A Tripartite Efflux Pump Involved in Gastrointestinal Colonization by Klebsiella pneumoniae Confers a Tolerance Response to Inorganic Acid

Abstract

In vivo assays showed that a ⌬eefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the ⌬eefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.Klebsiella pneumoniae is an opportunistic pathogen responsible for many nosocomial infections, and most clinical strains exhibit high levels of resistance to a wide variety of structurally unrelated antibiotics. Epidemiological studies have shown that, whatever the infection site, the first stage in nosocomial infections due to K. pneumoniae consists of the colonization of the patient's gastrointestinal (GI) tract (6,16). This pathogen therefore has to sense and respond to numerous different environments in order to survive and, consequently, to persist in the GI tract of the host. The first major barrier encountered following oral consumption is stomach acidity. The bacteria then enter the small intestine, where they encounter stresses associated with volatile fatty acids, variations in pH and osmolarity, and competition with endogenous flora. Using signature-tagged mutagenesis (STM) in a murine model, we previously identified 13 genes encoding factors required for the in vivo colonization of the GI tract by K. pneumoniae LM21 (14). All of these mutants were selected for their inability to colonize the murine intestinal tract under competitive conditions. One of the identified genes potentially encoded a protein involved in a cryptic efflux pump, EefABC, previously described in Enterobacter aerogenes (18). This pump is thought to belong to the resistance-nodulation-division (RND) family and to be composed of an inner membrane protein, EefB, a periplasmic intermediate, EefA, and an outer membrane protein, EefC (18). No specific function has been attributed to this structure so far, whose expression is repressed by the H-NS repressor in the heterologous host Escherichia coli (18). In addition to the export of antimicrobials, several recent publications have highlighted the natural physiological role of efflux pumps in exporting noxious substances out of the bacterial cell, thereby allowing the bacteria to survive in hostile environments and facilitating intestinal colonization (11,13,24,30). In this report, we characterized the eef-like operon of K. pneumoniae and investigated the role of the potentially enc...

Membrane Efflux and Influx Modulate both Multidrug Resistance and Virulence of Klebsiella pneumoniae in a Caenorhabditis elegans Model

“…They also have shown that the K. pneumoniae AcrAB efflux system is involved in resistance to the host antimicrobial peptides present in the lung, one of the first barriers of the innate immune system against infections. This finding strongly suggests the participation of efflux systems of K. pneumoniae in its virulence, as did the results provided by Coudeyras et al that showed that the potential efflux pump EefABC of K. pneumoniae conferred an advantage to this bacterial species to colonize the digestive tract in a murine model (4).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Nearly all tested clinical tigecycline-nonsusceptible isolates (25 out of 26) from China contained mutations in ramR and/or acrR, with about onethird of the isolates carrying the simultaneous overexpression of ramA-acrB and rarA-oqxB (301). The expression of eefABC appears to be induced by an acidic or hyperosmolar environment but not by bile salts (793). The CpxAR system and the LysR-type OxyR regulator are also involved in the positive control of the expression of the acrB, acrD, and/or eefB efflux gene (794,795).…”

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

“…Multidrug efflux pumps of bacterial pathogens are involved in intrinsic and acquired resistance to antimicrobial compounds, including those naturally present at mucosal surfaces; the pumps enable bacteria to grow on such surfaces and can thus be considered as being involved in colonization (65). Moreover, multidrug efflux pumps can discharge molecules involved in the quorum-sensing-regulated expression of virulence determinants (66 (Fig.…”