to Enterobacter asburiae Martin Rottman
a;
a b
, Yahia Benzerara a , Be¤atrice Hanau-Berc ot b , Chantal Bizet c , Alain Philippon d , Guillaume Arlet a Service de Bacte¤riologie, Ho“pital Tenon, UFR Saint Antoine, Paris, France Service de Bacte¤riologie, Ho“pital Lariboisie're, Institut Pasteur, Paris, France c Collection de l’Institut Pasteur, Institut Pasteur, Paris, France d Service de Bacte¤riologie, Ho“pital Cochin, Paris, France First published online 12 April 2002
Abstract The amplification and sequence of ampC genes in Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei and Enterobacter intermedius bring the number of known cephalosporinase sequences from the genus Enterobacter to seven. Expression in Escherichia coli of the ampC genes from E. asburiae, E. hormaechei and E. intermedius established the functional nature of these genes. ampC from E. asburiae shows 96.5% identity to blaACT 1 encoding a plasmid-borne cephalosporinase previously believed to derive from Enterobacter cloacae. The reassignment of ACT-1 ancestry to E. asburiae is confirmed by the 95.5% identity between ampR upstream of blaACT 1 and ampR from E. asburiae. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : AmpC L-lactamase, chromosomal; AmpC L-lactamase, plasmid-encoded; AmpR transcriptional regulator; Enterobacter; Enterobacter asburiae
1. Introduction Enterobacter cloacae is a major nosocomial pathogen, the third most prevalent bacterium isolated in intensive care settings [1]. E. cloacae naturally exhibits resistance to ¢rst and second generation cephalosporins and to aminopenicillins, a mechanism mediated by the production of AmpC, a chromosomal Ambler class C L-lactamase [2]. In E. cloacae, ampC is divergently transcribed from an intercistronic promoter region shared with ampR. The transcription of ampC is under the control of the transcriptional regulator encoded by ampR [3]. The ampR^ampC genetic arrangement found in E. cloacae also exists in other bacteria with chromosomal inducible L-lactamases, in particular in Enterobacter aerogenes [4]. The constitutive hyperproduction of AmpC is of major concern since it confers resistance to most L-lactam antibiotics, sparing * Corresponding author. Present address: Service de Bacte¤riologie, Ho“pital Raymond Poincare¤, 104 Bd Raymond Poincare¤, 92380 Garches, France. Tel.: +33 (1) 47-10-79-46; Fax: +33 (1) 47-10-79-49. E-mail address : [email protected] (M. Rottman).
only carbapenems and ‘fourth generation’ cephalosporins (i.e. cefepime). This phenotype commonly occurs under antibiotic treatment, and Enterobacter isolates resistant to third generation cephalosporins account for over 30% of the isolate from intensive care units [5]. Still, this chromosomal resistance mechanism typically does not spread from strain to strain. These last decades, many authors reported outbreaks of infections due to Klebsiella pneumoniae harboring plasmidencoded cephalosporinases and the spread of this resistance mechanism to bacterial species naturally susceptible to cephamycins. While some of the plasmid-encoded class C L-lactamases are still not linked to known bacterial lineages, plasmid-encoded cephalosporinases derived from Morganella morganii, Citrobacter freundii and Hafnia alvei have been reported. These chromosomal ancestors have a nucleotide identity of over 95% with their plasmid-encoded descendants. MIR-1 [6] and ACT-1 [7] are plasmid-encoded cephalosporinases admitted to derive from E. cloacae but blaMIR 1 and blaACT 1 only share 85% identity with the ampC gene of E. cloacae. To this day the only plasmid-borne blaAmpC gene reported to be under the con-
0378-1097 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 1 3 - 4
FEMSLE 10445 14-5-02
88
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
trol of an ampR regulatory gene is derived from M. morganii [8]. The genetic variability of the ampC gene within wild-type E. cloacae strains has been studied [9], and current sequence analysis shows more than 95% nucleotide sequence identity with the exception of a single strain from Japan [10]. Very little is known about the genetic variability of the ampC gene among di¡erent species of a same genus, and E. cloacae and E. aerogenes [4] are the only examples of known ampC sequences from di¡erent species of a given enterobacterial genus. There are currently 13 recognized species in the genus Enterobacter and E. cloacae and E. aerogenes are the only species routinely isolated from human clinical specimens, the other species being mostly isolated from environmental or vegetal sources [11]. The putative L-lactamases from Enterobacter other than E. cloacae or E. aerogenes have so far only been studied in terms of the modal minimal inhibitory concentrations (MICs) of strains to L-lactam compounds, and their basic biochemical characteristics [12,13]. We have undertaken the study of ampC genes from di¡erent species belonging to the Enterobacter genus.
2. Materials and methods 2.1. Strains and sequence data The Enterobacter type strains studied were kindly provided by the Collection de l’Institut Pasteur (Institut Pasteur, Paris, France). The MIR-1 plasmid (pMG230) conjugated to Escherichia coli strain C600 was a gift from G.A. Jacoby [6]. Phenotypic identi¢cation was performed
using the commercial API 50 CHE, API 20 E and ID 32 E biochemical gallery systems from Biome¤rieux (Marcy l’Etoile, France). The pCR-Blunt (KanR) vector (Invitrogen, Cergy-Pontoise, France) was used for cloning and expression of the ampli¢ed ampC genes in TOP10 E. coli strain (Invitrogen). All nucleotide sequences are listed in Table 1. 2.2. L-Lactams susceptibility MICs of L-lactam antibiotics, alone or in association with clavulanate (2 Wg ml31 ) or tazobactam (4 Wg ml31 ), were determined by the agar dilution method with Mueller^Hinton agar (Bio-Rad, Marnes-la-Coquette, France). Inocula of 104 colony forming units per spot were delivered with a multipoint inoculator. The resistance and susceptibility patterns were determined by the disk di¡usion method on Mueller^Hinton agar. Plates were examined after overnight incubation at 37‡C. 2.3. Inducibility The inducibility of the L-lactamase was tested by a double disk agar di¡usion assay with cefotaxime (30 Wg antibiotic disk load) and ticarcillin (75 Wg) versus cefoxitin (30 Wg) and imipenem (10 Wg) as inducing compounds [14]. Antibiotic susceptibility assay disks and Mueller^Hinton agar were purchased from Bio-Rad. The inducibility of the L-lactamase assayed showed as an antagonistic image between inducing and non-inducing L-lactams, with a reduced inhibitory diameter of the non-inducing L-lactam facing the inducing L-lactam.
Table 1 Sequences used for phylogenetic analysis and identity matrices calculations Label
Gene
EMBL accession number
Origin
Reference
Ec P99 GN7471 MIR-1a ACT-1a Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1a Y.ent S.mar H.alv P.stu FOX-1a E.asb ACT-1
ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampC ampR ampR
X07274 AB016611 M37839 U58495 AJ311172 AJ311363 AJ416709 AJ311364 AJ311365 AF211348 X03866 AF124205 AF055067 Y16410 X63149 AJ271368 AJ270942 Y17315 X77455 AJ421143 AF362955
E. cloacae P99 E. cloacae GN7471 K. pneumoniae plasmid pMG230 K. pneumoniae MCQ-95 E. asburiae type strain E. dissolvens type strain E. cancerogenus type strain E. hormaechei type strain E. intermedius type strain E. aerogenes C. freundii strain ‘OS60’ E. coli strain ‘E102’ M. morganii strain ‘GUI-1’ Salmonella enteritidis strain ‘KF92’ Yersinia enterocolitica strain ‘IP97’ Serratia marcescens strain ‘SLS73’ H. alvei Providentia stuartii strain ‘VDG96’ K. pneumoniae strain ‘BA32’ E. asburiae type strain upstream blaACT 1
[9] [10] [29] [7] This This This This This [4] [30] NR [8] [8] [31] NR [14] NR [23] This NR
NR : not currently referenced. Plasmid-encoded gene.
a
FEMSLE 10445 14-5-02
study study study study study
study
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
89
Table 2 Primers used for Enterobacter spp. gene ampli¢cation and sequence Reference sequence labelsa
Name
Sense
Sequenceb;c
Ec P99 (ampC) MIR-1, ACT-1 (ampC) Eaero (ampC) GN7471 (ampC) GN7471, Ec P99 (ampC) Ec P99 (ampC) MIR-1, ACT-1 (ampC) Eaero (ampC) GN7471 (ampC)
EBAC1 MirAct1 EaeroU JapU ampCU EBAC2 MirAct2 EaeroL JapL
5PC3P 5PC3P 5PC3P 5PC3P 5PC3P 3PC5P 3PC5P 3PC5P 3PC5P
(327) -TTG ACT CGC TAT TAC GGA AGA T(327) -TTG AAC TGC TAT TAC GGA AGA T(1) -ATG ATG AAA AAA TCC CTT ACA G(379) -GCC ACA GTC AAA TCC AAC AG(33) -MYK ATG ATG ANW AAA TCC CT(1153) -GCA ATG TTT TAC TGT AGC GC(1153) -GCA ATG TTT TAC TRC AGC GC(1159) -TTA CTG CAG CGC CTC AAG AA(1145) -CTG CAG AGC GCT CAG AAT AC-
ACT-1 (ampR) ACT-1 (ampR)
ampRFWD ampRREV
5PC3P 3PC5P
(1) -ATG ACG CGC AGC TAT CTC CCT(20) (881) -TGG TGG GGA AGA TGC AGA AAT AA- (860)
(36) (36) (21) (359) (16) (1133) (1133) (1141) (1127)
a
Gene sequence basing the design of the primer. Accession numbers and references are listed in Table 1. Numbers in parentheses refer to the position relative to the ¢rst nucleotide of the start codon of the reference genes. c M = A+C, Y = C+T, K = G+T, W = A+T, R = A+G. b
2.4. Enzymes Restriction endonucleases, T4 DNA polymerase (New England Biolabs, Ozyme, St Quentin en Yvelines, France) and T4 DNA ligase (Amersham Pharmacia Biotech, Saclay, France) were used according to manufacturers’ recommendations. 2.5. PCR Genomic DNA was obtained from exponential cultures in Mueller^Hinton medium, pelleted and washed in sterile water before being applied to a QIAamp column (Qiagen, Courtaboeuf, France) following the manufacturer’s protocol for bacterial DNA. Primers were speci¢cally designed to amplify the entire ampC gene from several Enterobacter-related cephalosporinases listed in Table 3. Additional primers were designed based on the ampR sequence found upstream of the blaACT 1 gene. Primers (listed in Table 2) were synthetized by OligoExpress (Montreuil, France). Ampli¢cations were performed with Taq DNA polymerase (Roche Molecular Biochemicals, Meylan, France) and Dynazyme EXT (Finnzymes, Ozyme) following the manufacturers’ recommendations. The PE 480 thermocycler (Perkin Elmer, France) was programmed as follows: initial denaturation at 94‡C for 6 min, followed by 35 cycles of ampli¢cation (94‡C for 60 s, 50‡C for 90 s and 72‡C for 120 s), with a ¢nal extension step of 600 s at 72‡C with mineral oil overlay of the samples. 2.6. Cloning of PCR products The PCR products were modi¢ed with T4 DNA polymerase according to the manufacturer’s protocol for blunt-ending restriction fragments, and ligated in the pCR-Blunt vector. Recombinant plasmids were transformed into the chemocompetent E. coli TOP10 by heat shock. Transformants were selected on the basis of resistance to amoxicillin (50 Wg ml31 ) and kanamycin (50 Wg
ml31 ) and were further characterized by analysis of their antibiotic susceptibility pattern. The size of the insert in the recombinant plasmid was estimated by PCR, EcoRI restriction enzyme digestion and electrophoresis in 1% agarose gels. 2.7. Sequencing and sequence analysis Double-stranded DNA sequencing was performed by Genome-Express (Montreuil, France). Sequence accession numbers are listed in Table 1. The BLASTN [15] program of the NCBI was used for database searches and the ClustalW [16] program was used to align multiple DNA and translated protein sequences. Phylogenetic analysis was performed using the PHYLogeny Inference Package [17]. All these programs where run at http://bioweb.pasteur.fr using the PISE interface [18]. Trees were displayed with Treeview32 [19]. Bioedit v5.0.9 [20] was employed for general purpose sequence editing and analysis.
3. Results and discussion 3.1. L-Lactams susceptibility MICs for L-lactam antibiotics of the Enterobacter type strains are listed in Table 3. The inducible phenotype (antagonism between cefotaxime and cefoxitin or imipenem) displayed by E. cloacae and E. aerogenes was also found in Enterobacter asburiae, Enterobacter cancerogenus and Enterobacter dissolvens. A lack of inducibility was observed with Enterobacter intermedius. An isolated resistance to cephalothin was found in Enterobacter amnigenus, Enterobacter gergoviae, Enterobacter nimipressuralis and Enterobacter sakazakii. Other Enterobacter species tested did not express resistance to cephalothin nor to amoxicillin, in concordance with previously reported data [12,13]. Transformant E. coli TOP10 expressing the cloned ampC genes from E. aerogenes, E. asburiae, Enterobacter hormae-
FEMSLE 10445 14-5-02
90
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
Table 3 MICs of 13 L-lactam antibiotics (in Wg ml31 ) alone or with clavulanate (2 Wg ml31 ) or tazobactam (4 Wg ml31 ) for Enterobacter sp. Strains E. E. E. E. E. E. E. E. E. E.
a
asburiae amnigenus cancerogenusa dissolvensa gergoviae hormaechei intermedius nimipressuralis pyrinus sakazakii
AMX
AMC
TIC
TCC
PIP
TZP
CEF
FOX
CTX
CAZ
FEP
ATM
IPM
s 128 8 64 128 4 4 128 4 4 8
s 128 8 128 s 128 4 4 64 4 4 4
4 4 4 4 16 4 4 4 4 4
4 2 4 4 8 4 4 4 4 4
8 1 1 4 8 1 1 1 2 1
4 1 1 2 8 1 1 1 1 1
s 128 128 s 128 s 128 128 8 64 16 16 64
128 16 s 128 128 8 4 16 2 16 4
0.25 0.12 0.06 0.12 1 0.06 0.25 0.06 0.25 0.06
0.25 0.25 0.25 0.25 1 0.25 0.25 0.25 0.25 0.25
0.03 0.03 0.06 0.03 0.25 0.03 0.06 0.03 0.06 0.03
0.125 0.03 0.03 0.06 0.25 0.03 0.06 0.03 0.03 0.03
1 0.25 0.06 0.06 0.125 0.06 0.06 0.03 0.03 0.03
AMX : amoxicillin ; AMC : amoxicillin clavulanate ; TIC: ticarcillin ; TCC: ticarcillin clavulanate; PIP : piperacillin ; TZP: piperacillin tazobactam; CEF : cephalothin; FOX: cefoxitin; CAZ: ceftazidime; FEP: cefepime; ATM: aztreonam; IPM: imipenem. a Inducible AmpC (double disk agar di¡usion assay).
chei and E. intermedius displayed identical antibiotic susceptibility patterns (constitutive resistance to amoxicillin and amoxicillin clavulanate, cephalothin and cefoxitin). Reported cases of E. hormaechei infections [21,22] describe antibiotic susceptibility patterns similar to E. cloacae. The lack of AmpC expression in the E. hormaechei type strain is not due to a deletion or a mutation of the ampC gene since it was functional in E. coli when plasmid-borne and under the control of the pCR-Blunt promoter. 3.2. ampC and ampR ampli¢cation Previously unknown ampC genes were successfully ampli¢ed from ¢ve of the Enterobacter type strains, notably all the strains resistant to amoxicillin, cephalothin and cefoxitin as well as the one from E. hormaechei. No detectable PCR product could be obtained from the six re-
maining species using our strategy (E. amnigenus, E. gergoviae, E. nimipressuralis, E. sakazaki, E. pyrinus and Enterobacter kobei), some of which have never been shown to possess a L-lactamase [12,13]. The strains for which no results have been obtained either lack the ampC gene altogether or display excessive variability to be ampli¢ed with E. cloacae-derived primers. The sequence of the ampR gene from E. asburiae was obtained from the sequencing of the PCR product obtained by ampli¢cation of E. asburiae chromosomal DNA with the ampRFWD and ampRREV primers based on the ampR sequence upstream of blaACT 1 submitted to the sequence databases. 3.3. Sequence analysis The search for homologous sequences for each of the obtained sequences in the public databases showed that
Table 4 Identity matrix for ampC nucleotide sequencesa from enterobacteria Ec P99 GN7471 MIR-1 ACT-1 Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1 Y.ent S.mar H.alv P.stu FOX-1 Ec P99 GN7471 MIR-1 ACT-1 Eas-1 Edi-1 Eca-1 Eho-1 Ein-1 Eaero C.fre E.col M.mor DHA-1 Y.ent S.mar H.alv P.stu FOX-1 a
1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.816 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.850 0.859 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.849 0.850 0.902 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.848 0.850 0.909 0.965 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.781 0.786 0.814 0.799 0.803 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.784 0.772 0.788 0.787 0.794 0.777 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.937 0.819 0.849 0.847 0.850 0.780 0.775 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.795 0.792 0.801 0.806 0.811 0.773 0.772 0.789 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
0.727 0.720 0.729 0.730 0.739 0.694 0.697 0.732 0.727 1.000 ^ ^ ^ ^ ^ ^ ^ ^ ^
0.707 0.712 0.721 0.718 0.722 0.681 0.698 0.705 0.712 0.722 1.000 ^ ^ ^ ^ ^ ^ ^ ^
Sequences are cropped to a length of 1118 bp. Matrix computed using Bioedit.
FEMSLE 10445 14-5-02
0.655 0.674 0.672 0.681 0.682 0.647 0.645 0.653 0.679 0.681 0.701 1.000 ^ ^ ^ ^ ^ ^ ^
0.602 0.594 0.605 0.606 0.604 0.587 0.587 0.608 0.590 0.594 0.584 0.581 1.000 ^ ^ ^ ^ ^ ^
0.604 0.595 0.605 0.607 0.606 0.586 0.586 0.609 0.591 0.596 0.585 0.580 0.990 1.000 ^ ^ ^ ^ ^
0.574 0.586 0.583 0.583 0.585 0.564 0.557 0.581 0.573 0.575 0.585 0.564 0.555 0.557 1.000 ^ ^ ^ ^
0.532 0.515 0.523 0.523 0.531 0.490 0.504 0.528 0.525 0.537 0.496 0.493 0.506 0.507 0.478 1.000 ^ ^ ^
0.480 0.490 0.476 0.481 0.481 0.467 0.464 0.484 0.496 0.490 0.495 0.482 0.485 0.488 0.503 0.570 1.000 ^ ^
0.455 0.460 0.456 0.455 0.452 0.432 0.430 0.462 0.453 0.441 0.449 0.458 0.464 0.464 0.467 0.552 0.531 1.000 ^
0.529 0.525 0.515 0.509 0.512 0.500 0.503 0.519 0.505 0.516 0.496 0.477 0.517 0.516 0.489 0.553 0.507 0.449 1.000
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
91
Fig. 1. Unrooted radial tree of the ClustalW alignment of AmpC putative sequences of enterobacterial origin, computed with the Neighbor program applied to a matrix calculated with Protdist (PHYLIP package). The scale bar represents a distance of 0.1 amino acid substitutions per site.
although all sequences were previously unreported, they were close matches to other ampC entries. The AmpC from E. asburiae, E. cancerogenus, E. dissolvens, E. hormaechei and E. intermedius were respectively named Eas-1, Eca-1, Edi-1, Eho-1 and Ein-1 and their nucleotide sequences submitted to the EMBL nucleotide sequence database. The identity matrix for ampC nucleotide sequences from enterobacteria is displayed Table 4. Some of the sequences obtained in this study were partial since primers MirAct2 and EBAC2 overlapped the last nine 3P bases from the E. cloacae P99 sequence, and primer EaeroU matched the ¢rst 22 5P bases of the previously published E. aerogenes ampC sequence. The neighbor-joining analysis of amino acid sequences from enterobacterial chromosomal AmpC yielded a tree shown radially Fig. 1. The plasmid-encoded cephalosporinase FOX-1 [23] of unknown origin was included as an outside reference. E. aerogenes was close to C. freundii and E. coli, while all other Enterobacter-related sequences clustered together. Within this major Enterobacter cluster, all known E. cloacae ampC sequences were within 98% nucleotide identity (only AmpC from E. cloacae P99 is shown) except for E. cloacae GN7471. AmpC from this strain belonged to a cluster gathering Eas-1 and the plasmid-encoded ACT-1 and MIR-1. The homogeneity of the E. cloacae species has been questioned from phenotypic as well as DNA hybridization standpoints [24], and the breaking-up of E. cloacae into di¡erent species (most recently E. hormaechei [25], E. kobei [26], soon Enterobacter cowanii [27]) is in
progress. AmpC from strain GN7471 was ¢rst reported in 1980 [28], a time at which the species boundaries within the Enterobacter genus were very di¡erent from what they currently are. The analysis of strain GN7471 with current molecular methods would be of interest to investigate ampC sequence polymorphism between E. cloacae strains or biogroups, or to ascertain its belonging to another species. The blaACT 1 gene sequence and the chromosomal ampC gene from E. asburiae have a 96.5% nucleotide sequence identity translating into a 98% amino acid sequence identity. To con¢rm that ACT-1 is derived from Eas-1, the sequence of the ampR gene from E. asburiae was obtained and ampR from E. asburiae was found to be 95.5% identical to ampR upstream of blaACT 1 , the closest deposited sequence. The blaACT 1 gene has been shown to be capable of mobilization from plasmid to chromosome in epidemic strains [7], we hypothesize that the original mobilization event from the chromosome of E. asburiae most likely occurred in the environment rather than in patient care settings where E. asburiae is unlikely to be present [11].
Acknowledgements This work was supported in part by a grant from the French Ministe're de la Recherche (Re¤seau be¤ta-lactamase).
FEMSLE 10445 14-5-02
92
M. Rottman et al. / FEMS Microbiology Letters 210 (2002) 87^92
References [1] Sanders Jr., W. and Sanders, C. (1997) Enterobacter spp. : pathogens poised to £ourish at the turn of the century. Clin. Microbiol. Rev. 10, 220^241. [2] Ambler, R.P. (1980) The structure of L-lactamases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 289, 321^331. [3] Honore, N., Nicolas, M.H. and Cole, S.T. (1986) Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO J. 5, 3709^3714. [4] Preston, K.E., Radomski, C.C. and Venezia, R.A. (2000) Nucleotide sequence of the chromosomal ampC gene of Enterobacter aerogenes. Antimicrob. Agents Chemother. 44, 3158^3162. [5] National Nosocomial Infections Surveillance System (2000) Semi-annual Report. Aggregated Data from the National Nosocomial Infections Surveillance (NNIS) System (http://www.cdc.gov/ncidod/hip/ NNIS/DEC2000sar.PDF). [6] Papanicolaou, G.A., Medeiros, A.A. and Jacoby, G.A. (1990) Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34, 2200^ 2209. [7] Bradford, P.A., Urban, C., Mariano, N., Projan, S.J., Rahal, J.J. and Bush, K. (1997) Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41, 563^569. [8] Barnaud, G., Arlet, G., Verdet, C., Gaillot, O., Lagrange, P.H. and Philippon, A. (1998) Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrob. Agents Chemother. 42, 2352^2358. [9] Galleni, M., Lindberg, F., Normark, S., Cole, S., Honore, N., Joris, B. and Frere, J.M. (1988) Sequence and comparative analysis of three Enterobacter cloacae ampC beta-lactamase genes and their products. J. Biochem. 250, 753^760. [10] Kuga, A., Okamoto, R. and Inoue, M. (2000) ampR gene mutations that greatly increase class C beta-lactamase activity in Enterobacter cloacae. Antimicrob. Agents Chemother. 44, 561^567. [11] Abbot, S. (1999) Klebsiella, Enterobacter, Citrobacter and Serratia. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 475^480. American Society for Microbiology, Washington, DC. [12] Muytjens, H.L. and van der Ros-van de Repe, J. (1986) Comparative in vitro susceptibilities of eight Enterobacter species, with special reference to Enterobacter sakazakii. Antimicrob. Agents Chemother. 29, 367^370. [13] Pitout, J.D., Moland, E.S., Sanders, C.C., Thomson, K.S. and Fitzsimmons, S.R. (1997) Beta-lactamases and detection of beta-lactam resistance in Enterobacter spp.. Antimicrob. Agents Chemother. 41, 35^39. [14] Nadjar, D., Rouveau, M., Verdet, C., Donay, L., Herrmann, J., Lagrange, P.H., Philippon, A. and Arlet, G. (2000) Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. FEMS Microbiol. Lett. 187, 35^40.
[15] Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. [16] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [17] Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) version 3.5c, Distributed by the author. Department of Genetics, University of Washington, Seattle, WA. [18] Letondal, C. (2001) A Web interface generator for molecular biology programs in Unix. Bioinformatics 17, 73^82. [19] Page, R.D. (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357^358. [20] Hall, T. (1999) BioEdit : a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95^98. [21] Wenger, P.N. et al. (1997) An outbreak of Enterobacter hormaechei infection and colonization in an intensive care nursery. Clin. Infect. Dis. 24, 1243^1244. [22] Davin-Regli, A., Bosi, C., Charrel, R., Ageron, E., Papazian, L., Grimont, P.A., Cremieux, A. and Bollet, C. (1997) A nosocomial outbreak due to Enterobacter cloacae strains with the E. hormaechei genotype in patients treated with £uoroquinolones. J. Clin. Microbiol. 35, 1008^1010. [23] Gonzalez Leiza, M., Perez-Diaz, J.C., Ayala, J., Casellas, J.M., Martinez-Beltran, J., Bush, K. and Baquero, F. (1994) Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants. Antimicrob. Agents Chemother. 38, 2150^2157. [24] Lindh, E. and Ursing, J. (1991) Genomic groups and biochemical pro¢les of clinical isolates of Enterobacter cloacae. APMIS 99, 507^ 514. [25] O’Hara, C.M., Steigerwalt, A.G., Hill, B.C., Farmer III, J.J., Fanning, G.R. and Brenner, D.J. (1989) Enterobacter hormaechei, a new species of the family Enterobacteriaceae formerly known as enteric group 75. J. Clin. Microbiol. 27, 2046^2049. [26] Kosako, Y., Tamura, K., Sakazaki, R. and Miki, K. (1996) Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae. Curr. Microbiol. 33, 261^265. [27] Inoue, K., Sugiyama, K., Kosako, Y., Sakazaki, R. and Yamai, S. (2000) Enterobacter cowanii sp. nov., a new species of the family Enterobacteriaceae. Curr. Microbiol. 41, 417^420. [28] Minami, S., Inoue, M. and Mitsuhashi, S. (1980) Puri¢cation and properties of a cephalosporinase from Enterobacter cloacae. Antimicrob. Agents Chemother. 18, 853^857. [29] Jacoby, G.A. and Tran, J. (1999) Sequence of the MIR-1 beta-lactamase gene. Antimicrob. Agents Chemother. 43, 1759^1760. [30] Lindberg, F. and Normark, S. (1986) Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene. Eur. J. Biochem. 156, 441^445. [31] Seoane, A., Francia, M.V. and Garcia Lobo, J.M. (1992) Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica. Antimicrob. Agents Chemother. 36, 1049^1052.
FEMSLE 10445 14-5-02