Respiratory infections caused by Klebsiella pneumoniae are characterized by high rates of mortality and morbidity. Management of these infections is often difficult, due to the high frequency of strains that are resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. In the present study, we investigated the role of the K. pneumoniae AcrRAB operon in antimicrobial resistance and virulence by using isogenic knockouts deficient in the AcrB component and the AcrR repressor, both derived from the virulent strain 52145R. We demonstrated that the AcrB knockout was more susceptible, not only to quinolones, but also to other antimicrobial agents, including β-lactams, than the wild-type strain and the AcrR knockout. We further showed that the AcrB knockout was more susceptible to antimicrobial agents present in human bronchoalveolar lavage fluid and to human antimicrobial peptides than the wild-type strain and the AcrR knockout. Finally, the AcrB knockout exhibited a reduced capacity to cause pneumonia in a murine model, in contrast to the wild-type strain. The results of this study suggest that, in addition to contributing to the multidrug resistance phenotype, the AcrAB efflux pump may represent a novel virulence factor required for K. pneumoniae to resist innate immune defense mechanisms of the lung, thus facilitating the onset of pneumonia.

Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa , a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD , encoding an N -acetyl-anhydromuramyl- l -alanine amidase, affecting the levels of ampC -activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis. IMPORTANCE Understanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced by Pseudomonas aeruginosa , a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all three P. aeruginosa AmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combating P. aeruginosa infections by simultaneously targeting virulence and antibiotic resistance.

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from two K. pneumoniae clinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene, yjhA , in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reduced in vitro fitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance in K. pneumoniae but cannot restore the fitness of the microorganism.

A total of 10 metallo-β-lactamase-producing isolates of six different species, including Brevundimonas diminuta ( n = 3), Rhizobium radiobacter ( n = 2), Pseudomonas monteilii ( n = 1), Pseudomonas aeruginosa ( n = 2), Ochrobactrum anthropi ( n = 1), and Enterobacter ludwigii ( n = 1), were detected in the sewage water of a hospital. The presence of bla _VIM-13 associated with a Tn 1721 -class 1 integron structure was detected in all but one of the isolates ( E. ludwigii , which produced VIM-2), and in two of them ( R. radiobacter ), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-β-lactamase genes.

Pseudomonas aeruginosa undergoes phase variation in the expression of the phosphorylcholine (ChoP) epitope, a structure crucial for the virulence of several respiratory pathogens. In this study, ChoP expression analysis comparing organisms from acute and chronic infections revealed that expression of ChoP at 37°C was higher among strains from chronic infections. Coimmunoprecipitation experiments and mass spectrometry analysis demonstrated that ChoP was on the protein elongation factor Tu. The presence of ChoP at the surface was confirmed by immunofluorescence and flow cytometry analysis of intact bacteria. Pretreatment of bronchial epithelial cells or mice with a platelet-activating factor receptor (PAFR) antagonist reduced adhesion and invasion of the ChoP-positive P. aeruginosa isolates. Results of this study suggest that ChoP expression may represent a novel phenotype expressed by the chronic infection isolates that could mediate P. aeruginosa colonization of the epithelial airway by means of the interaction with the PAFR.

To investigate the contribution of LamB in Klebsiella pneumoniae antimicrobial resistance, we determined the MICs of various antibiotics and the frequency of mutation to increased cefoxitin or meropenem resistance of the strains CSUB10S (expressing only OmpK36), CSUB10R (lacking OmpK35 and OmpK36), and their derived isogenic insertion-duplication mutants deficient in LamB. Expression of LamB was indispensable in order for CSUB10S to lose OmpK36 and become resistant to cefoxitin, while in CSUB10R, LamB deficiency promoted increased resistance to carbapenem.