Sylvie Baucheron

The AcrB Multidrug Transporter Plays a Major Role in High-Level Fluoroquinolone Resistance in Salmonella enterica Serovar Typhimurium Phage Type DT204

Salmonella enterica serovar Typhimurium phage type DT204 strains isolated from cattle and animal feed in Belgium were characterized for high-level fluoroquinolone resistance mechanisms [MICs to enrofloxacin (Enr) and ciprofloxacin (Cip), 64 and 32 microg/ml, respectively]. These strains isolated during the periods 1991-1994, and in 2000 were clonally related as shown by pulsed-field gel electrophoresis (PFGE). Selected strains studied carried several mutations in the quinolone target genes, i.e., a double mutation in the quinolone resistance-determining region (QRDR) of gyrA leading to amino acid changes Ser83Ala and Asp87Asn, a single mutation in the QRDR of gyrB leading to amino acid change Ser464Phe, and a single mutation in the QRDR of parC leading to amino acid change Ser80Ile. Moreover, Western blot analysis showed overproduction of the AcrA periplasmic protein belonging to the AcrAB-ToIC efflux system. This suggested active efflux as additional resistance mechanism resulting in a multiple antibiotic resistance (MAR) phenotype, which was measurable by an increased level of resistance to the structurally unrelated antibiotic florfenicol in the absence of the specific floR resistance gene. The importance of the AcrAB-TolC efflux system in high-level fluoroquinolone resistance was further confirmed by inactivating the acrB gene coding for the multidrug transporter. This resulted in a 32-fold reduction of resistance level to Enr (MIC = 2 microg/ml) and actually in a susceptible phenotype according to clinical breakpoints. Thus, AcrB plays a major role in high-level fluoroquinolone resistance, even when multiple target gene mutations are present. The same effect was obtained using the recently identified efflux pump inhibitor (EPI) Phe-Arg-naphthylamide also termed MC207,110. Among several fluoroquinolones tested in combination with EPI, the MIC of Enr was reduced most significantly. Thus, using EPI together with fluoroquinolones such as Enr may be promising in combination therapy against high-level fluoroquinolone-resistant S. enterica serovar Typhimurium.

AcrAB-TolC Directs Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium DT104

ABSTRACT Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S. enterica serovar Typhimurium DT104 strains.

ramR Mutations Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

ABSTRACT In the sequenced genome of Salmonella enterica serovar Typhimurium strain LT2, an open reading frame (STM0580) coding for a putative regulatory protein of the TetR family is found upstream of the ramA gene. Overexpression of ramA results in increased expression of the AcrAB efflux pump and, consequently, multidrug resistance (MDR) in several bacterial species. The inactivation of the putative regulatory protein gene upstream of ramA in a susceptible serovar Typhimurium strain resulted in an MDR phenotype with fourfold increases in the MICs of unrelated antibiotics, such as quinolones/fluoroquinolones, phenicols, and tetracycline. The inactivation of this gene also resulted in a fourfold increase in the expression of ramA and a fourfold increase in the expression of the AcrAB efflux pump. These results indicated that the gene encodes a local repressor of ramA and was thus named ramR. In contrast, the inactivation of marR, marA, soxR, and soxS did not affect the susceptibilities of the strain. In quinolone- or fluoroquinolone-resistant strains of serovar Typhimurium overexpressing AcrAB, several point mutations which resulted in amino acid changes or an in-frame shift were identified in ramR; in addition, mutations interrupting ramR with an IS1 element were identified in high-level fluoroquinolone-resistant serovar Typhimurium DT204 strains. One serovar Typhimurium DT104 isolate had a 2-nucleotide deletion in the putative RamR binding site found upstream of ramA. These mutations were confirmed to play a role in the MDR phenotype by complementing the isolates with an intact ramR gene or by inactivating their respective ramA gene. No mutations in the mar or sox region were found in the strains studied. In conclusion, mutations in ramR appear to play a major role in the upregulation of RamA and AcrAB and, consequently, in the efflux-mediated MDR phenotype of serovar Typhimurium.

Salmonella enterica serotype Typhimurium DT 104 antibiotic resistance genomic island I in serotype paratyphi B.

We have identified Salmonella genomic island I (SGI1) in an isolate of Salmonella enterica serotype Paratyphi B. This antibiotic-resistance gene cluster, which confers multidrug resistance, has been previously identified in S. enterica serotype Typhimurium phage types DT 104 and DT 120 and in S. enterica serotype Agona.

Plasmid-Mediated Florfenicol Resistance Encoded by the floR Gene in Escherichia coli Isolated from Cattle

ABSTRACT A florfenicol resistance gene almost identical to floRof Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coliisolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.

Ciprofloxacin-resistant Salmonella Kentucky in Travelers

To the Editor: Ciprofloxacin is the treatment of choice of severe nontyphoidal Salmonella infections in adults. Resistance to ciprofloxacin has been found exceptionally in nontyphoidal Salmonella enterica isolates and only in serotypes Typhimurium, Choleraesuis, and Schwarzengrund (1–8). Such isolates have been collected from humans and animals in Europe, Asia, and North America. We report the emergence of ciprofloxacin-resistant isolates of S. Kentucky since 2002 in French travelers returning from northeast and eastern Africa. From 2000 through 2005, 197 S. Kentucky isolates from humans (1 per patient) were serotyped, from 69,759 total S. enterica isolates serotyped at the French National Reference Centre for Salmonella. Antimicrobial drug susceptibility was determined for 186 isolates by the disk-diffusion method with 32 antimicrobial drugs, as previously described (9). Resistance to several drugs, amoxicillin (18%), gentamicin (16%), nalidixic acid (21%), sulfonamides (24%), and tetracycline (24%), has been observed from 2000 through 2005. A total of 17 (9%) ciprofloxacin-resistant S. Kentucky strains were isolated. A resistant isolate that was untypable by conventional serotyping (rough) but that had a pulsed-field gel electrophoresis (PFGE) profile associated with serotype Kentucky, was included in this study. Ciprofloxacin MIC levels in these isolates, determined by standard agar doubling dilution as previously described (2), were 4–16 mg/L. The first ciprofloxacin-resistant strain was isolated in December 2002 from a French tourist who had gastroenteritis during a Nile cruise in Egypt. In 2004 and 2005, 17 ciprofloxacin-resistant isolates were identified in unrelated adults who lived in different cities of France at different times of the year. The 16 patients we contacted acquired the infection during or immediately after travel to Egypt (10 patients), Kenya and Tanzania (3), or Sudan (1). In 2 cases, gastroenteritis occurred 2 months after travel to Egypt. None of the investigated cases were fatal or life-threatening. The 18 ciprofloxacin-resistant isolates (17 serotype Kentucky and 1 rough) displayed various susceptibility patterns, from single resistance to quinolones to multiple resistance (up to 9 antimicrobial agents). To identify mutations responsible for ciprofloxacin resistance, the quinolone resistance–determining regions (QRDRs) of gyrA, gyrB, parC, and parE were amplified by PCR and sequenced as described previously (3,9), except that different forward primers for gyrB (5´-TTATCGACGCCGCGCGTGCGC-3´) and parE (5´-CGCGTAACTGCATCGGGTTC-3´) were used. The 18 ciprofloxacin-resistant isolates had different double mutations in gyrA leading to amino acid substitutions, Ser83Phe and Asp87Gly (8 isolates), Ser83Phe and Asp87Asn (5), and Ser83Phe and Asp87Tyr (5), but had identical mutations in parC (resulting in Ser80Ile). An additional substitution was observed in ParC, Thr57Ser. This substitution, however, did not appear to be associated with quinolone resistance because it was also identified in nalidixic acid–susceptible isolates. No isolates had substitutions in the QRDRs of GyrB and ParE. All isolates tested by PCR for the plasmid-mediated quinolone resistance–conferring gene qnrA (9) were negative. In the presence of the efflux pump inhibitor Phe-Arg-β-naphthylamide, the MICs of ciprofloxacin were reduced from 4-fold to 16-fold, which suggests that an active efflux mechanism was present (2). The involvement of the AcrAB-TolC efflux system was determined by measuring AcrA expression with a method previously described (5). A moderate production of AcrA (3- to 4-fold increase when compared with the baseline production of AcrA in reference strain 98K) was observed in all but 1 ciprofloxacin-resistant isolate. This isolate overproduced (6-fold) AcrA, which correlated with a higher ciprofloxacin MIC (16 mg/L). The 18 ciprofloxacin-resistant isolates and 14 ciprofloxacin-susceptible S. Kentucky isolates used for comparison were genotyped by PFGE with XbaI restriction and PulseNets running conditions, as described previously (9). Each profile that differed by >1 clear band >50 kb was considered a distinct profile. The 18 resistant isolates displayed 9 profiles that differed by 1 to 3 bands (Dice correlation coefficient 55%) (Figure). Profile X1c was predominant (7 [39%] of 18). The 6 pansusceptible isolates tested displayed 5 different patterns unrelated to those of resistant isolates. Use of a second restriction enzyme, SpeI, for the resistant isolates of X1 cluster enhanced discrimination. No clear correlations between combined PFGE patterns, gyrA mutations, and probable country of infection were observed. Figure Dendrogram generated by BioNumerics version 4.1 (Applied Maths, Sint-Martens-Latem, Belgium) showing the results of cluster analysis on the basis of XbaI pulsed-field gel electrophoresis (PFGE) of Salmonella enterica serotype Kentucky isolates. Similarity ... Since S. Kentucky is infrequently isolated from human, animal, or environmental sources in France, these isolates must have been acquired abroad. Unfortunately, no investigations have been thus far conducted to identify the source of the contamination in probable countries of infection. Poultry products may be of particular interest because poultry is the main animal reservoir of S. Kentucky. Another possible source in East Africa is pork because a recent report identified quinolone-resistant (ciprofloxacin MIC >0.125 mg/L) S. Kentucky isolates in slaughtered pigs in Ethiopia (10). After identifying the source of the contamination, appropriate control measures should be implemented in the affected countries to control the spread of these isolates.

Nonenzymatic Chloramphenicol Resistance Mediated by IncC Plasmid R55 Is Encoded by a floR Gene Variant

ABSTRACT The IncC plasmid R55, initially described in the 1970s and isolated from Klebsiella pneumoniae, confers nonenzymatic chloramphenicol resistance. The gene coding for this resistance was cloned and sequenced and shows 95 to 97% nucleotide identity with the recently reportedfloR gene from Salmonella enterica serovar Typhimurium DT104 and from Escherichia coli animal isolates, respectively, conferring cross-resistance to florfenicol.

Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and blaCMY-2 genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.

Use of Recombinant BP26 Protein in Serological Diagnosis of Brucella melitensis Infection in Sheep

ABSTRACT Previously a Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and B. melitensisRev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in Escherichia coli in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (n = 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with B. melitensis H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and B. melitensis Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that aB. melitensis Rev.1 bp26 gene deletion mutant should be constructed to ensure this differentiation.

High-level resistance to fluoroquinolones linked to mutations in gyrA, parC, and parE in Salmonella enterica serovar Schwarzengrund isolates from humans in Taiwan

Recent reports suggest high-level fluoroquinolone (FQ) resistance is emerging in Salmonella enterica serovar Typhimurium, S. enterica serovar Choleraesuis, and S. enterica serovar Schwarzengrund in different parts of the world ([1][1]-[9][2]). In this study we analyzed high-level FQ resistance