Implication of Porins in β-Lactam Resistance of Providencia stuartii

Abstract

An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to ␤-lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with ␤-lactam molecules. Determination of ␤-lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem Ͼ Ͼ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to ␤-lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to ␤-lactam antibiotics.Providencia stuartii is an opportunistic pathogen involved in community-acquired as well as hospital-acquired infectious diseases. Clinical strains of P. stuartii are mostly isolated from urinary tract infections of patients with long-term indwelling urinary catheters and, in fewer cases, from respiratory and skin infections (1, 2). P. stuartii is reported as one of the most resistant species in the family of Enterobacteriaceae (3). P. stuartii strains show high levels of resistance to the majority of antibiotic classes but were found to remain susceptible to carbapenems (3, 4). P. stuartii produces a chromosomally encoded cephalosporinase, AmpC, which causes the natural resistance to aminopenicillins and narrow-spectrum cephalosporins (5). The productions of different extended-spectrum ␤-lactamases (6, 7, 8, 9, 10, 11) and metallo-␤-lactamases (12, 13, 14) have been reported in association with resistance to carbapenems in Providencia spp. Other enzymatic mechanisms of antibiotic resistance identified in P. stuartii include acetyl aminotransferases targeting aminoglycoside antibiotics and an integronencoded erythromycin esterase involved in the resistance to macrolides (15, 16). Moreover, mutations of the gyrA gene leading to a modification of target site were described in resistance to fluoroquinolones (17). In contrast, little is known about the involvement of membrane proteins in antibiotic resistance of P. stuartii. One study suggested a role of outer membrane porins in antibiotic permeability for Proteus, Morganella, and Providencia strains by selection of mutants resistant to cefoxitin, a cephalosporin of the second generation (18). However, the authors sugg...

Providencia stuartii form biofilms and floating communities of cells that display high resistance to environmental insults

“…Current interest in porins mostly stems from their involvement in antibiotic uptake [36] and in the emergence of antibiotic resistance [37]. But as the first door opened toward the exterior, they also play a number of additional roles in bacterial survival, homeostasis and pathogenesis, adhesion to surfaces and host cells [38], and sometimes penetration into these [39]. Porins are therefore good candidates for playing a role in limiting excessive urea accumulation in the periplasm.…”

Outbreak of Carbapenem-Resistant Providencia stuartii in an Intensive Care Unit

“…The OMP profile of the CRPS showed 2 bands probably corresponding to Omp A and OmpPst1 of 40 and 37 kDa, respectively, as described elsewhere. 12 The presence of this protein on the SDS gel suggests that it was expressed, but we cannot rule out a potential conformational modification in these OMPs that could affect carbapenem permeability. Thus, the carbapenem-resistant phenotype was due to de-repression of chromosomally encoded AmpC and ESBL production, probably coupled with one of the following mechanisms: conformational OMP alteration, modification of the penicillin-binding proteins, or efflux-pump expression, which were not investigated in this study.…”

“…The OMP profile of the CRPS showed 2 bands corresponding to the 2 major porins of 37 and 40 kDa, as described elsewhere. 12 All CRPS showed a single PFGE pattern belonging to a unique clone. discussion CRPS isolates represent a serious clinical threat because these organisms are intrinsically resistant to last-resort agents such as polymyxins and tigecycline.…”

Characterization of extended-spectrum β-lactamase (ESBL) producing non-typhoidal Salmonella (NTS) from imported food products

“…The broth mating conjugation method was performed by the method of Tran et al (Tran et al, 2010). MacConkey (BD, Franklin Lakes, NJ) and LB agar plates containing NaN 3 (300 mg/mL, Sigma Chemical Co., St. Louis, MO) with ampicillin (100 μg/mL, USB, Cleveland, OH) or ceftriaxone (64 μg/mL, Sigma Chemical Co.) were used to select transconjugants harboring genes conferring β-lactam resistance.…”