Klebsiella pneumoniae Major Porins OmpK35 and OmpK36 Allow More Efficient Diffusion of β-Lactams than Their Escherichia coli Homologs OmpF and OmpC

Abstract

Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum ␤-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged ␤-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of ␤-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by ␤-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of ␤-lactams through its wide porin channels. IMPORTANCEIn Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae. We found that the larger channel porin of this organism, OmpK35, produces a significantly larger channel than its Escherichia coli homolog, OmpF. This makes unmodified K. pneumoniae strains more susceptible to relatively large antibiotics, such as the third-and fourth-generation cephalosporins. Also, even the acquisition of powerful ␤-lactamases is not likely to make them fully resistant in the presence of such an effective influx process, explaining why so many clinical isolates of this organism lack porins. K lebsiella pneumoniae is currently one of the most important Gram-negative nosocomial pathogens, which are often carbapenem resistant (1), and its wide dissemination is helped by its capacity to survive in the hospital environment (2), presumably aided by the production of thick capsules. A remarkable feature of multidrug-resistant isolates of this species is the absence of porin(s) (for an early report, see reference 3, and for reviews, see references 4 and 5). K. pneumoniae produces two classical trimeric porins, OmpK35 and OmpK36, which are homologs of OmpF and OmpC of Escherichia col...

Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen

“…Our findings of increased MICs in OmpK35 mutants are consistent with those of others [102] but we show here that the more permeable OmpK35 is not important in the mammalian host. Rather, the much less permeable OmpK36 (equivalent to E coli OmpC) [102] is the bottleneck for large anionic antibiotics.…”

“…Diffusion of β-lactam antibiotics through non-specific porins such as OmpK35 and OmpK36 is dependent on size, charge and hydrophobicity [86, 87], with bulky negatively charged compounds diffusing at a lower rate than small zwitterions of the same molecular weight [88]. OmpK35 is much less expressed in high osmolarity nutrient-rich conditions than OmpK36, which has the narrower porin channel of the two (Fig S12) [9] and large negatively charged β-lactams such as third-generation cephalosporins and carbapenems diffuse more efficiently through OmpK35 than OmpK36 [78, 89]. Here we confirm the significantly increased MICs, commonly attributed to mutations in these two major porins [10, 90, 91], in three K. pneumoniae strains (the widely-published ATCC strain 13883 and two locally isolated clinical strains (Tables 2 and S4); and unequivocally identify the primary role of OmpK36 in carbapenem resistance.…”

“…High level antimicrobial resistance has been ascribed to similar variations in L3 of OmpK36 homologues in Enterobacter aerogenes , Escherichia coli and Neisseria gonorrhoeae (Fig S15) [97-101]. In comparison with their E. coli homologues (OmpF and OmpC), OmpK35 and OmpK36 permit greater diffusion of β-lactams [102]. Specifically, OmpK35 appears to be highly permeable to third-generation cephalosporins such as cefotaxime due to its particular L3 domain, which is also seen in Omp35 in E. aerogenes but not in other species, and has been proposed as an explanation for the high proportion of K. pneumoniae clinical isolates that lack this porin [102, 103].…”

“…In comparison with their E. coli homologues (OmpF and OmpC), OmpK35 and OmpK36 permit greater diffusion of β-lactams [102]. Specifically, OmpK35 appears to be highly permeable to third-generation cephalosporins such as cefotaxime due to its particular L3 domain, which is also seen in Omp35 in E. aerogenes but not in other species, and has been proposed as an explanation for the high proportion of K. pneumoniae clinical isolates that lack this porin [102, 103]. Our findings of increased MICs in OmpK35 mutants are consistent with those of others [102] but we show here that the more permeable OmpK35 is not important in the mammalian host.…”

Insertion Sequence-Dependent <i>OmpK</i>36 Mutation Associated Ertapenem Resistance in Clinical <i>Klebsiella pneumoniae</i>

“…Regardless of whether the isolate was ESBL-or non-ESBL-producing, the carbapenem-resistant isolates carried bla DHA-1 and an insertion in both ompK35 and ompK36 genes, exhibiting the highest MIC to carbapenems ( and a significant decrease in proinflammatory cytokine levels [45]. Further, the loss of OmpK35 is associated with resistance to lipophilic (benzylpenicillin) and large (cefepime) compounds [46]. In contrast to ompK35 deletion, ompK36 deletion caused an increased resistance to cefoxitin and cefepine associated with bla gene [47].…”