Cefepime/sulbactam as an enhanced antimicrobial combination therapy for the treatment of MDR Gram-negative infections.

BACKGROUND: ß-Lactam (BL)/ß-lactamase inhibitor (BLI) combinations are widely used for the treatment of Gram-negative infections. Cefepime has not been widely studied in combination with BLIs. Sulbactam, with dual BL/BLI activity, has been partnered with very few BLs. We investigated the potential of cefepime/sulbactam as an unorthodox BL/BLI combination against MDR Gram-negative bacteria. METHODS: In vitro activity of cefepime/sulbactam (1:1, 1:2 and 2:1) was assessed against 157 strains. Monte Carlo simulation was used to predict the PTA with a number of simulated cefepime combination regimens, modelled across putative cefepime/sulbactam breakpoints (≤16/≤0.25 mg/L). RESULTS: Cefepime/sulbactam was more active (MIC50/MIC90 8/8-64/128 mg/L) compared with either drug alone (MIC50/MIC90 128 to >256 mg/L). Activity was enhanced when sulbactam was added at 1:1 or 1:2 (P<0.05). Reduction in MIC was most notable against Acinetobacter baumannii and Enterobacterales (MIC 8/8-32/64 mg/L). Pharmacokinetic/pharmacodynamic modelling highlighted that up to 48% of all isolates and 73% of carbapenem-resistant A. baumannii with a cefepime/sulbactam MIC of ≤16/≤8 mg/L may be treatable with a high-dose, fixed-ratio (1:1 or 1:2) combination of cefepime/sulbactam. CONCLUSIONS: Cefepime/sulbactam (1:1 or 1:2) displays enhanced in vitro activity versus MDR Gram-negative pathogens. It could be a potential alternative to existing BL/BLI combinations for isolates with a cefepime/sulbactam MIC of 16/8 mg/L either as a definitive treatment or as a carbapenem-sparing option.

Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

Introductio n. Pseudomonas aeruginosa is an important Gram-negative pathogen that is intrinsically multidrug-resistant (MDR) and frequently associated with healthcare-associated outbreaks. With increasing resistance to antibiotics and with very few novel drugs under development, clinicians often use combinations to treat critically ill patients.Aim. The aim of this study was to evaluate the ability of epigallocatechin (EGCG) to restore the activity of aztreonam against clinical MDR strains of P. aeruginosa.Methodology. Checkerboard and time-kill kinetic assays were performed to assess synergy in vitro and the Galleria mellonella model of infection was used to test the efficacy of the combination in vivo. Accumulation assays were performed to gain insight into the mechanism of action.Results. The results demonstrate that synergy between aztreonam and EGCG exists [fractional inhibitory concentration indices (FICIs) 0.02-0.5], with the combination affording significantly (P=<0.05) enhanced bacterial killing, with a >3 log10 reduction in colony-forming units ml-1 at 24 h. EGCG was able to restore susceptibility to aztreonam to a level equal to or below the breakpoint set by the European Committee for Antimicrobial Susceptibility Testing. In G. mellonella, the combination was superior to monotherapy, with increased larval survival observed (94 % vs ≤63 %). We also demonstrated the relatively low toxicity of EGCG to human keratinocytes and G. mellonella larvae. Accumulation assay data suggest that the mechanism of synergy may be due to EGCG increasing the uptake of aztreonam.Conclusion. EGCG was able to restore the activity of aztreonam against MDR P. aeruginosa. The data presented support further evaluation of the aztreonam-EGCG combination and highlight its potential for use in clinical medicine.

Characterization of a colistin-resistant Avian Pathogenic Escherichia coli ST69 isolate recovered from a broiler chicken in Germany.

In recent years, several plasmids harbouring genes encoding phosphoethanolamine transferases conferring colistin resistance have been described in multiple Enterobacteriaceae species. Avian Pathogenic E. coli (APEC) causes colibacillosis and is responsible for a considerable proportion of the disease burden in commercial poultry flocks, and may be linked to zoonotic infections in humans. Here, we describe the genotypic and phenotypic characteristics of a multidrug-resistant APEC ST69 isolate (APECA2), recovered in 2016 from a diseased broiler at post-mortem examination in Germany. The isolate was resistant to several antibiotics of human and veterinary importance, including colistin. The mcr-1 gene was detected on a mobile genetic element located on an IncHI2/ST4 plasmid, which was characterized using long-read Nanopore and short-read Illumina sequencing of purified plasmid. Isolate APECA2 displayed resistance to chicken serum and harbours numerous virulence genes. This study highlights the public health importance of enhanced antimicrobial resistance surveillance and strict antimicrobial stewardship in human and veterinary healthcare.

Novel Antibacterials: Alternatives to Traditional Antibiotics.

With the advent of the global antimicrobial resistance (AMR) crisis, our arsenal of effective antibiotics is diminishing. The widespread use and misuse of antibiotics in human and veterinary medicine, compounded by the lack of novel classes of antibiotic in the pharmaceutical pipeline, has left a hole in our antibiotic armamentarium. Thus, alternatives to traditional antibiotics are being investigated, including two major groups of antibacterial agents, which have been extensively studied, phytochemicals and metals. Within these groups, there are several subclasses of compound/elements, including polyphenols and metal nanoparticles, which could be used to complement traditional antibiotics, either to increase their potency or extend their spectrum of activity. Alone or in combination, these antibacterial agents have been shown to be effective against a vast array of human and animal bacterial pathogens, including those resistant to licensed antibacterials. These alternative antibacterial agents could be a key element in our fight against AMR and provide desperately needed options, to veterinary and medical clinicians alike.

Effects of In vivo Emergent Tigecycline Resistance on the Pathogenic Potential of Acinetobacter baumannii.

Multidrug-resistant lineages of Acinetobacter baumannii (MDRAB) are important nosocomial pathogens. As tigecycline remains active against most MDRAB we sought to investigate whether tigecycline resistance impacts biological fitness. The effects of treatment-emergent tigecycline resistance were investigated in vitro and in vivo using two pre- (AB210; W6976) and post-therapy (AB211; W7282) clinical pairs, recovered from individual patients, where tigecycline resistance was associated with up-regulated efflux activity. All isolates belonged to the same epidemic UK lineage. Significant differences were observed in end-point survival proportions between AB210 and AB211, but not between W6976 and W7282, using the Galleria mellonella infection model. Isolate AB211 outcompeted AB210 in vivo, in contrast to isolate W7282, which was outcompeted by its pre-therapy counterpart, W6972. Whole-genome sequencing of isolates W6976 and W7282 revealed a mutation in the adeABC regulatory gene, adeS in W7282; resulting in a Ser-8 → Arg substitution. Previous whole-genome comparison of AB210 and AB211 also identified a non-synonymous mutation in adeS, among several other lesions in genes involved in biofilm formation and DNA mismatch repair; consistent with the phenotypic differences described here. In conclusion, the differing effects on the wider phenotype were not predictable from the antibiograms or clonal lineage, despite a common mechanism of tigecycline resistance.

In vitro and In vivo Activity of Theaflavin-Epicatechin Combinations versus Multidrug-Resistant Acinetobacter baumannii.

INTRODUCTION: Acinetobacter baumannii is an important human nosocomial pathogen; most clinical isolates are multidrug-resistant (MDR). Infections caused by A. baumannii often lead to high morbidity and mortality, with limited treatment options. Owing to the small number of anti-Gram-negative antibiotics in the development pipeline, researchers are looking to other natural compounds. The aim of this study was to determine the in vitro kill kinetics, in vivo efficacy and toxicity of theaflavin-epicatechin combinations against MDR A. baumannii. METHODS: Kill-kinetic assays were performed in Mueller-Hinton 2 broth over 24 h. Toxicity of the compound in the insect model, Galleria mellonella was investigated. The effect of theaflavin-epicatechin combinations on mortality and morbidity were assessed in Acinetobacter baumannii-infected G. mellonella. Larvae were scored for morbidity (melanisation: scale; 0-4) and mortality over 96 h. RESULTS: Kill-kinetic assays revealed that monotherapy had bacteriostatic activity over 24 h, whereas theaflavin-epicatechin combinations were bactericidal (a >3 log reduction in bacterial numbers at 24 h compared with the starting inoculum). Both polyphenols were non-toxic to G. mellonella at concentrations of up to 1000 mg/kg. In vivo treatment assays showed that the combination significantly increased (t test; p ≤ 0.05) larval survival at 96 h to 86% [±17 standard deviation percentage points (pp)] compared to monotherapy with theaflavin (52% ± 14 pp), epicatechin (44% ± 25 pp) or PBS (31% ± 13 pp). Morbidity was also lower in larvae treated with the combination, compared with monotherapy. CONCLUSION: Polyphenol combinations produce effective antibacterial action against A. baumannii and show great potential for the treatment of infections caused by MDR A. baumannii.

Deletions in a ribosomal protein-coding gene are associated with tigecycline resistance in Enterococcus faecium.

Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125 mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8 mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5 mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.

In vitro and in vivo activities of tigecycline-colistin combination therapies against carbapenem-resistant Enterobacteriaceae.

We assessed the activity of tigecycline (TGC) combined with colistin (COL) against carbapenem-resistant enterobacteria. Synergy occurred in vitro against the majority of isolates, with the exception of Serratia marcescens. In a simple animal model (Galleria mellonella), TGC-COL was superior (P < 0.01) in treating Escherichia coli, Klebsiella pneumoniae, and Enterobacter infections, including those with TGC-COL resistance. Clinical studies are needed to determine whether TGC-COL regimens may be a viable option.

In vitro activity of the novel monosulfactam BAL30072 alone and in combination with meropenem versus a diverse collection of important Gram-negative pathogens.

The problem of antimicrobial resistance is exemplified by multidrug-resistant (MDR) isolates of Gram-negative species. Of particular concern are expanded-spectrum cephalosporin-resistant isolates of Enterobacteriaceae, epidemic lineages of Acinetobacter baumannii producing OXA-type carbapenemases, and MDR Pseudomonas aeruginosa. In this study, the in vitro activity of the novel monosulfactam BAL30072 was investigated both alone and in combination with meropenem against a diverse collection of commonly encountered Gram-negative pathogens. Thirty-one isolates were studied, including type strains and clinical isolates with defined mechanisms conferring resistance to various antimicrobial agents including to carbapenems, colistin and tigecycline. BAL30072 minimum inhibitory concentrations (MICs) were determined in the presence and absence of meropenem (1:1, w/w) by agar dilution. Potential synergy or antagonism between BAL30072 and meropenem was investigated using standard chequerboard assays. Versus MDR A. baumannii strains producing class D oxacillinases, BAL30072 MICs were all ≤4 mg/L with the exception of the isolate belonging to the UK 'Burn' lineage. BAL30072 exhibited MIC values of 0.5 mg/L to >64 mg/L towards the five P. aeruginosa strains. Against three meropenem-susceptible Escherichia coli, including the CTX-M-15 extended-spectrum ß-lactamase-producer, BAL30072 exhibited MICs of 0.25-2 mg/L; higher MICs were recorded against some of the Enterobacteriaceae isolates tested. The in vitro data suggest that BAL30072 has a potential role in the treatment of infections due to Gram-negative pathogens, including those with important resistances to other agents. In addition, BAL30072 shows powerful synergistic activity in combination with meropenem, potentially expanding its coverage for the treatment of infections caused by problematic species.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.

OBJECTIVES: The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates. METHODS: The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility. RESULTS: Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression. CONCLUSIONS: Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

In vivo efficacy of telavancin/colistin combination therapy in a Galleria mellonella model of Acinetobacter baumannii infection.

Treatment of Acinetobacter baumannii infections is challenging owing to widespread multidrug resistance and the lack of novel agents. There is now considerable interest in the potential of unorthodox combination therapies such as colistin and glycopeptides (e.g. vancomycin and teicoplanin), since potent synergy can be demonstrated in vitro. A simple invertebrate model (Galleria mellonella) has been developed to assess the in vivo activity of antimicrobial therapies and was used to investigate the efficacy of colistin combined with the lipoglycopeptide telavancin in the treatment of A. baumannii infection. Galleria mellonella larvae were inoculated with 105 CFU/larvae of A. baumannii type strain ATCC 19606 or the multidrug-resistant clinical isolate AB210. Infected caterpillars were treated with either telavancin (10 mg/kg), colistin (2.5 mg/kg) or a telavancin/colistin combination. Larvae were incubated at 37 °C for up to 96 h and were scored daily. Survival curves were plotted and analysed using the log-rank test. The telavancin/colistin combination was effective in the treatment of larvae infected with both strains but was superior to colistin monotherapy in the treatment of A. baumannii AB210 (P<0.001). The combination of telavancin and colistin was effective in a simple invertebrate model of A. baumannii infection. This is in agreement with a previous in vitro study and provides preliminary in vivo evidence that such a combination might be useful therapeutically.

Evaluation of three selective chromogenic media, CHROMagar ESBL, CHROMagar CTX-M and CHROMagar KPC, for the detection of Klebsiella pneumoniae producing OXA-48 carbapenemase.

Three selective chromogenic culture media (CHROMagars ESBL, CTX-M and KPC) were evaluated for their ability to support the growth of nine Klebsiella pneumoniae isolates producing OXA-48 carbapenemase in combination with other ß-lactamases. CHROMagar ESBL and CHROMagar KPC were the most sensitive media, supporting growth of all isolates with a detection limit as low as < 100 CFU/ml. Five isolates failed to grow on CHROMagar CTX-M, and five were recovered on CHROMagar KPC only at counts > 10(6) CFU/ml. Both CHROMagar ESBL and CHROMagar KPC may be useful for enhanced isolation of K pneumoniae producing OXA-48-like carbapenemases.

In vitro activity of telavancin in combination with colistin versus Gram-negative bacterial pathogens.

The treatment of Gram-negative infections is increasingly compromised by the spread of resistance. With few agents currently in development, clinicians are now considering the use of unorthodox combination therapies for multidrug-resistant strains. Here we assessed the in vitro activity of the novel lipoglycopeptide telavancin (TLV) when combined with colistin (COL) versus 13 Gram-negative type strains and 66 clinical isolates. Marked synergy was observed in either checkerboard (fractional inhibitory concentration index [FICI], <0.5; susceptibility breakpoint index [SBPI], >2) or time-kill assays (>2-log reduction in viable counts compared with starting inocula at 24 h) versus the majority of COL-susceptible enterobacteria, Stenotrophomonas maltophilia, and Acinetobacter baumannii isolates, but only limited effects were seen against Pseudomonas aeruginosa or strains with COL resistance. Using an Etest/agar dilution method, the activity of TLV was potentiated by relatively low concentrations of COL (0.25 to 0.75 µg/ml), reducing the MIC of TLV from >32 µg/ml to ≤ 1 µg/ml for 35% of the clinical isolates. This provides further evidence that glycopeptide-polymyxin combinations may be a useful therapeutic option in the treatment of Gram-negative infections.

A novel variant, NDM-5, of the New Delhi metallo-ß-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom.

A new variant of the New Delhi metallo-enzyme (NDM) carbapenemase was identified in a multidrug-resistant Escherichia coli ST648 isolate recovered from the perineum and throat of a patient in the United Kingdom with a recent history of hospitalization in India. NDM-5 differed from existing enzymes due to substitutions at positions 88 (Val → Leu) and 154 (Met → Leu) and reduced the susceptibility of E. coli TOP10 transformants to expanded-spectrum cephalosporins and carbapenems when expressed under its native promoter.

Whole-genome comparison of two Acinetobacter baumannii isolates from a single patient, where resistance developed during tigecycline therapy.

OBJECTIVES: The whole genomes of two Acinetobacter baumannii isolates recovered from a single patient were sequenced to gain insight into the nature and extent of genomic plasticity in this important nosocomial pathogen over the course of a short infection. The first, AB210, was recovered before tigecycline therapy and was susceptible to this agent; the second, AB211, was recovered after therapy and was resistant. METHODS: DNA from AB210 was sequenced by 454 GS FLX pyrosequencing according to the standard protocol for whole-genome shotgun sequencing, producing ∼250 bp fragment reads. AB211 was shotgun sequenced using the Illumina Genetic Analyzer to produce fragment reads of exactly 36 bp. Single nucleotide polymorphisms (SNPs) and large deletions detected in AB211 in relation to AB210 were confirmed by PCR and DNA sequencing. RESULTS: Automated gene prediction detected 3850 putative coding sequences (CDSs). Sequence analysis demonstrated the presence of plasmids pAB0057 and pACICU2 in both isolates. Eighteen putative SNPs were detected between the pre- and post-therapy isolates, AB210 and AB211. Three contigs in AB210 were not covered by reads in AB211, representing three deletions of ∼15, 44 and 17 kb. CONCLUSIONS: This study demonstrates that significant differences were detectable between two bacterial isolates recovered 1 week apart from the same patient, and reveals the potential of whole-genome sequencing as a tool for elucidating the processes responsible for changes in antibiotic susceptibility profiles.

Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system.

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg(2+) induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A.

AdeABC-mediated efflux and tigecycline MICs for epidemic clones of Acinetobacter baumannii.

OBJECTIVES: Tigecycline non-susceptibility in individual Acinetobacter baumannii isolates has been associated with up-regulation of the resistance-nodulation-division (RND)-type efflux system, AdeABC. We sought to relate variation in the expression of this system to differences in modal tigecycline MIC among prevalent A. baumannii clones. The role of AdeABC in the emergence of tigecycline resistance during therapy was also investigated for two representatives of the prevalent UK lineage, OXA-23 clone 1. METHODS: Clonal type was defined by PFGE and expression of adeABC by real-time RT-PCR. Laboratory mutants were selected in vitro by exposing a susceptible clinical isolate to increasing tigecycline concentrations. The adeB gene was inactivated by the directed integration of a suicide plasmid containing an internal fragment of the target gene. RESULTS: Higher modal tigecycline MICs for particular clones correlated with elevated expression of adeABC. Expression of this operon was also increased in the two post-therapy, tigecycline-resistant clinical isolates and in a laboratory mutant as compared with their pre-exposure, tigecycline-susceptible counterparts. Interruption of adeB in a tigecycline-resistant clinical isolate restored full susceptibility to tigecycline. CONCLUSIONS: Differences in expression of adeABC contribute to both inter- and intra-clone variation in tigecycline MICs. Tigecycline resistance can arise during therapy, mediated by up-regulation of AdeABC.

Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment.

Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage lambda Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.

Tigecycline resistance in Serratia marcescens associated with up-regulation of the SdeXY-HasF efflux system also active against ciprofloxacin and cefpirome.

OBJECTIVES: Efflux by RND-type transporters is known to confer resistance to tigecycline in several Enterobacteriaceae species and we examined the potential of this mechanism in Serratia marcescens using a clinical isolate and laboratory-selected mutants. METHODS: Expression of RND-type efflux pump genes was analysed by real-time RT-PCR. Laboratory mutants were selected by exposure to either tigecycline or tetracycline in vitro. Efflux pump genes were inactivated by suicide plasmids containing the R6K gamma origin of replication. RESULTS: Higher tigecycline MICs correlated with elevated expression of the RND-type efflux pump genes sdeXY. Inactivation of sdeY or the outer membrane component gene hasF reduced MICs of tigecycline, tetracycline, ciprofloxacin and cefpirome to below those for strain NCTC 10211. A tetracycline-selected laboratory mutant also showed increases in sdeXY expression and tigecycline MIC. CONCLUSIONS: Up-regulation of endogenous SdeXY-HasF-mediated efflux is associated with tigecycline resistance in S. marcescens along with MIC rises for tetracycline, ciprofloxacin and cefpirome. Inactivation of this efflux system reduced MICs of those compounds to below those for strain NCTC 10211.