Contribution of the Multidrug Efflux Transporter CmeABC to Antibiotic Resistance in Different Campylobacter Species

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011, USA.
Foodborne Pathogens and Disease (Impact Factor: 1.91). 09/2009; 7(1):77-83. DOI: 10.1089/fpd.2009.0354
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CmeABC, a multidrug efflux system in Campylobacter jejuni, plays an important role in the resistance to different antimicrobials and toxic compounds. Although this efflux system has been well characterized in C. jejuni and to a less extent in C. coli, it is unknown if CmeABC homologs are functional in other Campylobacter spp. In this study, the cmeABC homologs were identified and functionally characterized in five Campylobacter species including C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus. Our results indicated that cmeABC is present in all five Campylobacter spp. and the genomic organization of this efflux operon is similar among the Campylobacter spp. Insertional mutagenesis of cmeB increased the susceptibilities of all the five Campylobacter spp. to structurally diverse antimicrobials. Together, these results indicated that the CmeABC efflux system is conserved at both the genomic and functional levels in all five Campylobacter spp. examined in this study, further highlighting the significant role of CmeABC in Campylobacter pathobiology.

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    • "Primers used in the construction and complementation of mutants are listed in Table 6. The chloramphenicol (cat) and kanamycin (aphA-3) resistance cassettes were PCR amplified using Ex-Taq (Takara Bio Inc.) from plasmids pUOA18 and pMW10 with cat and aphA3 primers, respectively, as described in a previous study [38]. PCR products were digested with the appropriate restriction enzymes (Table 6, Figure 1). "
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    ABSTRACT: Background Macrolide is the drug of choice to treat human campylobacteriosis, but Campylobacter resistance to this antibiotic is rising. The mechanisms employed by Campylobacter jejuni to adapt to erythromycin treatment remain unknown and are examined in this study. The transcriptomic response of C. jejuni NCTC 11168 to erythromycin (Ery) treatment was determined by competitive microarray hybridizations. Representative genes identified to be differentially expressed were further characterized by constructing mutants and assessing their involvement in antimicrobial susceptibility, oxidative stress tolerance, and chicken colonization. Results Following the treatment with an inhibitory dose of Ery, 139 genes were up-regulated and 119 were down-regulated. Many genes associated with flagellar biosynthesis and motility was up-regulated, while many genes involved in tricarboxylic acid cycle, electron transport, and ribonucleotide biosynthesis were down-regulated. Exposure to a sub-inhibitory dose of Ery resulted in differential expression of much fewer genes. Interestingly, two putative drug efflux operons (cj0309c-cj0310c and cj1173-cj1174) were up-regulated. Although mutation of the two operons did not alter the susceptibility of C. jejuni to antimicrobials, it reduced Campylobacter growth under high-level oxygen. Another notable finding is the consistent up-regulation of cj1169c-cj1170c, of which cj1170c encodes a known phosphokinase, an important regulatory protein in C. jejuni. Mutation of the cj1169c-cj1170c rendered C. jejuni less tolerant to atmospheric oxygen and reduced Campylobacter colonization and transmission in chickens. Conclusions These findings indicate that Ery treatment elicits a range of changes in C. jejuni transcriptome and affects the expression of genes important for in vitro and in vivo adaptation. Up-regulation of motility and down-regulation of energy metabolism likely facilitate Campylobacter to survive during Ery treatment. These findings provide new insight into Campylobacter adaptive response to antibiotic treatment and may help to understand the mechanisms underlying antibiotic resistance development.
    BMC Microbiology 06/2013; 13(1):133. DOI:10.1186/1471-2180-13-133 · 2.73 Impact Factor
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    • "have multiple resistance mechanisms that are associated with target mutations and resistance-nodulation-cell division (RND) and non-RND efflux pumps (Lin et al. 2005b; Pumbwe et al. 2005; Akiba et al. 2006). CmeABC is involved in the extrusion of structurally diverse antimicrobials and contributes to intrinsic and acquired resistance to various antimicrobials (Lin et al. 2002; Guo et al. 2010). Its function has also been defined for pure phenolic compounds and extracts of plant phenolics (Klančnik et al. 2012). "
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    ABSTRACT: We tested extracts from Alpinia katsumadai seeds for anti-Campylobacter activity and investigated the roles of the CmeABC and CmeDEF efflux pumps in Campylobacter resistance to these natural phenolics. Additionally, we investigated an A. katsumadai ethanolic extract (AlpE) and other plant extracts as putative efflux pump inhibitors on Campylobacter isolates and mutants in efflux pump genes. AlpE showed antimicrobial activity against sensitive and multidrug-resistant Campylobacter isolates. CmeB inactivation resulted in the greatest reduction in resistance, while cmeF and cmeR mutations produced only moderate effects on minimal inhibitory concentrations (MICs). The chemical efflux pump inhibitors additionally reduced MICs in isolates and mutants, confirming that active efflux is an important mechanism in resistance to AlpE, with additional contributions of other efflux systems. A notable decrease in resistance to tested antimicrobials in the presence of subinhibitory concentrations of AlpE confirms its modifying activity in Campylobacter spp. AlpE is important anti-Campylobacter source of antimicrobial compounds with resistance-modifying activity. At least two of the efflux systems are involved in the resistance to A. katsumadai antimicrobial seed extracts. This is the first report of antimicrobial and resistance-modifying activity of AlpE from A. katsumadai seeds, demonstrating its potential in the control of Campylobacter in the food chain.
    Journal of Applied Microbiology 08/2012; 113(5):1249-62. DOI:10.1111/j.1365-2672.2012.05424.x · 2.48 Impact Factor
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    ABSTRACT: In industrialized countries, campylobacteriosis is the most common cause of human gastroenteric infection. It is mainly caused by Campylobacter jejuni and Campylobacter coli, but the pathogenic role of other species, such as Campylobacter hyointestinalis, remains unclear. The predominant antimicrobial agents for treating Campylobacter infections are erythromycin and ciprofloxacin. However, during the past few decades resistant C. jejuni and C. coli strains have rapidly emerged. The aim of this thesis was to study the various resistance mechanisms of C. jejuni, C. coli and C. hyointestinalis towards antimicrobial agents. When susceptibility to 12 different antimicrobials was tested, it was concluded that Finnish C. hyointestinalis strains remain susceptible to most antimicrobials of veterinary importance. All the 24 reindeer strains included in the study proved susceptible, but resistance to streptomycin and sulphonamides was observed in 32% and 24% of the 25 bovine strains, respectively. The difference in the susceptibility profile of the bovine and reindeer strains most probably reflects the veterinary use of these substances in bovine husbandry, but not in reindeer. Unlike C. jejuni and C. coli, C. hyointestinalis is inherently resistant to nalidixic acid, but the mutations in the quinolone resistance-determining region (QRDR) of gyrA involved in the acquired quinolone resistance of C. jejuni were not present in C. hyointestinalis. An efflux pump system, CmeABC, has been established to play an important role in the resistance of C. jejuni and C. coli to various antimicrobial agents. The effect of putative efflux pump inducers and inhibitors on the minimum inhibitory concentration (MIC) values of C. jejuni, C. coli and C. hyointestinalis was evaluated. It was concluded that the most potent was an efflux pump inhibitor, PAβN, which decreased inherent resistance of C. hyointestinalis to nalidixic acid 2- to 8-fold, and of C. jejuni and C. coli to rifampicin 8- to 64-fold. It also increased inherent susceptibility of C. jejuni and C. coli to erythromycin 8- to 32-fold. Another inhibitor, NMP, had similar but smaller effect on erythromycin, rifampicin and tetracycline MIC values of C. jejuni and C. coli. The two tested putative efflux pump inducers, sodium salicylate and bile salts, did not have any major effect on the MIC values of the studied antimicrobials. Since target mutation is an important resistance mechanism in C. jejuni and C. coli, the mutation frequencies of C. jejuni and C. coli strains were determined. The obtained values varied from hypomutable to strongly hypermutable. The relatively large proportion (25%) of hypermutable strains may facilitate the adaptation of C. jejuni and C. coli to selective environments. When originally ciprofloxacin-susceptible C. jejuni strains were subjected to low-level (0.125 or 1 mg/l) ciprofloxacin, the MIC values for these strains increased up to 32 mg/l. The high MIC values persisted even when the strains were repeatedly subcultured in the absence of ciprofloxacin. Subsequent sequencing of the QRDR revealed multiple peaks at nucleotide positions 256 and 267, but some variants had ciprofloxacin MIC levels of up to 16 mg/l, even though no peaks corresponding to mutated nucleotides in the QRDR were observed; this suggests the presence of a QRDR-independent resistance mechanism in these strains. Teollistuneissa maissa, kuten Suomessa, suolisto-oireita ja ripulia yleisimmin ihmisille aiheuttava bakteeri on kampylobakteeri. Yleisimmät tautia aiheuttavat kampylobakteerilajit ovat Campylobacter jejuni ja Campylobacter coli, mutta muiden tautia aiheuttavien lajien, kuten Campylobacter hyointestinaliksen, patogeeninen merkitys on vielä selvittämättä. Kampylobakteeri-infektiota hoidetaan tarvittaessa antimikrobisilla lääkkeillä, kuten siprofloksasiinilla tai erytromysiinillä. Viimeisen parinkymmenen vuoden aikana bakteerien vastustuskyky antimikrobisille aineille on kuitenkin voimakkaasti lisääntynyt. Tämän tutkimuksen tarkoituksena oli tutkia C. jejunin, C. colin ja C. hyointestinaliksen resistenssimekanismeja. Tutkittaessa C. hyointestinalis -kantojen (24 kantaa poroista ja 25 naudoista) herkkyyttä 12 eri mikrobilääkkeelle havaittiin, että laji on edelleen hyvin herkkä suurelle osalle lääkkeistä. Sulfonamideille resistenttejä kantoja oli 12 % ja streptomysiinille resistenttejä kantoja oli 16 %, kaikkien resistenttien kantojen ollessa peräisin naudoista. Porojen lääkeherkkyyttä voidaan selittää sillä, että päinvastoin kuin nautoja, poroja tuskin koskaan lääkitään mikrobilääkkeillä. C. hyointestinalis on luontaisesti resistentti nalidiksiinihapolle. Resistenssimekanismin selvittämiseksi sekvensoitiin QRDR-geeni, jonka mutaatiot liittyvät hankittuun nalidiksiini- ja siprofloksasiiniresistenssiin C. jejunilla. Näillä mutaatioilla ei kuitenkaan voitu selittää C. hyointestinaliksen luontaista nalidiksiinihapporesistenssiä. C. jejunissa ja C. colissa on karakterisoitu pumppujärjestelmä, CmeABC, joka pystyy pumppaamaan bakteereista ulos antimikrobisia aineita ja siten parantamaan bakteerien mikrobilääkekestävyyttä. Tämän pumpun toimintaa estämään on kehitetty kaksi ainetta, PAβN ja NMP, joiden tehokkuutta tässä tutkimuksessa testattiin. Havaittiin, että PAβN selvästi vähentää C. jejunin ja C. colin kestävyyttä rifampisiinille ja erytromysiinille sekä C. hyointestinaliksen kestävyyttä nalidiksiinihapolle. NMP puolestaan vähentää C. jejunin erytromysiini-, rifampisiini- ja tetrasykliinikestävyyttä. Todettiin, että näiden kahden aineen vaikutustapa on erilainen ja että PAβN:n vaikutus on voimakkaampi. Monet kampylobakteerien resistenssimekanismeista perustuvat mutaatioihin, mistä syystä tutkittiin C. jejuni- ja C. coli -kantojen mutaatiofrekvenssit. Havaittiin, että kantojen välillä oli suuria eroja mutatoitumisalttiudessa. Kannoista verrattain suuri osa oli erittäin alttiita mutaatioille, mikä saattaa helpottaa niiden adaptoitumista selektiivisiin ympäristöihin. Kun siprofloksasiinille herkkiä C. jejuni -kantoja altistettiin vähäiselle määrälle siprofloksasiinia (0,125 mg/l tai 1 mg/l), lisääntyi näiden kantojen siprofloksasiinikestävyys jopa 32 mg:aan/l. Tämä resistenssi säilyi, vaikka kannat myöhemmin siirrettiin alustalle, jossa ei siprofloksasiinia ollut. Sekvensoitaessa resistentin kannan QRDR-alue, saatiin viitteitä siitä, että kanta sisälsi alapopulaatioita, joissa oli erilaisia siprofloksasiinille kestävyyttä lisääviä mutaatioita. Kuitenkin joissain tapauksessa kannan kestävyys saattoi olla jopa 16 mg/l vaikka sekvensoinnissa ei paljastunut yhtään mutaatiota QRDR-alueella. Tämä viittasi siihen, että näiden kantojen siprofloksasiiniresistenssimekanismi oli QRDR-mutaatioista riippumaton.
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