Interplay Between Membrane Permeability and Enzymatic Barrier Leads to Antibiotic-Dependent Resistance in Klebsiella Pneumoniae.

The interplay between membrane permeability alterations and the enzymatic barrier contributes to Klebsiella pneumoniae multidrug resistance. We assessed the specific effect of the efflux levels of the main efflux pumps (AcrAB and OqxAB), alone and associated with the loss of the main porins (OmpK35 and OMPK36), on the activity of various antibiotics by constructing a set of K. pneumoniae isogenic strains, including strains with plasmid-mediated ß-lactamases (DHA-1, CTX-M-15, and OXA-48). The two pumps contributed to intrinsic chloramphenicol resistance and AcrAB to that of nalidixic acid and cefoxitin, whereas they had no impact on the activity of the other 11 antibiotics tested. We confirmed the expulsion of these three antibiotics by the two overproduced pumps and that of tigecycline by overproduced AcrAB, and showed that overproduced AcrAB also expelled ertapenem, piperacillin, ceftolozane, and ceftazidime. The sole loss of porins did not significantly affect the activity of the tested antibiotics, except ertapenem. The effect of efflux increases and porin loss on ß-lactam activity was the highest in plasmid-mediated ß-lactamase-producing strains. Thus, DHA-1-producing strains became non-susceptible (NS) to (i) ertapenem when there was an increase in efflux or porin loss, (ii) imipenem and ceftazidime+avibactam when the two mechanisms were associated, and (iii) temocillin when AcrAB was overproduced. The CTX-M-15-producing strains became NS to (i) ertapenem when there was no porin, (ii) ceftolozane+tazobactam when there was either overproduced OqxAB or porin loss, and (iii) temocillin when AcrAB was overproduced. OXA-48-producing strains known to be NS to temocillin were also NS to ceftolozane and they became NS to imipenem when the two pumps were overproduced or there was porin loss. Overall, this study shows that the balance between influx and efflux differentially modulates the activity of the tested antibiotics, an important point for evaluating the activity of future antibiotics or new combinations.

Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae.

OBJECTIVES: In Klebsiella pneumoniae, overexpression of the AcrAB efflux pump and the more recently described OqxAB efflux pump has been linked to an antibiotic cross-resistance phenotype, but the mechanisms of regulation are largely unknown. Moreover, while AcrAB has been shown to participate in K. pneumoniae virulence, the contribution of OqxAB has not yet been assessed. METHODS: In the present study we investigated a K. pneumoniae clinical isolate (KPBj1 E+), displaying cross-resistance to quinolones, chloramphenicol and cefoxitin, and its phenotypic revertant (KPBj1 Rev, susceptible to antibiotics) by using whole-genome sequencing, RT-PCR, complementation and a Caenorhabditis elegans virulence model. RESULTS: We detected a point mutation in the oqxR repressor gene of KPBj1 E+, which overexpressed genes rarA, encoding a transcriptional regulator, and oqxB, but not acrB. Complementation with wild-type oqxR restored antibiotic susceptibility and normalized rarA and oqxB expression levels. Whole-genome sequencing showed that KPBj1 Rev had lost the entire rarA-oqxABR locus, situated close to an integration hot spot of phage P4. This large deletion seemed responsible for the significantly lower virulence potential of strain KPBj1 Rev compared with KPBj1 E+. Moreover, we found that KPBj1 E+ ΔacrB was significantly less virulent than its parental strain. CONCLUSIONS: This work demonstrates the role of the overexpression of efflux pump OqxAB, due to a mutation in gene oqxR, in the antibiotic resistance phenotype of a clinical isolate, and suggests that the presence of AcrAB, associated with overexpression of OqxAB, is required for high virulence potential.

Multilocus sequence typing and rtxA toxin gene sequencing analysis of Kingella kingae isolates demonstrates genetic diversity and international clones.

BACKGROUND: Kingella kingae, a normal component of the upper respiratory flora, is being increasingly recognized as an important invasive pathogen in young children. Genetic diversity of this species has not been studied. METHODS: We analyzed 103 strains from different countries and clinical origins by a new multilocus sequence-typing (MLST) schema. Putative virulence gene rtxA, encoding an RTX toxin, was also sequenced, and experimental virulence of representative strains was assessed in a juvenile-rat model. RESULTS: Thirty-six sequence-types (ST) and nine ST-complexes (STc) were detected. The main STc 6, 14 and 23 comprised 23, 17 and 20 strains respectively, and were internationally distributed. rtxA sequencing results were mostly congruent with MLST, and showed horizontal transfer events. Of interest, all members of the distantly related ST-6 (n = 22) and ST-5 (n = 4) harboured a 33 bp duplication or triplication in their rtxA sequence, suggesting that this genetic trait arose through selective advantage. The animal model revealed significant differences in virulence among strains of the species. CONCLUSION: MLST analysis reveals international spread of ST-complexes and will help to decipher acquisition and evolution of virulence traits and diversity of pathogenicity among K. kingae strains, for which an experimental animal model is now available.

Cost and effectiveness of intrapartum group B streptococcus polymerase chain reaction screening for term deliveries.

OBJECTIVE: To estimate the cost and consequences of intrapartum polymerase chain reaction (PCR) screening on early-onset group B streptococcal (GBS) disease compared with the antenatal lower vagina culture screening recommended in France. METHODS: This was a single-institution study comparing the intrapartum PCR screening strategy implemented in 2010 with antenatal culture strategy in place in 2009. Early-onset GBS disease in newborns was monitored exhaustively. We estimated direct costs, including screening test costs and hospital costs, for deliveries of healthy newborns compared with those infected with GBS. Costs in 2009 and 2010 were compared on an intention-to-treat basis. RESULTS: Term deliveries were 2,761 and 2,814 in 2009 and 2010, respectively. Among the screened mothers, the vaginal GBS colonization rate was 11.7% based on antenatal GBS culture screening in 2009 compared with 16.7% in 2010 using the intrapartum PCR testing. The overall probabilities of neonatal GBS disease were 0.9% compared with 0.5%, and the average total cost per delivery was $1,759±1,209 in 2009 compared with $1,754±842 in 2010 (P=.9) in antenatal and intrapartum screening strategies, respectively. The number and severity of cases of early-onset GBS disease and the resulting hospital costs were higher in 2009. CONCLUSION: Polymerase chain reaction intrapartum screening strategy was cost-neutral when compared with the 2009 antenatal lower vagina culture screening, with a significant decrease in early-onset GBS disease.