Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient's wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient's K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions.

Human milk oligosaccharides and non-digestible carbohydrates prevent adhesion of specific pathogens via modulating glycosylation or inflammatory genes in intestinal epithelial cells.

Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) are known to inhibit the adhesion of pathogens to the gut epithelium, but the mechanisms involved are not well understood. Here, the effects of 2'-FL, 3-FL, DP3-DP10, DP10-DP60 and DP30-DP60 inulins and DM7, DM55 and DM69 pectins were studied on pathogen adhesion to Caco-2 cells. As the growth phase influences virulence, E. coli ET8, E. coli LMG5862, E. coli O119, E. coli WA321, and S. enterica subsp. enterica LMG07233 from both log and stationary phases were tested. Specificity for enteric pathogens was tested by including the lung pathogen K. pneumoniae LMG20218. Expression of the cell membrane glycosylation genes of galectin and glycocalyx and inflammatory genes was studied in the presence and absence of 2'-FL or NDCs. Inhibition of pathogen adhesion was observed for 2'-FL, inulins, and pectins. Pre-incubation with 2'-FL downregulated ICAM1, and pectins modified the glycosylation genes. In contrast, K. pneumoniae LMG20218 downregulated the inflammatory genes, but these were restored by pre-incubation with pectins, which reduced the adhesion of K. pneumoniae LMG20218. In addition, DM69 pectin significantly upregulated the inflammatory genes. 2'-FL and pectins but not inulins inhibited pathogen adhesion to the gut epithelial Caco-2 cells through changing the cell membrane glycosylation and inflammatory genes, but the effects were molecule-, pathogen-, and growth phase-dependent.

Colistin resistance increases 28-day mortality in bloodstream infections due to carbapenem-resistant Klebsiella pneumoniae.

Mortality due to K. pneumoniae bacteremia is on rise, particularly in regions with high rates of carbapenem and colistin resistance. We aimed to define risk factors for colistin resistance and its impact on mortality. Patients diagnosed with "carbapenem-resistant K. pneumoniae (CRKp)" bacteremia between 2014 and 2018 were divided into two groups as "colistin susceptible (ColS)" and "colistin resistant (ColR)" based on broth microdilution method. Retrospective case-control study was conducted to compare characteristics and outcomes. Multiple logistic regression model was used to define independent risk factors for acquired colistin resistance and Cox proportional hazard model for 28-day mortality. A total of 82 patients (39 ColS and 43 ColR) were included. Mean age was 61.5 years, and 50 (61%) were male. Colistin resistance was significantly increased with duration of hospital stay (p = 0.007) and prior colistin use (p = 0.007). Overall, the 28-day mortality rate was 66%. Age (p = 0.014) and colistin resistance significantly increased 28-day (p = 0.009) mortality. Microbiological response to treatment within 7 days favors survival. PFGE analysis revealed an outbreak with K. pneumoniae ST78 and ST45 clones. Patients treated with combined antimicrobials had significantly lower 28-day mortality (p = 0.045) in comparison to monotherapy. However, types of combinations did not show significant superiority on each other. Colistin resistance increases 28-day mortality in CRKp bacteremia. Although combined regimens are more effective than monotherapy, existing antibacterial combinations have no apparent superiority to each other. New treatment options are pivotal.

Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13.

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

Purified ß-glucans from the Shiitake mushroom ameliorates antibiotic-resistant Klebsiella pneumoniae-induced pulmonary sepsis.

Bacterial infection remains the main cause of acute respiratory distress syndrome and is a leading cause of death and disability in critically ill patients. Here we report on the use of purified ß-glucan (lentinan) extracts from Lentinus edodes (Shiitake) mushroom that can reduce infection by a multidrug-resistant clinical isolate of Klebsiella pneumoniae in a rodent pneumonia model, likely through immunomodulation. Adult male Sprague-Dawley rats were subjected to intra-tracheal administration of K. pneumoniae to induce pulmonary sepsis and randomized to three groups; vehicle control (Vehicle, n = 12), commercial lentinan (CL, n = 8) or in-house extracted lentinan (IHL, n = 8) were administered intravenously 1 h postinfection. Physiological parameters and blood gas analysis were measured, bacterial counts from bronchoalveolar-lavage (BAL) were determined, along with differential staining of white cells and measurement of protein concentration in BAL 48 h after pneumonia induction. Use of IHL extract significantly decreased BAL CFU counts. Both CL and IHL extractions reduced protein concentration in BAL. Use of IHL resulted in an improvement in physiological parameters compared to controls and CL. In conclusion, administration of lentinan to treat sepsis-induced lung injury appears safe and effective and may exert its effects in an immunomodulatory manner.

In-vitro antibacterial effect of tea polyphenols combined with common antibiotics on multidrug-resistant Klebsiella pneumoniae.

BACKGROUND: To investigate the bactericidal effect of tea polyphenols on multidrug resistant Klebsiella pneumoniae and its antibacterial efficacy in combination with common antibiotics, and to explore its mechanism. METHODS: The VITEK 2 Compact automatic microbial identification system was used to identify 20 strains of Klebsiella pneumoniae isolated from clinical isolates. The KB method was used to detect sensitivity of common drugs such as imipenem, piperacillin, piperacillin/tazobactam and cefepime, cefotaxime and ceftazidime; agar dilution method was used for detection of minimum inhibitory concentration (MIC) of tea polyphenols; the variation of the diameter of the antibacterial ring after different concentrations (0.25 MIC, 0.5 MIC) of tea polyphenols in combination with commonly used antibacterial drugs was detected to judge the feasibility of the inhibition of the multidrug-resistant Klebsiella pneumoniae by the combination of tea polyphenols and commonly used antibacterial drugs. Congo red and crystal violent staining was used to detect the impact on bacterial biofilm formation and extracellular mucus-like substance (slime). RESULTS: The K-B values of 20 strains of Klebsiella pneumoniae against common antibiotics were 6-14 mm; the MIC of tea polyphenols against 20 strains of multi-drug resistant Klebsiella pneumoniae was 1024 ug/mL; 0.5 MIC tea polyphenols can increase the diameter of the bacteriostatic ring of imipenem, piperacillin, piperacillin/tazobactam, cefepime, cefotaxime and ceftazidime by 3-28 mm. Tea polyphenols at sub-inhibitory concentrations significantly inhibited the production of Klebsiella pneumoniae biofilm and extracellular mucus (slime). CONCLUSIONS: Tea polyphenols could be used in combination with commonly used antibiotics (imipenem, piperacillin, piperacillin/tazobactam, cefepime, cefotaxime and ceftazidime) against multidrug-resistant Klebsiella pneumoniae. It has a synergistic bactericidal effect and affects the formation of bacterial outer membrane and the production of extracellular slime-like substances (slime).

Antibacterial activity of the crude extracts from medicinally important Thuja occidentalis.

The present research was carried out at the Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, KPK Pakistan. In this study crude methanolic extracts from Thuja occidentalis were tested for their antimicrobial activity against five different bacterial strains (Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Xanthomonas sp. and Staphylococcus aureus) at two different concentrations (10 and 20 mg/ml) using disc diffusion assay. The results showed that Klebsiella pneumoniae was most sensitive to crude extracted sample from leaves at both low and high concentrations measuring 37 and 57% zone of inhibition respectively and Xanthomonas and Staphylococcus aureus was found to be more resistant to the crude extracted samples from leaves at both concentrations. Similarly, Klebsiella pneumoniae was most sensitive to crude extracted samples from seeds at high concentration followed by E. coli at both concentrations. Staphylococcus aureus on the other hand was found to be more resistant to the crude extracted samples at low and high concentrations. These results suggested that extracts prepared from the leaves and seeds of Thuja occidentalis can be used as natural remedy for the treatment of various bacterial infections.

Antibacterial, antibiofilm, and antiquorum sensing activities of phytosynthesized silver nanoparticles fabricated from Mespilus germanica extract against multidrug resistance of Klebsiella pneumoniae clinical strains.

The aim of the present work was to investigate the antibacterial, antibiofilm, and antiquorum sensing activities of phytosynthesized silver nanoparticles (AgNPs) fabricated from Mespilus germanica extract against multidrug-resistant (MDR) Klebsiella pneumoniae strains. Fifty strains of K. pneumoniae were isolated from various clinical specimens. Biofilm-forming strains were identified using Congo red agar and polymerase chain reaction (PCR) techniques. Subsequently, the antibacterial activity of phytosynthesized AgNPs on MDR K. pneumoniae strains was investigated by broth microdilution assay and agar well-diffusion method. Finally (in the last step), the antibiofilm activity of phytosynthesized AgNPs was determined using microtiter plate assay and real-time PCR (RT-PCR) methods for the analysis of type 3 fimbriae (mrkA) and quorum-sensing system (luxS) gene expression. The results of this study showed that the phytosynthesized AgNPs had a spherical nanostructure with the mean size of 17.60 nm. The AgNPs exhibited dose-dependent antibacterial activity. The results of the microtiter plate and RT-PCR methods show that AgNPs inhibited the biofilm formation in MDR K. pneumoniae strains, and the expressions of mrkA and luxS genes were downregulated significantly in MDR strains after treatment with a subminimum inhibitory concentration of AgNPs. In conclusion, AgNPs effectively prevent the formation of biofilms and kill bacteria in established biofilms, which suggests that AgNPs might be a promising candidate for the prevention and treatment of biofilm-related infections caused by MDR K. pneumoniae strains.

Gut bacteria of Cuora amboinensis (turtle) produce broad-spectrum antibacterial molecules.

Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.

Treatment with andrographolide sulfonate provides additional benefits to imipenem in a mouse model of Klebsiella pneumoniae pneumonia.

Klebsiella pneumoniae is a primary cause of community-acquired and nosocomial respiratory infections, and K. pneumoniae resistance to the current treatment approach with carbapenem is worsening. Andrographolide is a natural diterpenoid from Andrographis paniculata that was shown to exert anti-inflammatory activity. We herein show that pretreatment with a water-soluble andrographolide sulfonate significantly attenuate lung injury and infiltration of inflammatory cells. Interestingly, mice receiving combined treatment with andrographolide sulfonate displayed perfect survival rate than the mice treatment with imipenem alone, and monocyte chemotactic protein 5 (MCP-5) level was decreased further. These findings suggest that andrographolide sulfonate could as a potential synergist for antibiotic treatment of bacteria-induced inflammation.

Common hydrotherapy practices and the prevalence of burn wound bacterial colonisation at the University Teaching Hospital in Lusaka, Zambia.

BACKGROUND: In many parts of the world, hydrotherapy plays an important role in the management of patients with wounds including burns. Different centers practice hydrotherapy differently. At the University Teaching Hospital in Lusaka, Zambia, burn patients use a common bathtub for cleaning their wounds which theoretically increases the risk of cross-infection, an important source of morbidity and mortality. There is currently no evidence that hydrotherapy as practiced at our institution leads to cross infection among patients with burns. OBJECTIVE: The objective was to determine if our hydrotherapy practice and water plays a role in cross-infection and what organisms cause this infection. METHODS: This was a prospective analytical study. Patients meeting the selection criteria were recruited. Swabs from the burn wounds were collected on admission (day 0), day 4 and day 7. Weekly swabs of the bathtub were also collected, after the tub had been cleaned and declared ready for the next patient. Weekly water samples were also collected. Selected results, for Staphylococcus aureus and Klebsiella pneumoniae, were subjected to further analysis and PCR. Results were analyzed using statistics software, SPSS version 23. RESULTS: In this study, there were 96 participants of which 51 (53.1%) were males and 45 (46.9%) were females. Age distribution ranged from 5months to 91 years. The modal age range was 1 to 2 years. The modal burn percentage was 6%-10%, followed by 11%-15%. Hot water was the cause of burns in 65.6%. S. aureus and K. pneumoniae were the most common organisms isolated. Others were enteric organisms. In terms of readily available antibiotics, there was more sensitivity to Amikacin and Chloramphenicol than Ciprofloxacin (our commonly used antibiotic). The bathtub also had S. aureus and K. pneumoniae, besides enteric organisms. Sixty five point four percent (65.4%) of the Klebsiella were ESBL (Extended Spectrum Beta Lactamase) producers. The tub had samples that were both ESBL producers as well as widely resistant Klebsiella by other means. No growth was obtained from the water samples. Seventy-two point nine percent (72.9%) of the patients were discharged, 19.8% died, while 7.3% left against medical advice. CONCLUSION: Hydrotherapy as currently practiced at the University Teaching Hospital does contribute significantly to cross-infection among burn patients with widely resistant organisms. The main ones are S. aureus and K. pneumoniae. Switching care to a shower mechanism might help eliminate this problem as the study demonstrates that no bacteria were found in the water samples.

Exogenous fatty acids alter phospholipid composition, membrane permeability, capacity for biofilm formation, and antimicrobial peptide susceptibility in Klebsiella pneumoniae.

Klebsiella pneumoniae represents a major threat to human health due to a combination of its nosocomial emergence and a propensity for acquiring antibiotic resistance. Dissemination of the bacteria from its native intestinal location creates severe, complicated infections that are particularly problematic in healthcare settings. Thus, there is an urgency for identifying novel treatment regimens as the incidence of highly antibiotic-resistant bacteria rises. Recent findings have highlighted the ability of some Gram-negative bacteria to utilize exogenous fatty acids in ways that modify membrane phospholipids and influence virulence phenotypes, such as biofilm formation and antibiotic resistance. This study explores the ability of K. pneumoniae to assimilate and respond to exogenous fatty acids. The combination of thin-layer chromatography liquid chromatography-mass spectrometry confirmed adoption of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of K. pneumoniae. Membrane permeability was variably affected as determined by two dye uptake assays. Furthermore, the availability of many PUFAs lowered the MICs to the antimicrobial peptides polymyxin B and colistin. Biofilm formation was significantly affected depending upon the supplemented fatty acid.

Pharmacological and phytochemical analysis of Bergenia ciliata leaf and rhizome extracts.

Antibacterial, antifungal, antioxidant, cytotoxic, and anti-haemolytic activity of various rhizome extracts of Bergenia ciliata were evaluated in this study. The results showed inhibition of the growth of all selected bacterial and fungal strains in comparison with standard antibiotics. The antioxidant activities of Bergenia ciliata extracts were evaluated against DPPH, H2O2, ABTS, total antioxidant capacity and reducing power assays. The order of antioxidant activity of various extracts were methanol> ethanol>n-hexane> aqueous>chloroform. The cytotoxicity ( brine shrimp assay) and anti-haemolytic activities of plant extracts were also promising and varies in dose depended manner. The phytochemical analysis of rhizome extracts of Bergenia ciliata revealed presence of various secondary metabolites which might be responsible for the antimicrobial, antioxidant, cytotoxic and anti-haemolytic activities.

Antimicrobial polymers as therapeutics for treatment of multidrug-resistant Klebsiella pneumoniae lung infection.

Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens in hospital-acquired infections. It is often resistant to multiple antibiotics (including carbapenems), and can cause severe pneumonia. In search of effective antimicrobials, we recently developed polyionenes that were demonstrated to be potent against a broad-spectrum of microbes in vitro. In this study, polyionenes containing rigid amide bonds were synthesized to treat multidrug-resistant (MDR) K. pneumoniae lung infection. The polyionene exhibited broad-spectrum activity against clinically-isolated MDR bacteria with low minimum inhibitory concentrations (MICs). It also demonstrated stronger antimicrobial activity against 20 clinical strains of K. pneumoniae and more rapid killing kinetics than imipenem and other commonly used antibiotics. Multiple treatments with imipenem and gentamycin led to drug resistance in K. pneumoniae, while repeated use of the polymer did not cause resistance development due to its membrane-disruption antimicrobial mechanism. Additionally, the polymer showed potent anti-biofilm activity. In a MDR K. pneumoniae lung infection mouse model, the polymer demonstrated lower effective dose than imipenem with negligible systemic toxicity. The polymer treatment significantly alleviated lung injury, markedly reduced K. pneumoniae counts in the blood and major organs, and decreased mortality. Given its potent in vivo antimicrobial activity, negligible toxicity and ability of mitigating resistance development, the polyionene may be used to treat MDR K. pneumoniae lung infection. STATEMENT OF SIGNIFICANCE: Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens in hospital-acquired infections, is often resistant to multiple antibiotics including carbapenems and can cause severe pneumonia. In this study, we report synthesis of antimicrobial polymers (polyionenes) and their use as antimicrobial agents for treatment of K. pneumoniae-caused pneumonia. The polymers have broad spectrum antibacterial activity against clinically isolated MDR bacteria, and eliminate MDR K. pneumoniae more effectively and rapidly than clinically used antibiotics. The polymer treatment also provides higher survival rate and faster bacterial removal from the major organs and the blood than the antibiotics. Repeated use of the polymer does not lead to resistance development. More importantly, at the therapeutic dose, the polymer treatment does not cause acute toxicity. Given its in vivo efficacy and negligible toxicity, the polymer is a promising candidate for the treatment of MDR K. pneumoniae-caused pneumonia.

Influence of Bacterial Biofilm Polysaccharide Structure on Interactions with Antimicrobial Peptides: A Study on Klebsiella pneumoniae.

Balneotherapy is a clinically effective complementary approach in the treatment of low-grade inflammation- and stress-related pathologies. The biological mechanisms by which immersion in mineral-medicinal water and the application of mud alleviate symptoms of several pathologies are still not completely understood, but it is known that neuroendocrine and immunological responses­including both humoral and cell-mediated immunity­to balneotherapy are involved in these mechanisms of effectiveness; leading to anti-inflammatory, analgesic, antioxidant, chondroprotective, and anabolic effects together with neuroendocrine-immune regulation in different conditions. Hormesis can play a critical role in all these biological effects and mechanisms of effectiveness. The hormetic effects of balneotherapy can be related to non-specific factors such as heat­which induces the heat shock response, and therefore the synthesis and release of heat shock proteins­and also to specific biochemical components such as hydrogen sulfide (H2S) in sulfurous water and radon in radioactive water. Results from several investigations suggest that the beneficial effects of balneotherapy and hydrotherapy are consistent with the concept of hormesis, and thus support a role for hormesis in hydrothermal treatments.

Combination of essential oil and ciprofloxacin to inhibit/eradicate biofilms in multidrug-resistant Klebsiella pneumoniae.

AIM: This study aimed to test biofilm inhibition activities of each of essential oils (EOs), main compounds of EOs and enzymes against pathogenic Klebsiella pneumoniae. METHODS AND RESULTS: The effect of seven EOs and three enzymes was tested on formation and eradication of K. pneumoniae biofilm. Peppermint oil showed a robust biofilm inhibitory effect, causing inhibition that ranged from 69·2 to 98·2% at 5 µl ml-1 . Thyme oil was found to have the best biofilm eradication ability, causing eradication that ranged from 80·1 to 98·0% at 10 µl ml-1 . The most effective EOs were analysed by GC/MS, to determine the major chemical constitutes of each oil. Pure menthol was found to cause 75·3-97·5% biofilm inhibition at 2·5 µg ml-1 , whereas thymol caused 85·1-97·8% biofilm eradication at 5 µg ml-1 . However, moderate inhibition activity was detected for α-amylase and bromelain, while poor activity was detected for ß-amylase. Ciprofloxacin combination with thyme oil and thymol was found to enhance antibiotic activity, and affect biofilm cell viability. The observed inhibitory/eradication activity on K. pneumoniae biofilms was confirmed by scanning electron microscopy. CONCLUSIONS: Thyme and peppermint EOs, and their active components are promising antibiofilm agents alone and/or in combination with ciprofloxacin to inhibit/eradicate biofilms of K. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented results suggest the potential application of EOs against infections, caused by biofilm-producing K. pneumoniae, to prevent biofilm formation or decrease their resistance threshold. Moreover, the combination of EOs with ciprofloxacin minimizes the antibiotic concentration used and accordingly the potential accompanying toxic side effects.

Survival of bactericidal antibiotic treatment by tolerant persister cells of Klebsiella pneumoniae.

PURPOSE: Persister cells, a subpopulation of tolerant cells within the bacterial culture, are commonly thought to be responsible for antibiotic therapy failure and infection recurrence. Klebsiella pneumoniae is a notorious human pathogen for its increasing resistance to antibiotics and wide involvement in severe infections. In this study, we aimed to investigate the persister subpopulation of K. pneumoniae. METHODOLOGY: The presence of persisters in K. pneumoniae was determined by treatment with high concentrations of antibiotics, used alone or in combination. The effect of low level of antibiotics on persister formation was investigated by pre-exposure of cells to antibiotics with low concentrations followed by higher doses. The dependence of persister levels on growth phase was determined by measuring the survival ability of cells along the growth stages upon exposure to a high concentration of antibiotic. Analysis on persister type was carried out by persister elimination assays.Results/Key findings. We show that K. pneumoniae produces high levels of tolerant persister cells to survive treatment by a variety of high concentrations of bactericidal antibiotics and persister formation is prevalent among K. pneumoniae clinical strains. Besides, we find that persister cells can be induced by low concentrations of antibiotics. Finally, we provide evidence that persister formation is growth phase-dependent and Type II persisters dominate the persister subpopulation during the entire exponential phase of K. pneumoniae. CONCLUSION: Our study describes the formation of tolerant persister cells that allow survival of treatment by high concentrations of antibiotics in K. pneumoniae.

Findings from an outbreak of carbapenem-resistant Klebsiella pneumoniae emphasize the role of antibiotic treatment for cross transmission.

PURPOSE: In January 2015, we noticed by rectal swab analyses that seven of 23 patients at an early rehabilitation ward had been colonized with carbapenem-resistant Klebsiella pneumoniae (CKP). Here, we describe risk factors for CKP acquisition. METHODS: In the present study, the outbreak is described and risk factors for CKP acquisition are examined, e.g., antibiotic treatment. Microbiological analyses including corresponding results were examined to study when colonization with CKP occurred and whether patients had suffered from diarrhea. To examine whether spread of bacteria was clonal, multi-locus sequence typing as well as Xbal macrorestriction and pulsed-field gel electrophoresis was performed. The presence of carbapenmase was examined by PCR analysis. Through univariate analysis of risk factors in the small study sample, the role of antibiotic consumption, isolation procedures, patient's age, gender, and Barthel index on colonization was elucidated. RESULTS: Clonal spread of the novel sequence type (ST)2255 was identified. Additionally, one patient was colonized with Escherichia coli and Serratia marcescens, both resistant to carbapenems, while a further patient carried another carbapenem-resistant E. coli strain. In all isolates, carbapenemase gene bla OXA-48 was found to be located on a conjugative plasmid (60 kb), suggesting in vivo transmission from CKP to E. coli and S. marcescens. Univariate tests indicated that antibiotic treatment was the only risk factor showing a significant association with being colonized by CKP. In addition, the likelihood of diarrhea appeared to be higher in this group. Antibiotic treatment was associated with CKP colonization, whereas patients´ age, gender, Barthel index at admission, and residence with a CKP-colonized roommate were not. Diarrhea also seemed to support to distribution of CKP. CONCLUSIONS: In this small outbreak, antibiotic treatment seemed to be the predominant risk factor for monoclonal transmission of bla OXA-48 positive CKP.

Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae.

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance.

Measuring and modelling the response of Klebsiella pneumoniae KPC prey to Bdellovibrio bacteriovorus predation, in human serum and defined buffer.

In worldwide conditions of increasingly antibiotic-resistant hospital infections, it is important to research alternative therapies. Bdellovibrio bacteriovorus bacteria naturally prey on Gram-negative pathogens, including antibiotic-resistant strains and so B. bacteriovorus have been proposed as "living antibiotics" to combat antimicrobially-resistant pathogens. Predator-prey interactions are complex and can be altered by environmental components. To be effective B. bacteriovorus predation needs to work in human body fluids such as serum where predation dynamics may differ to that studied in laboratory media. Here we combine mathematical modelling and lab experimentation to investigate the predation of an important carbapenem-resistant human pathogen, Klebsiella pneumoniae, by B. bacteriovorus in human serum versus buffer. We show experimentally that B. bacteriovorus is able to reduce prey numbers in each environment, on different timescales. Our mathematical model captures the underlying dynamics of the experimentation, including an initial predation-delay at the predator-prey-serum interface. Our research shows differences between predation in buffer and serum and highlights both the potential and limitations of B. bacteriovorus acting therapeutically against K. pneumoniae in serum, informing future research into the medicinal behaviours and dosing of this living antibacterial.