Klebsiella pneumoniae exhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants.

In this study, we characterized a Klebsiella pneumoniae strain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes in Enterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 isolates with different resisto- and morpho-types were distinguished from the patient's urine and tissue samples. Whole genome sequencing revealed that all isolates were clonally identical belonging to the K. pneumoniae high-risk sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic "hyper-splitting" in a K. pneumoniae SCV and its potential clinical significance.

Case Report: Hip arthroplasty after fracture-related joint infection caused by extensively drug-resistant Klebsiella pneumoniae.

This case-report focuses on a 23-year-old soldier suffering from a fracture-related hip joint infection (FRI) due to extensively drug-resistant Klebsiella pneumoniae and S. epidermidis. The patient underwent multiple septic revision surgeries including the removal of remaining shrapnel accompanied by last-resort antimicrobial therapy with cefiderocol and colistin. Additionally, the surgeries included repeated tissue sampling for microbiological and histopathological analysis. An antibiotic-loaded cemented filler containing cefiderocol was used to improve local antimicrobial therapy. The biopsies prior to and during hip replacement surgery confirmed successful microbe eradication. Hip arthroplasty restored hip joint function and significantly improved patient's quality of life. The utilization of a trabecular metal shell and a meta-diaphyseally anchored cementless hip stem ensured secure implant fixation and early patient mobilisation. An adjusted biofilm active oral antimicrobial therapy after arthroplasty intervention was continued to prevent early periprosthetic joint infection. This case emphasizes the difficulties of managing FRI and multidrug-resistant pathogens. It contributes valuable insight into navigating complex orthopedic cases while ensuring successful hip arthroplasty outcomes. In conclusion, early interdisciplinary collaboration, appropriate antimicrobial therapy along with tailored surgical interventions are crucial for managing such complex cases successfully.

Genomic analysis of the international high-risk clonal lineage Klebsiella pneumoniae sequence type 395.

BACKGROUND: Klebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. METHODS: We computationally analyzed a large collection of K. pneumoniae ST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. RESULTS: Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of "hybrid" resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. CONCLUSIONS: To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

Geno- and Phenotypic Characteristics of a Klebsiella pneumoniae ST20 Isolate with Unusual Colony Morphology.

Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens.

Extensively Drug-Resistant Klebsiella pneumoniae Counteracts Fitness and Virulence Costs That Accompanied Ceftazidime-Avibactam Resistance Acquisition.

The ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages. IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Antibiotic-Resistant Enterobacteriaceae in Wastewater of Abattoirs.

Antibiotic-resistant Enterobacteriaceae are regularly detected in livestock. As pathogens, they cause difficult-to-treat infections and, as commensals, they may serve as a source of resistance genes for other bacteria. Slaughterhouses produce significant amounts of wastewater containing antimicrobial-resistant bacteria (AMRB), which are released into the environment. We analyzed the wastewater from seven slaughterhouses (pig and poultry) for extended-spectrum ß-lactamase (ESBL)-carrying and colistin-resistant Enterobacteriaceae. AMRB were regularly detected in pig and poultry slaughterhouse wastewaters monitored here. All 25 ESBL-producing bacterial strains (19 E. coli and six K. pneumoniae) isolated from poultry slaughterhouses were multidrug-resistant. In pig slaughterhouses 64% (12 of 21 E. coli [57%] and all four detected K. pneumoniae [100%]) were multidrug-resistant. Regarding colistin, resistant Enterobacteriaceae were detected in 54% of poultry and 21% of pig water samples. Carbapenem resistance was not detected. Resistant bacteria were found directly during discharge of wastewaters from abattoirs into water bodies highlighting the role of slaughterhouses for environmental surface water contamination.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

A Klebsiella pneumoniae ST307 outbreak clone from Germany demonstrates features of extensive drug resistance, hypermucoviscosity, and enhanced iron acquisition.

BACKGROUND: Antibiotic-resistant Klebsiella pneumoniae are a major cause of hospital- and community-acquired infections, including sepsis, liver abscess, and pneumonia, driven mainly by the emergence of successful high-risk clonal lineages. The K. pneumoniae sequence type (ST) 307 lineage has appeared in several different parts of the world after first being described in Europe in 2008. From June to October 2019, we recorded an outbreak of an extensively drug-resistant ST307 lineage in four medical facilities in north-eastern Germany. METHODS: Here, we investigated these isolates and those from subsequent cases in the same facilities. We performed whole-genome sequencing to study phylogenetics, microevolution, and plasmid transmission, as well as phenotypic experiments including growth curves, hypermucoviscosity, siderophore secretion, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance for an in-depth characterization of this outbreak clone. RESULTS: Phylogenetics suggest a homogenous phylogram with several sub-clades containing either isolates from only one patient or isolates originating from different patients, suggesting inter-patient transmission. We identified three large resistance plasmids, carrying either NDM-1, CTX-M-15, or OXA-48, which K. pneumoniae ST307 likely donated to other K. pneumoniae isolates of different STs and even other bacterial species (e.g., Enterobacter cloacae) within the clinical settings. Several chromosomally and plasmid-encoded, hypervirulence-associated virulence factors (e.g., yersiniabactin, metabolite transporter, aerobactin, and heavy metal resistance genes) were identified in addition. While growth, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance were comparable to several control strains, results from siderophore secretion and hypermucoviscosity experiments revealed superiority of the ST307 clone, similar to an archetypical, hypervirulent K. pneumoniae strain (hvKP1). CONCLUSIONS: The combination of extensive drug resistance and virulence, partly conferred through a "mosaic" plasmid carrying both antibiotic resistance and hypervirulence-associated features, demonstrates serious public health implications.

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands.

A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements.

On-site blood culture incubation shortens the time to knowledge of positivity and microbiological results in septic patients.

INTRODUCTION: To determine whether on-site incubation of blood cultures at the intensive care unit (ICU) improves not only the time to incubation but also time to positivity, time to knowledge of positivity and time to results (identification and antibiotic susceptibility testing). METHODS: This observational single-centre study in ICU patients with severe sepsis and septic shock investigated the impact of blood culture incubation immediately on-site at the ICU (ICU group) by comparison with traditional processing in a remote laboratory (LAB group) on different time intervals of blood culture diagnostics from obtaining blood to clinician notification of final result. The effect of on-site incubation was evaluated in Kaplan-Meier estimates for the time to positivity, time to knowledge of positivity and time to microbiological results and a linear mixed model was built. RESULTS: A total of 3,549 blood culture sets from 657 ICU patients were analysed: 2,381 in the LAB group and 1,168 in the ICU group. Overall, 660 (18.6%) blood culture sets were positive and 2,889 (81.4%) sets remained negative. On-site incubation was associated with reduced time to knowledge of positivity (46.9 h [CI 43.4-50.8 h] vs. 28.0 h [CI 23.6-32.2 h], p < 0.001) and reduced time to result (61.4 h [CI 58.4-64.8 h] vs. 42.1 h [CI 39.1-47.5 h], p < 0.001). In blood cultures processed instantaneously at the ICU compared to incubation in the remote laboratory within 4 h, the time to microbiological result was significantly reduced by 8.5 h (p < 0.001). Pre-existing anti-infective therapy had no significant impact on diagnostic time intervals. CONCLUSIONS: Instantaneous incubation of blood cultures in the ICU compared to incubation in a remote laboratory significantly improves time to knowledge to positivity and time to result. These effects are even more pronounced during off-hours of the microbiological laboratory. The results underline the importance of 24/7 diagnostics to provide round-the-clock processing of blood culture samples in patients with sepsis and septic shock and an immediate to communication of the results to the clinicians.

Extensively drug-resistant Klebsiella pneumoniae ST307 outbreak, north-eastern Germany, June to October 2019.

From June to October 2019, 17 patients (six infected, 11 colonised) with an extensively drug-resistant (XDR) Klebsiella pneumoniae strain were notified from four Western Pomerania medical facilities. The XDR K. pneumoniae produced carbapenemases NDM-1 and OXA-48, and was only susceptible to chloramphenicol, tigecycline and cefiderocol. Synergistic activity was observed for the combination of aztreonam plus ceftazidime-avibactam. Genomic analyses showed all isolates belonged to K. pneumoniae sequence type 307. Control measures and further investigations are ongoing.

Clinically Approved Drugs Inhibit the Staphylococcus aureus Multidrug NorA Efflux Pump and Reduce Biofilm Formation.

Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 µM. Moreover, at 0.39 µM, nilotinib, in combination with 8 µg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations.

Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-ß-Naphthylamide from Random Mutagenesis.

Phe-Arg-ß-naphthylamide (PAßN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAßN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAßN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAßN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAßN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

Simple Ciprofloxacin Resistance Test and Determination of Minimal Inhibitory Concentration within 2 h Using Raman Spectroscopy.

Resistant bacteria are spreading worldwide, which makes fast antibiotic susceptibility testing and determination of the minimal inhibitory concentration (MIC) urgently necessary to select appropriate antibiotic therapy in time and, by this, improve patient's outcome and, at the same time, avoid inappropriate treatment as well as the unnecessary use of broad spectrum antibiotics that would foster further spread of resistant bacteria. Here, a simple and fast Raman spectroscopy-based procedure is introduced to identify antimicrobial susceptibilities and determine the MIC within only 2 h total analysis, marking a huge time savings compared to established phenotypic methods nowadays used in diagnostics. Sample preparation is fast and easy as well as comparable to currently established tests. The use of a dielectrophoresis chip allows automated collection of the bacteria in a micron-sized region for high-quality Raman measurement directly from bacterial suspensions. The new Raman spectroscopic MIC test was validated with 13 clinical E. coli isolates that show a broad range of ciprofloxacin resistance levels and were collected from patients with blood-stream infection. Micro-Raman spectroscopy was able to detect ciprofloxacin-induced changes in E. coli after only 90 min interaction time. Principal component analysis as well as a simple computed ratio of the Raman marker bands at 1458 and 1485 cm-1 show a clear concentration-dependent effect. The MIC values determined with the new Raman method are in good agreement with MICs obtained by reference methods (broth microdilution, Vitek-2, E-test) and can be used to provide a classification as sensitive, intermediate, or resistant using the clinical breakpoints provided by EUCAST.

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible.

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 µM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 µM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 µM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.

Intestinal decolonization of Enterobacteriaceae producing extended-spectrum ß-lactamases (ESBL): a retrospective observational study in patients at risk for infection and a brief review of the literature.

BACKGROUND: Multidrug-resistant Escherichia coli and other enteric bacteria producing extended-spectrum ß-lactamases (ESBL) have emerged as an important cause of invasive infection. Targeting the primary (intestinal) niche by decolonization may be a valuable approach to decrease the risk of relapsing infections and to reduce transmission of ESBL-producing enteric pathogens. METHODS: In a retrospective observational study we evaluated the efficacy of intestinal decolonization treatment using orally administered colistin or other non-absorbable agents given for 2 to 4 weeks in adult patients with previous relapsing infection and persistent intestinal colonization with ESBL-positive Enterobacteriaceae (ESBL-E). Eradication success was defined as negative rectal swab or stool culture at the end of treatment and at follow up-2 weeks after treatment discontinuation. RESULTS: First-line decolonization treatment led to eradication of ESBL-E in 19/45 patients (42%, 7/18 low-dose [4 × 1 million units] colistin, 3/12 high-dose [4 × 2 million units] colistin, 9/15 rifaximin [2 × 400 mg]), and secondary/salvage treatment was successful in 8/13 patients (62 %, 20 treatment episodes). Late follow-up showed that 7/13 patients (54%) with successful initial or salvage decolonization became recolonized within 3 months after post-treatment assessment while all eight of the patients failing initial or salvage decolonization treatment with late follow-up remained colonized. A narrative review of the literature confirms the limited efficacy of non-absorbable antibiotics including conventional selective digestive tract decolonization (SDD)-like combination regimens for eradicating multidrug-resistant enteric bacteria from the intestinal tract. CONCLUSIONS: At present, there is no clear evidence of a significant decolonization efficacy using single-drug treatment with oral non-absorbable antibiotics. More effective regimens are needed and a better definition of at risk patients is required for planning meaningful randomized controlled studies in this field.

Three Dimensional Checkerboard Synergy Analysis of Colistin, Meropenem, Tigecycline against Multidrug-Resistant Clinical Klebsiella pneumonia Isolates.

The spread of carbapenem-non-susceptible Klebsiella pneumoniae strains bearing different resistance determinants is a rising problem worldwide. Especially infections with KPC (Klebsiella pneumoniae carbapenemase) - producers are associated with high mortality rates due to limited treatment options. Recent clinical studies of KPC-blood stream infections revealed that colistin-based combination therapy with a carbapenem and/or tigecycline was associated with significantly decreased mortality rates when compared to colistin monotherapy. However, it remains unclear if these observations can be transferred to K. pneumoniae harboring other mechanisms of carbapenem resistance. A three-dimensional synergy analysis was performed to evaluate the benefits of a triple combination with meropenem, tigecycline and colistin against 20 K. pneumoniae isolates harboring different ß-lactamases. To examine the mechanism behind the clinically observed synergistic effect, efflux properties and outer membrane porin (Omp) genes (ompK35 and ompK36) were also analyzed. Synergism was found for colistin-based double combinations for strains exhibiting high minimal inhibition concentrations against all of the three antibiotics. Adding a third antibiotic did not result in further increased synergistic effect in these strains. Antagonism did not occur. These results support the idea that colistin-based double combinations might be sufficient and the most effective combination partner for colistin should be chosen according to its MIC.