Bacterial Strains

During 1998–2003, a total of 1,744 S. maltophilia isolates collected worldwide were forwarded to the SENTRY Program (Europe, USA, and Australia) and tested for antimicrobial drug susceptibility. A TMP/SMX resistance phenotype was demonstrated for 71. From these isolates, 25 nonclonal strains from patients in North America, Latin America, and Europe were analyzed by using molecular methods together with 30 representative isolates that were TMP/SMX-susceptible. Isolates were identified by using the Vitek System and confirmed by using API20NE (bioMérieux, Hazelwood, MO, USA).

Susceptibility Methods

Isolates were tested for susceptibility to TMP/SMX according to procedures of the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards [NCCLS]) (12,13) by using broth microdilution methods (TREK Diagnostics, Cleveland, OH, USA). MIC results were confirmed with TMP/SMX Etests was performed according to the manufacturer’s directions (AB Biodisk, Solna, Sweden).

Molecular Materials

PCR primers were purchased from Sigma-Genosys Ltd. (Pampisford, UK) and are listed in the Table. General reagents for DNA manipulation were obtained from Invitrogen (Groningen, the Netherlands). All other reagents were obtained from Sigma Chemical Co. or BDH (both of Poole, England, UK).

Strain Typing

Clonality among the S. maltophilia isolates was assessed by pulsed-field gel electrophoresis (PFGE) followed by XbaI digestion of genomic DNA. This assessment was conducted according to the standard 1-day protocol (16).

Plasmid Isolation

Bacterial plasmids were isolated by the alkaline lysis method described by Grinsted and Bennett (17). Essentially, an overnight 10-mL culture was centrifuged (12,000× g) and suspended in water (250 μL) before 200 μL of lysis solution (0.2 mol/L NaOH, 1% sodium dodecyl sulfate [SDS]) was added. After lysis, 125 μL of neutralizing solution (0.3 mol/L potassium acetate, 1 mmol/L EDTA) was added. After precipitation, the suspension was centrifuged (12,000× g) and washed twice with 500 μL of a 50/50 (v/v) phenol/chloroform solution. The DNA was precipitated from the solution with the addition of 0.7 volumes of iso-amyl alcohol. The DNA/RNA pellet was washed twice in 1 mL 70% ethanol before being dried. The DNA was dissolved in 30 μL with 0.1 U RNase.

Southern Hybridization

ISCR and sul2 PCR product amplified with primers CRF/CRFF-r were labeled with P³²-ATP by random primer extension by using a commercially available kit (Stratagene, Amsterdam, the Netherlands) according to the manufacturer’s instructions. Unincorporated nucleotides were removed by passing the labeled DNA through a Sephadex column (Nick column, Pharmacia Bio-tech, Uppsala, Sweden).

Agarose gels used for Southern transfer were denatured for 45 min in denaturing solution (0.5 mol/L NaOH, 1.5 mol/L NaCl) before being neutralized in 0.5 mol/L Tris-HCl, pH7.5, 1.5 mol/L NaCl for 30 min. DNA was then transferred to Hybond (Amersham, Buckinghamshire, England, UK) nylon membrane by vacuum by using a custom-made Southern blotting apparatus. The nylon filter was prehybridized for at least 2 h with a blocking solution (6× SSC [1× SSC is 0.15 mol/L NaCl plus 0.014 mol/L sodium citrate], 0.1% [w/v] polyvinylpyrrolidone 400, 0.1% Ficoll [v/v], 0.1% bovine serum albumin, 0.5% SDS, 150 μg/mL denatured calf thymus DNA) at 65°C. The labeled denatured probe was then added to the solution and incubated overnight at 65°C. Finally, the filter was washed (300 mL 2× SSC, 0.1% [w/v] SDS followed by 0.1× SSC 0.1% SDS) at 65°C. Autoradiographic images were recorded on Hyperfilm-MP (Pharmacia Bio-tech), which was exposed overnight with intensifying screens.

PCR Analysis

The presence of class 1 integrons in each strain was assessed by using class 1 specific primers. Gene cassettes embedded within the class 1 integrons were determined by using primers listed in the Table. Isolates were also screened for sul1, sul2, and sul3 by using sul1-F and -R, sul2-F and -R, and sul3-F and -R, respectively. Seven positive class 1 integron PCR products were chosen randomly, extracted from agarose gels after size separation, and sequenced with IntF, IntR, and custom-made oligonucleotide primers (Table).

The presence of ISCR elements in each strain was also determined by using primers CRF/CRFF-r designed to amplify the same 700-bp fragment internal to the open reading frames (ORFS) of ISCR1–5 (Table). Full-length ISCR2 elements were amplified with primers designed to target the ends of ISCR2. Primers used to amplify genes often associated with ISCR2 or ISCR3 are also given (Table). Because dhfr genes are associated with ISCR elements, we also performed molecular analysis of them.

PCRs were conducted in a final volume of 20 μL by using 10 μL ABgene Expand Hi-fidelity Master Mix (ABgene House, Surrey, England, UK). Primers were used at final concentrations of 10 μmol/L, and 1 μL of an overnight bacterial culture (optical density 1.0 at 600 nm) was added as source of DNA template. The cycling parameters were as follows: 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 68°C for 1–4 min, depending on the sequence to be amplified, and ending with a 5-min incubation at 68°C.

DNA Sequencing and Analysis

Sequencing was conducted on both strands by the dideoxyl-chain termination method with a Perkin-Elmer Biosystems 377 DNA sequencer (Perkin-Elmer, Waltham, MA, USA). Sequence analysis was performed with the Lasergene DNASTAR software package (SelectScience Ltd., Bath, England, UK). Sequence alignments were conducted with the ClustalW program (www.ebi.ac.uk/clustalw) and the PAM 250 matrix.

The sequence of ISCR2, together with the adjacent sul2 region and the novel ISCR9 and ISCR10, has been deposited in GenBank. The genetic locus ISCR2-glmM/sul2 from isolates 5232, 4647, 3800, and 2107 has been attributed the accession nos. AM182031, 182030, 182029, and 181666, respectively. ISCR9 and ISCR10 have been given the numbers AM182033 and AM182032, respectively.

TMP/SMX MICs

TMP/SMX MICs separated the isolates into an obvious bimodal distribution. The TMP/SMX-resistant isolates possessed MICs >32 mg/L, whereas the sensitive controls used as molecular comparators possessed TMP/SMX MICs ranging from 0.5 to 2 mg/L (Appendix Table).

Detection and Determination of Class 1 Integrons

Figure 1

Figure 1. Schematic diagram of class 1 integrons from Stenotrophomonas maltophilia isolates. A), isolates 1893 and 9431. B) isolates 489, 9189, and 12221; C) isolates 3438 and 3444. Arrows depict direction of transcription,...

Of the 25 TMP/SMX-resistant S. maltophilia isolates that we analyzed, 17 possessed the sul1 gene as part of the 3′ end of a class 1 integron. None of the TMP/SMX-susceptible S. maltophilia isolates yielded positive sul1 PCR products. PFGE analysis (data not shown) showed that only 2 isolates (9189 and 12221 from Chile) are clonally related (Appendix Table). To our knowledge, this is the first report of sul1-positive S. maltophilia isolates from North America and Europe. The sul1-positive isolates are widespread, being from Europe, North America, and South America. Most (5) were isolated from Brazil. The integrons associated with the sul1 gene vary in size; however, when 2 strains were isolated from the same country (e.g., 3438 and 3444, 9189 and 12221, and 98 and 14469), they possessed integrons of the same size, despite not being clonally related (Appendix Table). Seven of these integrons were randomly selected to examine their gene cassettes. The genetic context of the class 1 integrons and procured gene cassettes are shown in Figure 1. Strains 1893 (Germany) and 9431 (Brazil) possessed only the int and sul/qac genes. The class 1 integrons from strains 4891 (USA), 9189 (Chile), and 12221 (Chile) contained an embedded aacA4 gene cassette. The 2 Mexican strains (3438 and 3444) contained 2 aminoglycoside-modifying genes (aacA7 and aadA5) and an unknown ORF (Figure 1) yet were clonally unrelated, as judged by PFGE profiling. None of the integrons were the same as those characterized from strains isolated from Argentina (10).

Detection and Location of sul2 Genes

All 55 isolates (both TMP/SMX resistant and sensitive) were screened for sul2 genes with the primers listed in the Appendix Table. Nine of the isolates gave PCR products for sul2. None of the TMP/SMX-susceptible S. maltophilia isolates displayed positive sul2 PCR products. Sequence analysis showed 100% identity with previous sul2 sequences.

Given that sul2 is normally located on medium-to-large sized plasmids, plasmids were isolated and characterized for sul2 carriage. Plasmid DNA was prepared from each isolate and used as a template for PCRs by using the sul2 primer detection set. In every case, a product of the size expected of sul2 sequence amplification was obtained. The purity of each plasmid preparation was evaluated by attempted PCR amplification of the host cell chromosomal gyrA gene. In no case was an amplification product obtained when plasmid DNA was used as template; in contrast, a gyrA amplification product of the correct size was obtained from genomic DNA. These data were later confirmed by Southern hybridization that used the labeled sul2 gene as a probe (data not shown). Unsurprisingly, in most cases sul2 was found on a large plasmid of ≈120 kb; however, in 2 of 9 sul2-positive isolates, sul2 gene was chromosomally encoded.

Detection of ISCR Elements in TMP/SMX-sensitive and -resistant Strains

The sul2 gene and dhfr genes are often found on plasmids and in close association with class 1 integrons or ISCR mobile genetic elements (10,15,18,19). Accordingly, we investigated the 55 S. maltophilia isolates for ISCR elements. Seven of the 25 TMP/SMX-resistant isolates yielded PCR products of the expected size (≈700 bp) when the ISCR specific primers CRF/CRFF-r were used, and 6 of 23 TMP/SMX-sensitive S. maltophilia isolates also yielded the correct-sized amplification products.

To determine whether the locations of the ISCR sequences in the S. maltophilia isolates are chromosomal or plasmid mediated, plasmid DNA was prepared from each isolate and used as a template for ISCR-PCR and Southern hybridization analysis in a similar manner as described for sul2. In every case, a product of the size expected of ISCR sequence amplification was obtained. Hence, in those isolates that possess an ISCR element, the element is located on a plasmid (data not shown). The PCR ISCR amplification products were recovered, purified, and ligated into the cloning vector, PCR-Topo-2.1 (Invitrogen) and recombinant plasmids were recovered by transformation of Escherichia coli DH5α. One clone from each transformation was chosen for further study.

Sequence analysis showed that 5/7 amplicons obtained from TMP/SMX-resistant S. maltophilia isolates were identical to the equivalent sequence of ISCR2; the other 2 amplicons were identical to that of ISCR3 (Appendix Table). ISCR2 sequences were identified in isolates originating from North and South America, as well as from Europe. In contrast, the ISCR3 sequence was identified only in isolates that originated from Spain.

The ISCR-like elements carried by the sensitive isolates, while clearly related to ISCR1–5, differed markedly from known ISCR sequences (15). Two variants were found, which we have designated ISCR9 and ISCR10. The putative amino acid sequences of ISCR9 and ISCR10 are ≈95% identical to each other and display 30%, 48%, and 74% identity to ISCR2, ISCR,3 and ISCR5, respectively (Appendix Figure). These novel ISCRs are harbored in isolates from several different regions, including South American countries, the United States, and Turkey (Table).

Identification of Resistance Genes and Sequences Adjacent to ISCR Elements

Figure 2

Figure 2. Genetic context of various insertion element common region (ISCR2) elements. Schematic of various GenBank sequences that harbor the ISCR2 element. Open reading frames (ORFs) are depicted as open boxes with the...

ISCR2 is often associated with various antimicrobial resistance genes, not least, genes mediating TMP/SMX resistance (Figure 2) (15). These and other genes normally associated with ISCR2 were therefore analyzed; these included dhfrA10, dhfrA9, dhfrA20, floR, tetR, strA, sul2, and glmM encoding a truncated phosphoglucosamine mutase. Pairs of oligonucleotides were used (Table) to genetically characterize all those S. maltophilia isolates that possessed an ISCR element.

The floR gene was detected in isolates 2139 and 2170 (which also contains ISCR2) from Turkey and the United States, respectively, and in isolates 12044 and 12049 (which also contains ISCR3) from Spain. A truncated glmM allele (ΔglmM) was detected in all ISCR2-containing isolates, and sul2 was found in all ISCR2- and ISCR3-containing isolates. The dhfr, tetR, and strA genes were not detected.

Linkage of the ISCR element to ΔglmM, sul2, or floR was then investigated by PCR analysis, i.e., the oligonucleotide pair CRFF/sul2F is expected to generate a product if the ISCR sequence is close to sul2 and downstream of it (Figure 2). Using this strategy, we found that ISCR2 was linked to ΔglmM and sul2 in all isolates that possess ISCR2. The floR gene was also found to be linked to ISCR2, on the opposite side from ΔglmM and sul2, in isolates 2139 and 2170 (Figure 2). Linkage of ISCR3 to either sul2 or floR was not demonstrated.