Abstract
Two multidrug-resistant Salmonella enterica serovar Wien strains (SW468 and SW1107) were isolated in 2001 in Tunis. Both strains produced the β-lactamases TEM-1, SHV-2a, and CMY-4, whereas strain SW1107 also produced the β-lactamase CTX-M-3. The imipenem-resistant strain (SW468) was totally devoid of the OmpF-immunorelated porin. Imipenem resistance was shown as being related to porin loss and CMY-4 β-lactamase production.
For several years, the resistance of Salmonella enterica to expanded-spectrum cephalosporins has been increasingly reported, as has the diversity of β-lactamases involved in this resistance. Using a chronological approach, we can assume that the extended-spectrum β-lactamases observed between 1985 and 1990 concerned SHV and TEM derivatives (1, 9) whereas those described since 1990 concerned not only new class A β-lactamases, such as CTX-M type and PER enzymes (2, 4, 15), but also plasmid-mediated class C β-lactamases, namely DHA-1 (8) and CMY-2 (7, 13). Fortunately, all these plasmid-mediated class A and class C β-lactamases do not confer imipenem (IMI) resistance to the isolates. However, as presented in this paper, the association of different mechanisms of resistance led to IMI resistance in one of the two multidrug-resistant isolates of Salmonella enterica serovar Wien which were recently found in Tunis.
The two isolates (SW468 and SW1107) came from the blood of two neonates hospitalized in the military hospital of Tunis in January and March 2001, respectively. Both neonates were treated with IMI and fosfomycin just after blood sampling, but they died 1 or 2 days after starting empirical treatment. Strain SW468 was resistant to all β-lactam molecules as well as to tetracycline (TET), chloramphenicol (CMP), sulfonamides (SFA), gentamicin (GEN), kanamycin (KAN) and tobramycin (TOB). Strain SW1107 displayed the same antibiotic resistance pattern as strain SW468 except that it was susceptible to IMI and also resistant to netilmicin (NET), amikacin (AMI), trimethoprim (TMP), and cotrimoxazole (STX).
The presence of β-lactamases in these two strains was determined first by using the isoelectric focusing method as previously described (12). Three enzymes with pI values of 5.4, 7.6, and 9.2 were found in both strains SW468 and SW1107, and an additional enzyme with a pI value of 8.4 was found in strain SW1107. PCRs and the sequencing of amplified fragments carried out as previously described (14, 18) showed that both strains harbored the blaTEM-1B gene (controlled by the promoter Pa/Pb), coding for TEM-1 (pI, 5.4), and the blaSHV gene derivative, coding for the extended-spectrum β-lactamase SHV-2a (pI, 7.6). When the sequence of the fragment amplified by the degenerated ampC primers previously defined by Verdet et al. (20) was compared with those for the ampC genes available in GenBank, we observed strong homology with the blaCMY genes. By using primers specific for blaCMY genes (Table 1), we determined that the two strains produced the β-lactamase CMY-4.
TABLE 1.
Primers selected and used in PCR and sequencing reactions
| Gene | Primer (sequence)a | Annealing temp (°C) | Fragment size (bp) |
|---|---|---|---|
| blaCTX-M | CTX-C1 (5′ - ATG TGC AGC ACC AGT AAA GT - 3′) | 54 | 545 |
| CTX-C2 (5′ - ACC GCG ATA TCG TTG GTG G - 3′) | |||
| blaCMY | CASE-E1 (5′ - TGT CAA CAC GGT GCA AAT CA - 3′) | 55 | 1,347 |
| CASE-E2 (5′ - AGC AAC GAC GGG CAA AAT G - 3′) | |||
| CASE-E3∗ (5′ - CCG ATC CTA GCT CAA - 3′) | |||
| CASE-E4∗ (5′ - TAT ACG GCA GGC GGC - 3′) | |||
| CASE-E5∗ (5′ - GCG ATA TGT ACC AGG - 3′) | |||
| blaMIR-1 | MIR-A1 (5′ - TTC GCC GCA CCG ATG TC - 3′) | 54 | 1,087 |
| MIR-A2 (5′ - GGC GTC GAG GAT ACG GT - 3′) |
Asterisk denotes primer used only for sequencing.
With a pI value of 8.2, the fourth β-lactamase in strain SW1107 could correspond to a CTX-M enzyme or the MIR-1 enzyme. PCR with the primers specific for the blaMIR-1 gene (Table 1) was negative, whereas it was positive when using the primers specific for blaCTX-M genes (Table 1). The sequence analysis of the amplified fragment showed that strain SW1107 also produced the β-lactamase CTX-M-3.
As mating experiments failed, plasmids harbored by strains SW468 and SW1107 were extracted, purified, and used to transform Escherichia coli strain NM554 (Strr). Independently of the donor strain, the E. coli NM554 transformants selected on agar plates containing either ceftazidime (CAZ) (10 μg/ml) or cefoxitin (FOX) (50 μg/ml) displayed the same antibiotic resistance phenotype (Table 2). With regard to the β-lactam molecules whose activity was determined with the agar dilution method, they were susceptible only to cefepime (FEP), cefpirome (CPO), and IMI (Table 2). We demonstrated by using bla gene PCRs that this β-lactam phenotype was due to the transfer of the only β-lactamase, CMY-4 (Table 3). Concerning the other antibiotic families tested with the agar disk diffusion method, these E. coli NM554 transformants were found to be resistant to TET, CMP, and SFA but susceptible to aminoglycosides, TMP, and STX (Table 3). They were also as susceptible to nalidixic acid and fluroquinolones as strains SW468 and SW1107 (Table 2). When cefotaxime (CTX) (10 μg/ml) was used as the selector, the E. coli NM554 transformants obtained from strain SW468 displayed exactly the same antibiotic resistance pattern as the transformants selected on agar plates containing either CAZ or FOX, whereas those obtained from SW1107 displayed a new antibiotic resistance pattern. These transformants were resistant to penicillins and cephalothin (CEF), less susceptible to CTX (MIC, 16 μg/ml) and FEP (MIC, 8 μg/ml), and susceptible to the combination piperacillin-tazobactam (TZB), FOX, cefotetan, CAZ, CPO, and IMI (Table 2). In the presence of 2 μg of clavulanate/ml, MICs of CTX and FEP decreased to <0.006 μg/ml whereas that of CAZ decreased from 2 to 0.25 μg/ml (Table 2). We demonstrated by using bla gene PCRs that this β-lactam phenotype was due to the transfer of the only β-lactamase, CTX-M-3 (Table 3). The other antibiotics to which the transformants selected on CTX from strain SW1107 became resistant were GEN, KAN, TOB, NET, AMI, SFA, TMP, and STX (Table 3). Finally, no transformant from strain SW468 was selected on agar plates containing IMI (20 μg/ml).
TABLE 2.
MICs of β-lactam molecules and quinolones for clinical isolates of serovar Wien, recipient E. coli strains, and E. coli transformants
| Antibiotica | MIC (μg/ml)
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Serovar Wien clinical isolate
|
E. coli recipient
|
E. coli NM554 transformant
|
E. coli BZB 1170 (OmpF−) transformant
|
||||||||
| 186 | 468 | 1107 | NM554 | BZB1107 (OmpF−) | SW468b | SW1107c | SW1107d | SW468b | SW1107c | SW1107d | |
| Amoxicillin | 2 | >1,024 | >1,024 | 2 | 8 | 1,024 | 1,024 | 1,024 | >1,024 | >1,024 | >1,024 |
| Amoxicillin-CLA | 1 | >1,024 | >1,024 | 2 | 8 | 1,024 | 1,024 | 1,024 | >1,024 | >1,024 | >1,024 |
| Ticarcillin | 4 | >1,024 | >1,024 | 2 | 16 | 1,024 | 1,024 | 1,024 | 1,024 | 1,024 | >1,024 |
| Ticarcillin-CLA | 2 | >1,024 | >1,024 | 2 | 16 | 1,024 | 1,024 | 128 | 1,024 | 1,024 | 512 |
| Piperacillin | 2 | >1,024 | >1,024 | 1 | 0.5 | 64 | 64 | 512 | 32 | 32 | 256 |
| Piperacillin-TAZ | 1 | 1,024 | >1,024 | 0.5 | 0.5 | 16 | 16 | 0.5 | 4 | 4 | 2 |
| Cephalothin | 2 | >1,024 | 1,024 | 1 | 32 | 256 | 512 | 512 | >1,024 | >1,024 | >1,024 |
| Cefoxitin | 1 | 1,024 | 256 | 1 | 32 | 128 | 128 | 1 | 128 | 128 | 16 |
| Cefotetan | <0.06 | 512 | 256 | <0.06 | 4 | 64 | 32 | 0.12 | 256 | 256 | 8 |
| Cefotaxime | <0.06 | 512 | 1,024 | <0.06 | <0.06 | 32 | 64 | 16 | 64 | 64 | 128 |
| Cefotaxime-CLA | <0.06 | 512 | 128 | <0.06 | <0.06 | 32 | 32 | <0.06 | 64 | 64 | 0.12 |
| Ceftazidime | 0.25 | 512 | 256 | <0.06 | 0.5 | 128 | 256 | 2 | 128 | 128 | 4 |
| Ceftazidime-CLA | 0.25 | 512 | 256 | <0.06 | 0.5 | 128 | 128 | 0.25 | 128 | 128 | 1 |
| Cefepime | <0.06 | 32 | 512 | <0.06 | 0.12 | 0.5 | 1 | 8 | 4 | 4 | 256 |
| Cefepime-CLA | <0.06 | 32 | 64 | <0.06 | 0.06 | 1 | 1 | <0.06 | 4 | 4 | 4 |
| Cefpirome | <0.06 | 128 | 512 | <0.06 | <0.06 | 1 | 1 | 4 | 8 | 8 | 128 |
| Imipenem | 0.5 | 32 | 0.5 | 0.5 | 0.5 | 1 | 1 | 1 | 16 | 8 | 4 |
| Nalidixic acid | 4 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 1 | 2 |
| Norfloxacin | 0.06 | 0.12 | 0.06 | <0.03 | 0.06 | <0.03 | <0.03 | <0.03 | 0.06 | 0.06 | 0.06 |
| Ciprofloxacin | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 | <0.03 |
CLA, clavulanic acid (2 μg/ml); TAZ, tazobactam (4 μg/ml).
Transformants selected on agar plates containing ceftazidime, cefoxitine, or cefotaxime.
Transformants selected on agar plates containing ceftazidime or cefoxitine.
Transformants selected on agar plates containing cefotaxime.
TABLE 3.
bla gene and antibiotic susceptibility of serovar Wien clinical isolates, E. coli NM554, and E. coli BZB 1107 (OmpF−) recipients and transformantsa
| Strain | β-Lactam selector |
bla gene
|
Antibiotic susceptibility
|
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TEM-1B | SHV2a | CMY-4 | CTX-M-3 | GEN | KAN | TOB | NET | AMI | TET | CMP | SFA | TMP | STX | ||
| Recipient E. coli | |||||||||||||||
| NM 554 | − | − | − | − | S | S | S | S | S | S | S | S | S | S | |
| BZB 1107 (OmpF−) | − | − | − | − | S | R∗ | S | S | S | I∗ | S | S | S | S | |
| Clinical isolate | |||||||||||||||
| Serovar Wien 468 | + | + | + | − | R | R | R | S | S | R | R | R | S | S | |
| Serovar Wien 1107 | + | + | + | + | R | R | R | R | R | R | R | R | R | R | |
| Transf. E. coli NM554 x | |||||||||||||||
| Serovar Wien 468 | CAZ or FOX or CTX | − | − | + | − | S | S | S | S | S | R | R | R | S | S |
| Serovar Wien 1107 | CAZ or FOX | − | − | + | − | S | S | S | S | S | R | R | R | S | S |
| Serovar Wien 1107 | CTX | − | − | − | + | R | R | R | R | R | S | S | R | R | R |
| Transf. E. coli BZB 1107 (OmpF−) x | |||||||||||||||
| Serovar Wien 468 | CAZ or FOX or CTX | − | − | + | − | S | R∗ | S | S | S | R | R | R | S | S |
| Serovar Wien 1107 | CAZ or FOX | − | − | + | − | S | R∗ | S | S | S | R | R | R | S | S |
| Serovar Wien 1107 | CTX | − | − | − | + | R | R | R | R | R | I∗ | S | R | R | R |
+, positive reaction; −, negative reaction; R, resistant; S, susceptible; ∗, resistance marker of E. coli BZB 1107 (OmpF−) recipient; Transf., transformant.
This result strongly suggested that another or other mechanisms must be investigated to explain the IMI resistance displayed by strain SW468. Thus, the analysis of membrane proteins of strains SW468 and SW1107 was performed as previously described (6, 11, 16) in comparison with the membrane proteins of the following strains: E. coli strain BZB 1107 (ompF::Tn5) (Kmr), derived from the wild-type E. coli BE from the Biozentrum collection (11, 17), E. coli strain BZB 1107 containing plasmid pLG361 encoding wild-type OmpF (17), and a clinical isolate of serovar Wien (strain SW186) resistant only to TET, SFA, TMP, and STX. By using labeled polyclonal antibodies directed against the E. coli OmpF porin monomer, we observed (Fig. 1A) that strain SW186 displayed two immunorelated porins and strain SW1107 displayed only the upper product, whereas strain SW468 displayed no immunorelated porin. Since the internal loop 3 contributes to the electrostatic channel architecture which governs the diffusion efficacy of solutes through the porin (5, 10), and since it has been reported that some porins produced by β-lactam-resistant isolates harbor special mutations in loop L3, we used an antipeptide antibody which allows for the detection of these mutations (6, 17). The porin synthesized by strains SW186 and SW1107 expressed the corresponding antigenic loop site (Fig. 1B) showing the preservation of this functional domain. Immunodetection of OmpA, which plays a key role in the conservation of the membrane architecture, was positive for the three serovar Wien strains tested (Fig. 1C). These results suggest that the envelope is not pleiotropically altered and support a specific decrease of porin expression.
FIG. 1.
Immunodetection of outer membrane proteins. Labeling of membrane proteins of OmpF+ E. coli (lane 1), OmpF− E. coli (lane 2), and serovar Wien strains SW186 (lane 3), SW468 (lane 4), and SW1107 (lane 5) with anti-OmpF porin monomer antibodies (A), anti-loop3 antibodies (B), and anti-OmpA antibodies (C).
In order to evaluate the impact of porin loss in the β-lactam resistance pattern following β-lactamase transfer, plasmids extracted from strains SW468 and SW1107 were transferred into E. coli BZB 1107 (OmpF−). As indicated in Table 2, the β-lactam MICs for the E. coli BZB 1107 transformants having only the β-lactamase CMY-4 (Table 3), whether it came from SW468 or SW1107, were globally higher than those observed with the E. coli NM554 transformants possessing the same β-lactamase, except for TZB. The increase in MICs was marked enough to classify the CMY-4-producing E. coli BZB 1107 transformants into the intermediate or resistant category regarding CPO (MIC, 8 μg/ml) and IMI (MIC, 8 to 16 μg/ml). The E. coli BZB 1107 transformants having only the β-lactamase CTX-M-3 (Table 3) were susceptible to IMI, but MICs of FEP alone and in the presence of 2 μg of clavulanate/ml and of CPO were particularly increased (Table 2). The two types of E. coli BZB 1107 transformants remained susceptible to nalidixic acid and fluoroquinolones, whereas they acquired the same other antibiotic resistances (Table 3) as the two corresponding types of E. coli NM554 transformants.
To our knowledge, this is the first report on the presence of four β-lactamases in the same strain of S. enterica and the first description of the β-lactamase CMY-4 in this species.
Moreover, we demonstrated that strains SW468 and SW1107 showed an altered porin pattern. An OmpF-immunorelated porin was present in strain SW1107, which was susceptible to IMI, whereas it was absent in SW468, which was resistant to IMI. Two previous studies have shown that Klebsiella pneumoniae and E. coli isolates which produced a plasmid-mediated AmpC β-lactamase (ACT-1 and CMY-4, respectively) and lacked a major outer membrane protein were resistant to IMI (3, 19). Both strains, SW468 and SW1107, produced the CMY-4 β-lactamase, but only the one totally devoid of OmpF porin was resistant to IMI. From this result and previous studies, we inferred that the IMI resistance observed in strain SW468 resulted from both the production of CMY-4 β-lactamase and the loss of porin. This inference was reinforced by the fact that OmpF− E. coli BZB 1107 became resistant to IMI when it possessed the β-lactamase CMY-4.
Acknowledgments
We thank E. Bingen for having provided us with strain SW186.
This work was supported by a grant from the Direction de la Recherche des Etudes Doctorales, Ministère de L'Education Nationale, Paris, France, and by a grant from the French β-lactamase network (Ministère de la Recherche, Paris, France).
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