Rungtip Chuanchuen

Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps : Exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ

ABSTRACT Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosaexpresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Δ(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM.

The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.

Substrate-Dependent Utilization of OprM or OpmH by the Pseudomonas aeruginosa MexJK Efflux Pump

ABSTRACT MexJK requires OprM for erythromycin efflux but not for triclosan efflux. Deletion of 15 OprM family outer membrane proteins (OMPs) revealed that only the TolC homolog OpmH functions with MexJK for triclosan efflux. This is the first report of natural utilization of multiple OMPs by a given resistance nodulation cell division transporter/membrane fusion protein pair.

Class 1 Integrons and Salmonella Genomic Island 1 Among Salmonella enterica Isolated from Poultry and Swine

Two hundred eleven Salmonella enterica strains representing 35 serotypes isolated from healthy poultry (n=103) and swine (n=108) were used in this study. The occurrence and characteristics of class 1 integrons were investigated. Salmonella genomic islands (SGIs) and the horizontal transfer of integrons were assessed. One hundred eighty-six isolates (88%) were resistant to at least one antimicrobial and 140 isolates (66%) were multidrug resistant. The intI1 gene was present in 54 isolates (25.6%), of which 33 (15.6%) carried gene cassettes with sizes ranging from 0.7 to 2.3 kb. Sequence analysis revealed 11 distinct integron profiles in which resistance genes bla(PSE-1), dfrA1, dfrA12, aadA2, aadA4a, and silB were present. The gene cassette array dfrA12-aadA2 was the most prevalent among the isolates whereas most integrons were located on conjugative plasmids. SGI1 variants (SGI1-A and -F) were present in nine isolates belonging to serovars Albany, Emek, Kedougou, and Kingston.

Antimicrobial resistance of Campylobacter coli isolates from swine.

Eighty-three swine isolates of Campylobacter coli were tested for mechanisms underlying resistance to nalidixic acid, ciprofloxacin, erythromycin and tetracycline. Four isolates harbored class 1 integrons but none carried class 2 and 3 integrons. Most of the tetracycline-resistant isolates (97%) possessed tet(O). A Thr-86-Ile substitution in GyrA and an A-2230-G mutation in 23S rRNA were the main resistance mechanisms for quinolones and erythromycin, respectively.

Molecular Characterization of MexL, the Transcriptional Repressor of the mexJK Multidrug Efflux Operon in Pseudomonas aeruginosa

ABSTRACT The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexLA47D mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexLA47D oligomerization was confirmed using a genetic approach, and the MexLA47D mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexLA47D, binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions −84 and −20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted −10 and −35 promoter sequences for both mexL and mexJ, with partially overlapping −10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.

Antimicrobial resistance, virulence, and phylogenetic characteristics of Escherichia coli isolates from clinically healthy swine.

A total of 344 commensal Escherichia coli isolates from clinically healthy pigs were examined for antimicrobial resistance phenotypes, class 1 integrons, resistance genes, virulence gene profile, and phylogenetic groups. The majority of E. coli isolates were resistant to tetracycline (96.2%) and ampicillin (91.6%). Up to 98% were multidrug resistant. Seventy-three percent of the isolates carried class 1 integrons. Inserted-gene cassette arrays in variable regions included incomplete sat, aadA22, aadA1, dfrA12-aadA2, and sat-psp-aadA2, of which the aadA2 gene cassette was most prevalent (42.9%). Horizontal transfer was detected in eight E. coli isolates carrying class 1 integrons with dfrA12-aadA2 gene cassette array. Sixteen resistance genes were identified among the E. coli isolates with corresponding resistance phenotype. Ten virulence genes (including elt, estA, estB, astA, faeG, fasA, fedA, eaeA, paa, and sepA) were detected, of which fasA was most commonly found (98.3%). Most of the E. coli isolates belonged to phylogenetic group B1. Significantly positive associations were observed between some virulence genes and some resistance phenotypes and genotypes (p < 0.05). The results support a finding that commensal E. coli have a role as reservoirs for antimicrobial resistance-encoding genes and virulence determinants.

Antimicrobial Resistance and Virulence Genes in Salmonella enterica Isolates from Dairy Cows

One hundred sixty Salmonella enterica isolates from clinically healthy dairy cows were assayed for antibiotic susceptibilities, the presence of class 1 integrons, antimicrobial resistance genes and virulence genes, and conjugal transfer of antimicrobial resistance determinants. One hundred nine (68%) of the Salmonella isolates were resistant to at least 1 antibiotic, and 14 isolates (9%) were multiresistant. The most prevalent resistance observed was to streptomycin (64%). Class 1 integrons were detected in only two Salmonella isolates (serovar Singapore and Derby), and both integrons harbored the same cassette content aadA2. The Derby class 1 integrons were associated with Salmonella genomic island 1-A. Most commonly found resistance genes were strA and strB (9.2%). None of class 1 integrons were horizontally transferred, and the resistance genes were successfully transferred from six (5.5%) Salmonella strains. One hundred fifty-nine isolates (98.8%) were positive to the invasion gene invA, whereas the virulence plasmid-associated genes spvC and pefA were found in only two (1.3%) and one (0.6%) Salmonella isolates, respectively.

Characterization of antibiotic resistance in Vibrio spp. isolated from farmed marine shrimps (Penaeus monodon)

A total of 83 Vibrio isolates from farmed marine shrimps (Penaeus monodon) were tested for the presence of class 1, 2 and 3 integrons, SXT constin and tetracycline resistance-encoding genes. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were determined in fluoroquinolone-resistant Vibrio strains (n=17). Five isolates were found to carry class 1 integrons, of which only one contained the partial rumA gene in the variable region. All the Vibrio strains were devoid of class 2 and 3 integrons. Seven isolates harbored SXT constin. None of the Vibrio isolates were positive to the tet(K), tet(L), tet(M), tet(O) and tet(S) genes. Ten fluoroquinolone-resistant Vibrio strains carried a point mutation G-248-T in the gyrA QRDR, leading to a Ser-83-Ile substitution in GyrA, but none of these strains had mutations in the QRDR of the parC gene.

Role of the MexXY multidrug efflux pump in moderate aminoglycoside resistance in Pseudomonas aeruginosa isolates from Pseudomonas mastitis.

The contribution of the MexXY multidrug efflux system to aminoglycoside resistance was investigated in 18 clinical isolates of Pseudomonas aeruginosa obtained from dairy cows with Pseudomonas mastitis. All of the isolates expressed MexXY as determined by reverse transcription‐PCR. The loss of mexXY resulted in increased susceptibility (two‐ to 16‐fold decline in MIC) to aminoglycosides, confirming the contribution of this system in aminoglycoside resistance in these strains. As the impact of ΔmexXY varied, overexpression of MexXY alone is not sufficient for aminoglycoside resistance. Expression of mexXY also varied and did not strictly correlate with aminoglycoside insusceptibility. Transcription levels of mexY were independent on mutations in mexZ, suggesting the existence of additional regulatory mechanisms other than mexZ.