The prevalence of active drug efflux pump and porin alterations was investigated in Turkish nosocomial strains of Klebsiella pneumoniae exhibiting a multidrug-resistant phenotype. MICs of various antibiotics, including quinolones, chloramphenicol, tetracycline, and β-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine β-naphthylamide (PAβN). Thirty-nine percent of the strains exhibited a PAβN-modulated resistance for quinolones, chloramphenicol, and tetracycline. In these strains, a significant increase of chloramphenicol accumulation was gained in the presence of the efflux pump inhibitor PAβN or with the energy uncoupler carbonyl cyanide m -chlorophenylhydrazone. Moreover, high-level expression of the membrane fusion protein AcrA, which was immunodetected in most of those isolates, suggests that the AcrAB/TolC efflux machinery contributed to their antibiotic resistance. Studies of K. pneumoniae porins indicated that the majority of the strains, including extended-spectrum β-lactamase producers and efflux-positive ones, presented an alteration in their sorbitol-sensitive porin (OmpK35) expression. This is the first report showing the prominent role of active drug efflux in the antibiotic resistance of nosocomial K. pneumoniae strains from Turkey.

Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confirmed by combination disk synergy tests. Types of carbapenemases were investigated using specific primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available.

Colistin resistant Acinetobacter baumannii strains are of great concern worldwide. However, the role of efflux pumps in colistin resistance needs to be elucidated. We investigated the changes in colistin MICs of 29 colistin resistant A. baumannii isolates in response to resistance-nodulation-division (RND)-type efflux pump inhibitor (EPI) and the alterations in AdeR and AdeS two-component regulatory proteins previously associated with the overproduction of AdeAB. The EPI, 1-(1-naphthylmethyl)-piperazine (NMP), led to significant reductions in colistin MICs. At least one of the following amino acid substitutions was found in AdeS proteins from 18 of the isolates: L172P, A94V, V27I, V32I, G186V, and G164A. Besides, A136V and V120I alterations were identified in AdeR from five isolates. Therefore, EPI-responsive colistin resistance in our isolates is most likely due to the action of an RND-type efflux system. The underlying mechanism of resistance might be the result of certain AdeRS alterations, leading to AdeAB overexpression.

In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n=22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla_KPC, bla_IMP, bla_VIM, bla_NDM, and bla_OXA-48. The rates of bla_NDM, bla_OXA-48, and bla_KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla_NDM and bla_OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying bla_KPC, bla_NDM, and/or bla_OXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla_IMP and bla_VIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.