Characterization of IS26-composite transposons and multidrug resistance in
conjugative plasmids from Enterobacter cloacae
Chih-Ming Chen1, †, Wen-Liang Yu2,3,4, †, Mei Huang5, Jau-Jin Liu6, I-Chien Chen6, Huei-Fen
Chen6, and Lii-Tzu Wu6
1Division of Infectious Disease, Department of Internal Medicine, Tungs’ Taichung
MetroHarbor Hospital, Taichung, Taiwan
2Departments of Medical Research and 3Intensive Care Medicine, Chi-Mei Medical Center,
Yungkang City, Tainan, Taiwan
4Department of Medicine, Taipei Medical University, Taipei, Taiwan
5Division of Infectious Disease, Chang Bing Show Chwan Memorial Hospital, Changhua
County, Taiwan
6The Institute of Medical Science and Department of Microbiology, China Medical
University and Hospital, Taichung, Taiwan
Running title: IS26-composite transposon and resistance
†These authors contributed equally to this work.
Correspondence
Lii-Tzu Wu , The Institute of Medical Science and Department of Microbiology, China Medical University, 91 Hsueh-Shih Road, Taichung City 404, Taiwan, ROC
Tel: +886-4-22053366-2169; Fax: +886-4-22053764 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 3
E-mail address: [email protected] (L.T. Wu) 22
ABSTRACT
SHV-12 is the most widespread resistance determinant of Enterobacter cloacae in Taiwan; however, blaSHV-12 has rarely been mobilized. Six multidrug-resistant E. cloacae isolates were
collected. After conjugal transfer, plasmid profiling and analysis of incompatibility groups was performed to characterize the genetic context of blaSHV-12-containing fragments. The
presence of mobile genetic elements was demonstrated by PCR, cloning, sequencing, and bioinformatics analyses. Four different β-lactamase genes (blaTEM-1, blaSHV-12, blaCTX-M-3 and/or
blaCTX-M-14) were observed in the conjugative plasmid belonging to the IncHI2 (n=4), IncI1, or
IncP incompatibility groups. The IS26-blaSHV-12-IS26 locus was located in five different
genetic environments. A novel structural organization of a class 1 integron with the
aac(6')-IIc cassette truncated by IS26 was identified in one isolate. Thus, blaSHV-12 was obtained from
different plasmids through IS26-mediated homologous recombination. IS26 plays a vital role in the distribution of mobile resistance elements between different plasmids found in multidrug-resistant E. cloacae isolates.
Keywords blaSHV-12, Enterobacter cloacae, homologous recombination, IS26-blaSHV-12-IS26
locus 10 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 11
List of Abbreviations: CLSI, Clinical and Laboratory Standards Institute; CMUH, China
Medical University Hospital; ESBL, extended-spectrum β-lactamases; MDR, multidrug-resistant; MIC, minimal inhibitory concentration; PCR, polymerase chain reactions; PFGE, pulsed-field gel electrophoresis
40 41 42 43
INTRODUCTION
The emergence of multidrug resistance Enterobacter cloacae strains has become a global concern (1). The SHV-type, CTX-M-type extended-spectrum β-lactamases (ESBLs), and/or chromosomally encoded AmpC β-lactamases are widely distributed among E. cloacae, where they confer resistance to broad-spectrum β-lactam-containing antibiotics (2-6). As with most antibiotic resistance genes that are carried by plasmids and transposons and result in high clonal dissemination, extended-spectrum β-lactamase genes are located on large conjugative plasmids, thus facilitating intra- and inter-species spread (6, 7).
Previous studies have demonstrated that SHV-12 is the most frequently identified ESBL in several enterobacterial species, including E. cloacae, recovered from hospitalized patients in Taiwan (4, 8, 9). Genetic mechanisms involved in the acquisition of blaSHV have been
described, and IS26-mediated mobilization of blaSHV-containing fragments of Klebsiella
chromosome has been frequently observed (10-12). Among these gene-transferring mechanisms, a composite transposon containing blaSHV-5, duplicated in tandem and flanked by
IS26 copies on a 70-kb conjugative plasmid (pHNM1), was detected in an E. cloacae strain (13). Moreover, IS26 elements associated with integrons generated novel multidrug resistance loci in Salmonella genomic island 1 (14). Recently, blaSHV-12, flanked by two IS26
elements, has been reported in Pseudomonas aeruginosa (15), indicating that blaSHV-12 is no
longer limited to Enterobacteriaceae. These multidrug-resistant (MDR) regions harboring 18 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 19
several IS26 elements linked to antibiotic resistance genes and composite transposon structures prompted us to further investigate the IS26 structure surrounding blaSHV-12 and to
compare the IS26-blaSHV-12-IS26 locus flanking regions.
The present study was conducted to analyze the molecular basis for antibiotic resistance. The detailed genetic environments of IS26-blaSHV-12 in E. cloacae were studied to enable
tracking of mobility between plasmids, such as IS26-mediated mobilization, that result in further dissemination. 63 64 65 66 67 68 69 23
MATERIALS AND METHODS Bacterial strains and plasmid
Non-repetitive E. cloacae clinical isolates (n = 6) with blaSHV-12 were obtained from the
Microbiology Laboratory of the China Medical University Hospital (CMUH), which is located in central Taiwan, were included in this study. The isolates were identified on the basis of routine microbiologic methods, and species identification was confirmed using the VITEK system (BioMerieux Vitek, Inc., Hazelwood, MO, USA). Escherichia coli J53 (sodium azide-resistant), E. coli DH5, and E23 (non-ESBL and cefotaxime-susceptible E.
cloacae clinical isolate) were used as recipients in conjugation, transformation, and
electroporation experiments, respectively. The kanamycin-resistant plasmids, pBKCMV (Stratagene, La Jolla, CA, USA) and pOK-12 (16), were used as the vector in the molecular cloning experiments.
Confirmatory testing of the extended-spectrum -lactamase phenotype and
antimicrobial susceptibility testing
Double-disk synergy tests and Clinical and Laboratory Standards Institute (CLSI) minimal inhibitory concentration (MIC) testing were undertaken to confirm ESBL production, as described previously (17). Susceptibilities to various antimicrobial agents, including cefotaxime, cefotaxime/clavulanic acid, ceftazidime, ceftazidime/clavulanic acid, cefepime, cefepime/clavulanic acid, amoxicillin-clavulanic acid, imipenem, chloramphenicol, trimethoprim, and aminoglycosides, were tested by the disk diffusion method, as described 26 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 27
by the CLSI. Quality control was assured by testing E. coli ATCC 25922 and Klebsiella
pneumoniae 700603.
Polymerase chain reaction amplification and plasmid profiles
Polymerase chain reaction (PCR) amplification was used to identify the presence of blaTEM-1,
blaSHV-12, blaCTX-M, and blaAmpC, and the insertion sequence. Plasmid DNA was extracted using
a commercial plasmid DNA purification kit (Genemark, Taichung, Taiwan). The oligonucleotide primer sets specific for the -lactamase genes and insertion sequences used in the PCR assays are shown in Table 1 (18-24). The replicon typing method based on PCR amplification and sequence typing was undertaken, as previously described (25). The amplicons were purified using a commercially available kit (Roche Diagnostics, Mannheim, Germany) and sequenced using an ABI PRISM 377 sequencer analyser (Applied Biosystems, Foster City, CA, USA). Sequence analyses were performed online at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Plasmids bearing blaSHV-12
were extracted from transconjugants and digested with EcoRI (Takara Shuzo, Kyoto, Japan). The resulting fragments were separated by electrophoresis on a 1% agarose gel.
Transfer of resistance determinants
Transfer of the β-lactam resistance element from E. cloacae to E. coli J53 (Azir) was
achieved using solid mating assays. The donor cells (E. cloacae) were grown with shaking at 37 °C overnight in Lauria-Bertani medium containing 2 μg of cefotaxime/ml. The recipient cells (E. coli J53) were grown for 24 h at 37 °C in fresh LB broth (30 ml in 250-ml flasks) 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 31
containing 100 ug/ml of sodium azide. Two strains were separately inoculated with an initial
OD600 of 0.2, and grown until the OD600 was 1.0. The cells were mixed (donor:recipient
[1:10]) and centrifuged at 5000 × g for 5 min. The pellet was resuspended in a minimal volume of LB broth sufficient to suspend the cells. The cell suspension was dropped onto a piece of nylon membrane (4 cm2) on an LB agar plate. After incubating at 37 °C for 16 h, the
conjugation mixture was washed once with 10 mM MgSO4, then the transconjugants were
selected on trypticase soy agar plates (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 100 μg/mL of sodium azide and 2 μg/mL of cefotaxime (Sigma, St. Louis, MO, USA). Plasmids were extracted from E. coli J53 transconjugants using a Plasmid Miniprep Purification Kit (Amersham Biosciences, Uppsala, Sweden). Transformation of resistance determinants to E. cloacae cells was achieved by electroporation (26).
Cloning and sequencing of the genetic environment of blaSHV
The resistance plasmids of the transconjugants were partially digested with the Sau3AI restriction enzyme (Takara Shuzou), and the digested fragments were ligated into the BamHI site of pBKCMV. E. coli DH5 transformants were preselected on Luria-Bertani agar containing ampicillin (50 µg/mL) and subsequently selected on Luria-Bertani agar containing cefotaxime (2 µg/mL). The PCR assays, purifying DNA from an agarose gel, cloning strategy, and restriction endonuclease mapping of the recombinant clones were performed, as previously described (27). The DNA purification and recombinant clones were purified using 34 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 35
Plus Gel Elution Kit and Qiaquick Plasmid Purification Kit (Genemark GMbiolab Co, Taiwan), and the inserts were mapped and sequenced.
Southern blot hybridization
DNA samples were analyzed by electrophoresis at 50 V for 1 h in 1 w/v agarose gel,﹪ denatured, then the gel was neutralized on Whatman filter paper (Maidstone, UK), saturated sequentially with 10% w/v sodium dodecyl sulfate, denaturation solution (0.5 M NaOH and 1.5 M NaCl), and neutralization solution (0.5 M Tris-HCl [pH 7.5] and 1.5 M NaCl) for 15 min each. The DNA was transferred to a positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany) using an electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA, USA). A probe for blaSHV-12 was prepared by random labeling the
820-bp PCR product of blaSHV-12 with the digoxigenin (Digoxigenin Labeling and Detection
Kit; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Pulsed-field gel electrophoresis (PFGE)
Whole chromosomal DNA of clinical isolates embedded in agarose gel plugs (FMC Bioproduct, Rockland, ME, USA) were treated with proteinase K and restriction endonuclease XbaI according to the manufacturer's recommendations(New England Biolabs, Beverly, MA, USA). The restriction fragments were separated by electrophoresis in a CHEF-DR 3 apparatus (Bio-Rad Laboratories) for 24 h. SmaI-digested DNA from Staphylococcus
aureus NCTC 8325 were used as molecular size markers. The CHEF run conditions were as
follows: 1.0% SeaKem LE agarose; 12 °C; 21 h; 6 V/cm; and a 2.2-54.2-s linear switch ramp. 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 39
PFGEpatterns were compared using standard published criteria (28).
Nucleotide sequence accession numbers
The complete nucleotide sequences of the individual Sau3AI fragments can be found in GenBank under accession numbers DQ247972 (pE71), GU205813 (pE19), GU299861 (pE81), and GU206876 (pE80).
Ethics
This study was approved by the Institutional Research Committee of China Medical University and was deemed exempt from the Institutional Review Board.
42 149 150 151 152 153 154 155 156 157 43
RESULTS
Antibiotic susceptibility and characterization of plasmid -lactamases
Six E. cloacae isolates (E17, E19, E71, E80, E81, and E95) highly resistant to cefotaxime, ceftazidime, and/or cefepime were identified as ESBL-producers by double-disk synergy tests and MIC-based methods. PCR and subsequent sequence analysis revealed that all 6 isolates carried the encoding genes of TEM-1, SHV-12, and AmpC-like β-lactamase. In addition, 2 isolates produced the CTX-M-3 enzyme (E17 and E80) and 3 isolates produced the CTX-M-14 enzyme (E71, E81, and E95; Table 2).
Next, the blaTEM-1, blaSHV-12, and blaCTX-M -lactam resistance genes of E. cloacae were
transferred to E. coli J53 AzideR in conjugation experiments. The transconjugants with
IncHI2, IncI1, and IncP incompatibility group plasmids exhibited resistance to chloramphenicol, trimethoprim, aminoglycosides (data not shown), and several -lactams; however, the incompatibility group plasmids remained susceptible to imipenem (Table 2). The transconjugants (T17, T19, T71, T80, T81, and T95) were obtained by mating out in the presence of sodium azide and cefotaxime to confirm the presence of -lactamase genes of
blaSHV-12 on the plasmid DNA. The conjugation efficiencies were 10-5–10-8 recombinations per
donor cell (Table 2). The gene of blaAmpC was not transferred to the transconjugants, which
was confirmed by PCR analysis. The nucleotide sequences of 6 ampC (E17, E19, E71, E80, E81, and E95) showed close similarity to the native chromosomal ampC gene (blaEcloMHN1) of 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 47
Enterobacter cloacae MHN1 (99.9, 99.7, 99.7, 99.7, 99.6, and 99.6% identity, respectively).
Epidemiologic characterization
Among the 6 non-repetitive E. cloacae isolates, E17 and E19 were isolated from urine, E80 and E81 originated from blood, E71 was recovered from wound purulent drainage, and E95 was derived from a central venous catheter tip. Five different Xba1-digested PFGE profiles of the 6 E. cloacae isolates analyzed in this study were obtained according to the interpretation criteria of Tenover et al. (28; Fig. 1A). The DNA fingerprinting of the identified transconjugant plasmids had different EcoR1-digested patterns in 6 isolates (Fig. 1B). There was no evidence of clonal dissemination in this hospital during the period of the study.
The blaSHV-12 was flanked by two copies ofan identical IS26 element
To investigate the possible transfer mechanisms for blaSHV-12, the recombinant clones
encoding SHV-12 were selected from the first PCR screening. Six of the recombinant plasmids (pE17-1, pE19-1, pE71-2, pE80-3, pE81-1, and pE95-2) containing Sau3AI fragments of approximately 8.1, 8.9, 8.9, 9.5, 5.4, and 8.9 kb, respectively, were retained for further study. Sequence analysis of the Sau3AI fragment inserts revealed that blaSHV-12 was
flanked by two copies ofan identical IS26 element that were in the same orientation (Fig. 2). In pE17-1, pE19-1, pE71-2, pE80-3, and pE95-2, the IS26-blaSHV-12-IS26 locus had a similar
genetic structure, comprising the following five open reading frames: truncated fuculose-1-phosphate aldolase (fucA); putative tRNA synthase (ygbK); putative oxidoreductase (ygbJ); DeoR-type putative transcriptional regulator (deoR); and blaSHV-12. Analysis of pE81-1
50 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 51
revealed that the sequences downstream from blaSHV12 shared homology with IS903. The
ygbK-fucA gene is adjacent to IS26 by the other flanking genes secondary to an
IS26-mediated recombination event.
The IS26-blaSHV-12-IS26 composite transposon boundary by different genetic contexts
As shown in Fig. 2, different genetic environments were identified in the IS26-blaSHV-12-IS26
composite transposon. Specifically, the ygbK and 3’ truncated fucA (ΔfucA) genes were tandem duplications in two plasmids (pE19-1 and pE81-1). In pE17-1 and pE19-1, the sequence upstream of the second IS26 retained 100% DNA identity with a part of pKPS77 from K. pneumoniae KPS77, and was truncated in the 5’ end of IS26 (ΔIS26) by a transposase gene (tnpA). The IS26-blaSHV-12-IS26 composite transposon revealed the same
configuration in pE80-3 and pE95-2. The 106 bp downstream region of the first IS26 contained a resolvase gene (res), and the sequence upstream of the second IS26 contained a chloramphenicol acetyltransferase gene (catA2) followed by another IS26.
In pE71-2, the IS26-blaSHV-12 composite transposon truncated an aac(6')-IIc cassette in
the first position in a class 1 integron. The aac(6')-IIc cassette is found in a number of different GenBank entries. The intI1 gene, with its own promoter region and an adjacent recombination site (attI [GTTAGAC]), was found upstream of the aac(6')-IIc (Fig. 3), indicating that IS26-blaSHV-12 was indeed inserted in the gene cassette of class 1 integrons.
The region to the right-hand of IS26 is relative to the direction of the tnpA and between them 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 55
with a partial res gene.
Analysis of the exact insertion site did not demonstrate the presence of a duplicated target site at the external flanks of the two IS26 elements. The IS26-blaSHV-12 composite
transposon truncated an aac(6')-IIc cassette in the first position in a class 1 integron;
however, it lacked the usual class 1 integron-associated 3’- CS due the insertion of IS26. The insertion of IS26 presumably caused the deletions in aac(6’)-IIc in strain E71 (Fig. 3). In sequences downstream of the extreme right IS26 in pE71-2, a Tn3-based transposon was located, including its tnpA and res genes.
Transposition of the blaSHV by IS26-composite transposons into non-ESBL E. cloacae
strain
The pE71-2 plasmid was electroporated into a non-ESBL strain (E23) to test for transposition ability. A single colony of a test strain (E23-1) was picked from a selective plate with cefotaxime (2 g/ml) and grown overnight. The culture was diluted 1000-fold into fresh LB broth with antibiotics and grown to saturation (OD550 of approximately 5.0). E23-5 was
constructed by the dilution and growth E23-1 in fresh medium and was performed 5 times in which each cycle constituted approximately 10 generations. Using a blaSHV PCR DNA
fragment as a probe, one hybridizing band was seen at the start of the first culture isolate (E23-1) and approximately 2 hybridizing bands (3.6 and 4.5 kb) were seen at the end isolate (E23-6) of the repeat culture experiment, meaning that approximately 2 copies were
58 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 59
presented in the E23-6 genome (Fig.4). For comparison of genetic environments of the two hybridizing bands, we purified the DNA of BamHI band of the 3.6 and 4.5 kb from an agarose gel for cloning and sequencing. The BamHI fragments were cloned to plasmid pOK-12 and designated as pE23-3.6 and pE23-4.5. To sequence and analysis the two inserts of the 3.6 and 4.5 kb fragments from E23-6 genome revealed different genetic contexts in upstream of blaSHV-12. The results indicated the recombination through IS26 in mobilization by the
plasmid of pE71-2 and the schematic map is presented in Figure 5.
235 236 237 238 239 240 241 63
DISCUSSION
The 6 E. cloacae isolates had different PFGE and plasmid profiles, but 4 isolates carried an IncHI2 replicon. Large IncHI2-plasmids encode resistance genes associated with mobile genetic elements, which supported frequent events of acquisition, loss, and rearrangements of genetic material and was often identical in global clinical Enterobacteriaceae strains (7, 29, 30). The genes encoding SHV-12 and CTX-M-3 enzymes have been observed on the same plasmid among E. cloacae, E. coli, and K. pneumoniae isolates in Taiwan (2, 31, 32). Moreover, the present study showed that the blaSHV-12 and blaCTX-M-14 were located on the
transconjugant plasmid found in E. cloacae isolates, indicating that multiple plasmid-based β-lactamases are frequently found in E. cloacae with variations of the CTX-M subtype in Taiwan. In addition, the association between blaCTX-M-type and blaSHV-12 on IncHI2 plasmids was
identified. This assortment of multiple extended-spectrum β-lactamases genes on the plasmid could suggest the presence of larger transferable resistance island. Furthermore, PCR and DNA sequencing analysis of the AmpC alleles of 6 E. cloacae isolates revealed a 1,165-bp PCR fragment with 99.9% and 99.7% identity to blaEcloK995120.1 and blaEcloK9973, respectively; the
chromosomal ampC of E. cloacae has been reported in Korea (33). The results from DNA sequencing analysis suggested that 6 genes of ampC could originate from chromosomal
ampC of blaEcloMHN1(34).
In the present study we have further characterized the structure beyond the blaSHV-12/IS26
66 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 67
element in plasmids of different incompatibility groups. The blaSHV-12 was flanked by two
IS26 elements; two isolated plasmids (E80 and E95) also contained a third IS26 element close to the IS26-blaSHV-12-IS26 locus. A GenBank search revealed that this IS26-blaSHV-12
complex resembles three previously described plasmids, including the IncR plasmid in pKPS77 from K. pneumoniae KPS77 (35), the pUMB-9 plasmid from E. coli (EF370423, unpublished data), and the IncHI2 pEC-IMP from E. cloacae (EU855787; 7; Fig. 2). The plasmids, pKPN4, pUMB-9, and pEC-IMP, all have the same fragment of the K. pneumoniae chromosome (10-12) as we identified in our study, and the various replicons were truncated by an IS26 at the same position, which suggests a close evolutionary relationship among the IS26-blaSHV-12-IS26 locus of these plasmids.
In pE81-1, the sequence 3' of the blaSHV-12 contained only ygbJ, deoR, and IS903, an
insertion sequence often encountered in Enterobacteriaceae (36). PCR using the IS903 forward and reverse primers produced amplicons in all 6 of our strains and their transconjugants, suggesting that IS903-like elements are apparently common in plasmids of these strains (data not shown). A similar organization for SHV-12 was presented in the plasmid, p61.9, isolated from Salmonella enterica strain STYM61/9 in which a part of Tn1721 was observed between deoR and IS26 (FJ790886; Fig. 2). Although the recombination events, IS903 and Tn1721, remain to be elucidated, it may contribute to bla mobilization. 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 71
The conserved segment (5’-CS) of the class 1 integron is often truncated by IS26 (IS26/∆5’-CS), as previously described in IncL/M pSEM from Salmonella enterica serovar Typhimurium (37), the IncFII p1658/97 plasmid from E. coli (AF550679), and the IncL/M pACM1 from Klebsiella oxytoca (U90945, AY081221, AY309067, and AY309066). Another reported structure of In53 is comprised of a truncated integron contained in a composite transposon, Tn2000, flanked by two IS26 elements in opposite orientations (38). The class 1 integron associated with IS26 elements was also present in the corresponding region of a 318,782 bp plasmid, pEC-IMP, of which the IS26 was identified 741 bp downstream of the
intI1 (EU855787; Fig. 2), which was recently described in clinical E.cloacae isolates from
Taiwan (7). This is the first time that an IS26-blaSHV-12 fragment has been shown to be inserted
into the extreme 5’-end of the integron and truncated 3’ of aac(6')-IIc in plasmid pE71. A similar structure was recently reported by Dawes et al. (39) in E. coli strains of different serotypes, in which the horizontal transfer occurred in atypical integrons containing the dfrA-IS26 configuration. These atypical integrons containing the aac(6')-IIc-dfrA-IS26 or dfrA-dfrA-IS26 configuration in the conjugative plasmid suggests that the horizontal transfer of this gene arrangement has occurred in clinical enterobacteria. Few studies have analyzed the structure beyond the IS26-blaSHV-12-IS26 locus. This is the first description of an association between
the extended blaSHV-12 genetic environment with intI1-aac(6')-IIc-IS26 element.
Examinations of the six plasmid backbones revealed an IS26-composite transposon 74 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 75
element, which was inserted in a direct orientation, similar to a previous report about the enterobacterial plasmids pSEM (37). Sequences compared with the relevant flanks of IS26 elements did not include the duplication characteristics of IS26-mediated transposition. Moreover, the fact that either side of these IS26-flanked regions did not exhibit any marked target sequence specificity strengthens the hypothesis that this variant probably presents a genetic rearrangement. As demonstrated by Ford and Avison (12), IS26-mediated mobilization of bla-SHV-containing fragments of the K. pneumoniae chromosome has been a repeatedly occurring phenomenon due to independent events. However, for these strains, the precise genetic rearrangement awaits further investigation. Thus, to characterize the recombination through IS26 in mobilization by the plasmid of pE71-2 was demonstrated by Southern blot hybridization in this study. Taken together, the mechanism of IS26 transposon insertion is likely to be variable and occurs through homologous recombination in these clinical isolates.
In conclusion, four different β-lactamase genes, including blaTEM-1, blaSHV-12, blaCTX-M-3,
and/or blaCTX-M-14, were observed in the conjugative plasmid of clinical E.cloacae isolates in
Taiwan. This work further confirmed that the IS26-blaSHV-12-IS26 locus was located in
different genetic environments, including class 1 integrons, transposons, and IS903. These elements could play an important role in the spread of the blaSHV-12 and is suggestive of
multiple recombination events. In addition, the first unique configuration of the 5’-conserved 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 79
segment-intI1-aac(6')IIc-IS26-blaSHV-12 might have originated from a common class 1
integron truncated by the mobileelements, such as IS26-containing transposons. The different combination observed in our study suggests that the rearrangements were mediated by both homologous and IntI-contained recombination.
82 318 319 320 321 83
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Science Council, Taiwan (NSC 96-2621-B-339-001) and China Medical University (CMU-96-206).
DISCLOSURE
The authors declare that there are no conflicts of interest regarding the publication of this paper. 322 323 324 325 326 327 87
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FIGURE LEGENDS
Fig. 1. (a): Groups of XbaI pulsed-field gel electrophoresis (PFGE) patterns for isolates of 6
ESBL-producing E. cloacae (Lanes A-E: E17, E19 [E81], E71, E80, and E95). (b):DNA fingerprinting of transconjugant plasmids identified 6 different EcoRI-digested plasmids of strains E17, E19, E71, E80, E81, and E95 (lanes A-F). Lane M, Lambda/HindIII
Fig. 2. Schematic representation of the genetic context of the blaSHV-12 of E. cloacae. Sizes of
genes and intergenic regions are not drawn to scale. Open reading frames (ORFs) are shown, and their directions of transcription are presented as broad arrows. The blaSHV-12 is white, and
the IS26 elements are dark gray. The boxed area outlines the IS26-blaSHV-12-IS26 locus
regions. Gene names are derived from the closest Blast (40, 35).
Fig. 3. Nucleotide sequences of the immediate upstream and downstream region of IS26 in
pE71-2. The arrows represent genes and their orientation. The boxed sequence corresponds to the invert repeat of IS26, and GTTRRRY; the attl1 core site is denoted by a black bar.
Fig. 4. Southern blot analysis of the blaSHV insert in E23. Agarose gel (1%) electrophoresis of
the DNA fragments of blaSHV amplified by PCR and genomic DNA digested with BamHI
followed by southern blotting using the labelled PCR fragment as the probe (a and b). Lanes 1-4: 1 kb ladder marker, SHV-12 PCR product (0.85 kb), BamHI fragments of E71 and E23-1(a). Lanes 1-5: 1 kb ladder marker, SHV-12 PCR product (0.85 kb), BamHI fragments of E23-6, BamHI fragments of pE23-4.5 and BamHI fragments of pE23-3.6 (b).
Fig. 5. Schematic representation of the elements flanking blaSHV-12 in plasmid DNA fragments
460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 119
of pE23-3.6 and pE23-4.5 are shown. The genes and their coding orientations are indicated using the open horizontal arrows with the gene names above the corresponding boxes. The IS26 elements are represented as open box and inverted right and left repeats are shown by empty triangles. The coding orientations of the transposase genes in the inserted sequences of IS26 are shown with the black arrows.
122 480 481 482 483 484 485 123
