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The species included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the genus Enterobacter) have a high capacity to develop antimicrobial resistance (AMR), a health problem that is already among the leading causes of death and could kill 10 million people a year by 2050. The generation of new potentially therapeutic molecules has been insufficient to combat the AMR "crisis", and the World Health Organization (WHO) has stated that it will seek to promote the development of rapid diagnostic strategies. The physicochemical properties of metallic nanoparticles (MNPs) have made it possible to design biosensors capable of identifying low concentrations of ESKAPE bacteria in the short term; other systems identify antimicrobial susceptibility, and some have been designed with dual activity in situ (bacterial detection and antimicrobial activity), which suggests that, in the near future, multifunctional biosensors could exist based on MNPs capable of quickly identifying bacterial pathogens in clinical niches might become commercially available. This review focuses on the use of MNP-based systems for the rapid and accurate identification of clinically important bacterial pathogens, exhibiting the necessity for exhaustive research to achieve these objectives. This review focuses on the use of metal nanoparticle-based systems for the rapid and accurate identification of clinically important bacterial pathogens. [ABSTRACT FROM AUTHOR]

BackgroundDespite significant global progress in reducing neonatal mortality, bacterial sepsis remains a major cause of neonatal deaths. Klebsiella pneumoniae (K. pneumoniae) is the leading pathogen globally underlying cases of neonatal sepsis and is frequently resistant to antibiotic treatment regimens recommended by the World Health Organization (WHO), including first-line therapy with ampicillin and gentamicin, second-line therapy with amikacin and ceftazidime, and meropenem. Maternal vaccination to prevent neonatal infection could reduce the burden of K. pneumoniae neonatal sepsis in low- and middle-income countries (LMICs), but the potential impact of vaccination remains poorly quantified. We estimated the potential impact of such vaccination on cases and deaths of K. pneumoniae neonatal sepsis and project the global effects of routine immunization of pregnant women with the K. pneumoniae vaccine as antimicrobial resistance (AMR) increases.Methods and findingsWe developed a Bayesian mixture-modeling framework to estimate the effects of a hypothetical K. pneumoniae maternal vaccine with 70% efficacy administered with coverage equivalent to that of the maternal tetanus vaccine on neonatal sepsis infections and mortality. To parameterize our model, we used data from 3 global studies of neonatal sepsis and/or mortality-with 2,330 neonates who died with sepsis surveilled from 2016 to 2020 undertaken in 18 mainly LMICs across all WHO regions (Ethiopia, Kenya, Mali, Mozambique, Nigeria, Rwanda, Sierra Leone, South Africa, Uganda, Brazil, Italy, Greece, Pakistan, Bangladesh, India, Thailand, China, and Vietnam). Within these studies, 26.95% of fatal neonatal sepsis cases were culture-positive for K. pneumoniae. We analyzed 9,070 K. pneumoniae genomes from human isolates gathered globally from 2001 to 2020 to quantify the temporal rate of acquisition of AMR genes in K. pneumoniae isolates to predict the future number of drug-resistant cases and deaths that could be averted by vaccination. Resistance rates to carbapenems are increasing most rapidly and 22.43% [95th percentile Bayesian credible interval (CrI): 5.24 to 41.42] of neonatal sepsis deaths are caused by meropenem-resistant K. pneumoniae. Globally, we estimate that maternal vaccination could avert 80,258 [CrI: 18,084 to 189,040] neonatal deaths and 399,015 [CrI: 334,523 to 485,442] neonatal sepsis cases yearly worldwide, accounting for more than 3.40% [CrI: 0.75 to 8.01] of all neonatal deaths. The largest relative benefits are in Africa (Sierra Leone, Mali, Niger) and South-East Asia (Bangladesh) where vaccination could avert over 6% of all neonatal deaths. Nevertheless, our modeling only considers country-level trends in K. pneumoniae neonatal sepsis deaths and is unable to consider within-country variability in bacterial prevalence that may impact the projected burden of sepsis.ConclusionsA K. pneumoniae maternal vaccine could have widespread, sustained global benefits as AMR in K. pneumoniae continues to increase.

BackgroundKlebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate.ResultsCompared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose.ConclusionsThis research provides a new way to improve the efficiency of the biological route of isobutanol production.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a global threat. We therefore analysed the bacterial characteristics of CRKP infections and the clinical outcomes of patients with CRKP infections across different countries.MethodsIn this prospective, multicentre, cohort study (CRACKLE-2), hospitalised patients with cultures positive for CRKP were recruited from 71 hospitals in Argentina, Australia, Chile, China, Colombia, Lebanon, Singapore, and the USA. The first culture positive for CRKP was included for each unique patient. Clinical data on post-hospitalisation death and readmission were collected from health records, and whole genome sequencing was done on all isolates. The primary outcome was a desirability of outcome ranking at 30 days after the index culture, and, along with bacterial characteristics and 30-day all-cause mortality (a key secondary outcome), was compared between patients from China, South America, and the USA. The desirability of outcome ranking was adjusted for location before admission, Charlson comorbidity index, age at culture, Pitt bacteremia score, and anatomical culture source through inverse probability weighting; mortality was adjusted for the same confounders, plus region where relevant, through multivariable logistic regression. This study is registered at ClinicalTrials.gov, NCT03646227, and is complete.FindingsBetween June 13, 2017, and Nov 30, 2018, 991 patients were enrolled, of whom 502 (51%) met the criteria for CRKP infection and 489 (49%) had positive cultures that were considered colonisation. We observed little intra-country genetic variation in CRKP. Infected patients from the USA were more acutely ill than were patients from China or South America (median Pitt bacteremia score 3 [IQR 2-6] vs 2 [0-4] vs 2 [0-4]) and had more comorbidities (median Charlson comorbidity index 3 [IQR 2-5] vs 1 [0-3] vs 1 [0-2]). Adjusted desirability of outcome ranking outcomes were similar in infected patients from China (n=246), South America (n=109), and the USA (n=130); the estimates were 53% (95% CI 42-65) for China versus South America, 50% (41-61) for the USA versus China, and 53% (41-66) for the USA versus South America. In patients with CRKP infections, unadjusted 30-day mortality was lower in China (12%, 95% CI 8-16; 29 of 246) than in the USA (23%, 16-30; 30 of 130) and South America (28%, 20-37; 31 of 109). Adjusted 30-day all-cause mortality was higher in South America than in China (adjusted odds ratio [aOR] 4·82, 95% CI 2·22-10·50) and the USA (aOR 3·34, 1·50-7·47), with the mortality difference between the USA and China no longer being significant (aOR 1·44, 0·70-2·96).InterpretationGlobal CRKP epidemics have important regional differences in patients' baseline characteristics and clinical outcomes, and in bacterial characteristics. Research findings from one region might not be generalisable to other regions.FundingThe National Institutes of Health.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) frequently causes hospital-acquired infections and is associated with high morbidity and mortality. CRKP can have multiple resistance mechanisms and only a few can be routinely detected by commercial molecular or phenotypic assays making surveillance for CRKP particularly challenging. In this report, we identified and characterized an unusual non-carbapenemase-producing CRKP carrying a rare plasmid-borne inducible AmpC gene, blaDHA-1 . The isolate was recovered from blood culture of a 67-year-old female presenting with sepsis post bladder surgery and ureteral stent removal. The primary isolate displayed an indeterminate susceptibility pattern for ceftriaxone by broth microdilution, but was susceptible by disk diffusion with one colony growing within the zone of inhibition. The ceftriaxone resistant colony was sub-cultured and had a minimum inhibitory concentration (MIC) of 2 ug/ml for imipenem (intermediate) and a zone size of 18 mm for ertapenem (resistant), but remained susceptible to cefepime and meropenem. Further phenotypic characterization of this sub-cultured isolate showed carbapenemase activity. Whole genome sequencing (WGS) revealed the presence of two subpopulations of a K. pneumoniae (MLST sequence type 11) from the primary blood culture isolate: one pan-susceptible to beta-lactams tested and the other resistant to the 3rd generation cephalosporins and ertapenem. WGS analysis identified the resistant K. pneumoniae harboring IncFIB(K) and IncR plasmids and the presence of plasmid-borne beta-lactam resistance genes bla OXA-1 and bla DHA-1, an inducible AmpC gene. Additional resistance genes against quinolones (aac(6')-Ib-cr, oqxA, oqB), aminoglycoside (aph(3')-Ia), sulfonamide (sul1), and tetracycline (tet(A)) were also identified. DHA-1 positive K. pneumoniae have been previously identified outside the US, particularly in Asia and Europe, but limited cases have been reported in the United States and may be underrecognized. Our study highlights the importance of using both extended phenotypic testing and WGS to identify emerging resistance mechanisms in clinical Enterobacterales isolates with unusual antimicrobial resistance patterns.

We describe a case of endogenous endophthalmitis caused by sequence type 66-K2 hypervirulent Klebsiella pneumoniae in a diabetic patient with no travel history outside the United States. Genomic analysis showed the pathogen has remained highly conserved, retaining >98% genetic similarity to the original strain described in Indonesia in 1935.

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

Recent years have seen an unprecedented rise in the incidence of multidrug-resistant (MDR) Gram-negative bacteria (GNBs) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent, and treat infections by GNBs are urgently needed. Previously, we have reported that the Candida albicans hypha-regulated protein Hyr1 shares striking three-dimensional structural homology with cell surface proteins of Acinetobacter baumannii. Moreover, active vaccination with rHyr1p-N or passive immunization with anti-Hyr1p polyclonal antibody protects mice from Acinetobacter infection. In the present study, we use molecular modeling to guide design of monoclonal antibodies (mAbs) generated against Hyr1p and show them to bind to priority surface antigens of Acinetobacter and Klebsiella pneumoniae. The anti-Hyr1 mAbs block damage to primary endothelial cells induced by the bacteria and protect mice from lethal pulmonary infections mediated by A. baumannii or K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAbs as a cross-kingdom immunotherapeutic strategy against MDR GNBs.

In recent years, several bacterial strains have acquired significant antibiotic resistance and can, therefore, become difficult to contain. To counteract such trends, relational databases can be a powerful tool for supporting the decision-making process. The case of Klebsiella pneumoniae diffusion in a central region of Italy was analyzed as a case study. A specific relational database is shown to provide very detailed and timely information about the spatial–temporal diffusion of the contagion, together with a clear assessment of the multidrug resistance of the strains. The analysis is particularized for both internal and external patients. Tools such as the one proposed can, therefore, be considered important elements in the identification of infection hotspots, a key ingredient of any strategy to reduce the diffusion of an infectious disease at the community level and in hospitals. These types of tools are also very valuable in the decision-making process related to antibiotic prescription and to the management of stockpiles. The application of this processing technology to viral diseases such as COVID-19 is under investigation. [ABSTRACT FROM AUTHOR]

Gram-negative pathogens expressing serine β-lactamases (SBLs) and metallo-β-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all β-lactam antibiotics. Here we describe the discovery of a heteroaryl phosphonate scaffold that exhibits noncovalent cross-class inhibition of representative carbapenemases, specifically the SBL KPC-2 and the MBLs NDM-1 and VIM-2. The most potent lead, compound 16, exhibited low nM to low μM inhibition of KPC-2, NDM-1, and VIM-2. Compound 16 potentiated imipenem efficacy against resistant clinical and laboratory bacterial strains expressing carbapenemases while showing some cytotoxicity toward human HEK293T cells only at concentrations above 100 μg/mL. Complex structures with KPC-2, NDM-1, and VIM-2 demonstrate how these inhibitors achieve high binding affinity to both enzyme classes. These findings provide a structurally and mechanistically new scaffold for drug discovery targeting multidrug resistant Gram-negative pathogens and more generally highlight the active site features of carbapenemases that can be leveraged for lead discovery.

Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105  CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.

This report describes the first documented case of Klebsiella pneumoniae endogenous endophthalmitis with concurrent orbital cellulitis in a patient without a liver abscess. A 34-year-old, caucasian male with a history of intravenous drug abuse and type 1 diabetes mellitus was transferred from an outside hospital for progressively worsening pain and swelling in the right eye. Careful history, physical examination, and imaging were consistent with a diagnosis of endogenous endophthalmitis with concurrent orbital cellulitis. Vitreous cultures were positive for Klebsiella pneumoniae. Despite aggressive and early intervention with antibiotics and vitrectomy, the patient's condition continued to worsen and evisceration was required to adequately control the infection. Klebsiella pneumoniae endogenous endophthalmitis with concurrent orbital cellulitis is a rare and often blinding infection, despite appropriately aggressive intervention.

RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP β subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP β' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.

Infections due to colistin-resistant (Colr) Gram-negative rods (GNRs) and colistin-resistant Klebsiella pneumoniae isolates in particular result in high associated mortality and poor treatment options. To determine the risk factors for recovery on culture of Colr GNRs and ColrK. pneumoniae, analyses were chosen to aid decisions at two separate time points: the first when only Gram stain results are available without any bacterial species information (corresponding to the Colr GNR model) and the second when organism identification is performed but prior to reporting of antimicrobial susceptibility testing results (corresponding to the ColrK. pneumoniae model). Cases were retrospectively analyzed at a major academic hospital system from 2011 to 2016. After excluding bacteria that were intrinsically resistant to colistin, a total of 28,512 GNR isolates (4,557 K. pneumoniae isolates) were analyzed, 128 of which were Colr (i.e., MIC > 2 μg/ml), including 68 of which that were ColrK. pneumoniae In multivariate analysis, risk factors for Colr GNRs were neurologic disease, residence in a skilled nursing facility prior to admission, receipt of carbapenems in the last 90 days, prior infection with a carbapenem-resistant organism, and receipt of ventilatory support (c-statistic = 0.81). Risk factors for ColrK. pneumoniae specifically were neurologic disease, residence in a skilled nursing facility prior to admission, receipt of carbapenems in the last 90 days, receipt of an anti-methicillin-resistant Staphylococcus aureus antimicrobial in the last 90 days, and prior infection with a carbapenem-resistant organism (c-statistic = 0.89). A scoring system derived from these models can be applied by providers to guide empirical antimicrobial therapy in patients with infections with suspected Colr GNR and ColrK. pneumoniae isolates.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 μg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.

Antimicrobial properties of a long-chain, synthetic, cationic, and hydrophobic amino acid block copolymer are reported. In 5 and 60 min time-kill assays, solutions of K100 L40 block copolymers (poly(l-lysine·hydrochloride)100 -b-poly(l-leucine)40 ) at concentrations of 10-100 µg mL-1 show multi-log reductions in colony forming units of Gram-positive and Gram-negative bacteria, as well as yeast, including multidrug-resistant strains. Driven by association of hydrophobic segments, K100 L40 copolymers form viscous solutions and self-supporting hydrogels in water at concentrations of 1 and 2 wt%, respectively. These K100 L40 preparations provide an effective barrier to microbial contamination of wounds, as measured by multi-log decreases of tissue-associated bacteria with deliberate inoculation of porcine skin explants, porcine open wounds, and rodent closed wounds with foreign body. Based on these findings, amino acid copolymers with the features of K100 L40 can combine potent, direct antimicrobial activity and barrier properties in one biopolymer for a new approach to prevention of wound infections.

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included blaIMP, blaNDM, blaOXA-48, blaCTX-M, blaAmpC, and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log10-CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log10-CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae.

Gram-negative pneumonia is a dangerous illness, and bacterial dissemination to the bloodstream during the infection is strongly associated with death. Antibiotic resistance among the causative pathogens has resulted in diminishing treatment options against this infection. Hepcidin is the master regulator of extracellular iron availability in vertebrates, but its role in the context of host defense is undefined. We hypothesized that hepcidin-mediated depletion of extracellular iron during Gram-negative pneumonia protects the host by limiting dissemination of bacteria to the bloodstream. During experimental pneumonia, hepcidin was induced in the liver in an IL-6-dependent manner and mediated a rapid decline in plasma iron. In contrast, hepcidin-deficient mice developed a paradoxical increase in plasma iron during infection associated with profound susceptibility to bacteremia. Incubation of bacteria with iron-supplemented plasma enhanced bacterial growth in vitro, and systemic administration of iron to WT mice similarly promoted increased susceptibility to bloodstream infection. Finally, treatment with a hepcidin analogue restored hypoferremia in hepcidin-deficient hosts, mediated bacterial control, and improved outcomes. These data show hepcidin induction during pneumonia to be essential to preventing bacterial dissemination by limiting extracellular iron availability. Hepcidin agonists may represent an effective therapy for Gram-negative infections in patients with impaired hepcidin production or signaling.

ObjectivesTo study the effect of a lack of antioxidant defenses during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice.SettingLaboratory experiments.SubjectsC57Bl6 and glutathione peroxidase 1 knockout mice.InterventionMurine acute pneumonia model induced by Klebsiella pneumonia.Measurements and main resultsWe show here that despite a lack of one of the major antioxidant defense enzymes, glutathione peroxidase 1 knockout mice are protected during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. Furthermore, this protective effect was suppressed when antioxidant defenses were restored. Infected glutathione peroxidase 1 mice showed an early and significant, albeit transient, increase in the activity of the NOD-like receptor family, pyrin domain containing 3 inflammasome when compared with wild-type mice. The key role of the NOD-like receptor family, pyrin domain containing 3 inflammasome during acute pneumonia was confirmed in vivo when the protective effect was suppressed by treating glutathione peroxidase 1 mice with an interleukin-1 receptor antagonist. Additionally we report, in vitro, that increased concentrations of active caspase-1 and interleukin-1β are related to an increased concentration of hydrogen peroxide in bacterially infected glutathione peroxidase 1 macrophages and that restoring hydrogen peroxide antioxidant defenses suppressed this effect.ConclusionsOur findings demonstrate that, contrary to current thinking, an early intervention targeting NOD-like receptor family, pyrin domain containing 3 inflammasome activity induces a timely and efficient activation of the innate immune response during acute infection. Our findings also demonstrate a role for hydrogen peroxide in the mechanisms tightly regulating NOD-like receptor family, pyrin domain containing 3 activation.

Siderophores are small molecular iron chelators that are produced by microbes and whose most notable function is to sequester iron from the host and provide this essential metal nutrient to microbes. Recent studies have proposed additional, noncanonical roles for siderophores, including the acquisition of noniron metals and modulation of host functions. Recently, Holden et al. (V. I. Holden, P. Breen, S. Houle, C. M. Dozois, and M. A. Bachman, mBio 7:e01397-16, 2016, http://dx.doi.org/10.1128/mBio.01397-16) showed that siderophores secreted by Klebsiella pneumoniae during lung infection induce stabilization of the transcription factor HIF-1α, increase the expression of proinflammatory cytokines in the lung, and promote dissemination of K. pneumoniae to the spleen. Thus, their study demonstrated novel roles for siderophores in vivo, beyond iron sequestration. The interaction of siderophores with host cells further promotes the pathogenicity of K. pneumoniae and is likely relevant for other pathogens that also secrete siderophores in the host.

Klebsiella pneumoniae, one of the most important nosocomial pathogens, is becoming a major problem in health care because of its resistance to multiple antibiotics, including cephalosporins of the latest generation and, more recently, even carbapenems. This is largely due to the spread of plasmid-encoded extended-spectrum β-lactamases. However, antimicrobial agents must first penetrate the outer membrane barrier in order to reach their targets, and hydrophilic and charged β-lactams presumably diffuse through the porin channels. Unfortunately, the properties of K. pneumoniae porin channels are largely unknown. In this study, we made clean deletions of K. pneumoniae porin genes ompK35 and ompK36 and examined the antibiotic susceptibilities and diffusion rates of β-lactams. The results showed that OmpK35 and OmpK36 produced larger more permeable channels than their Escherichia coli homologs OmpF and OmpC; OmpK35 especially produced a diffusion channel of remarkably high permeability toward lipophilic (benzylpenicillin) and large (cefepime) compounds. These results were also confirmed by expressing various porins in an E. coli strain lacking major porins and the major multidrug efflux pump AcrAB. Our data explain why the development of drug resistance in K. pneumoniae is so often accompanied by the mutational loss of its porins, especially OmpK35, in addition to the various plasmid-carried genes of antibiotic resistance, because even hydrolysis by β-lactamases becomes inefficient in producing high levels of resistance if the bacterium continues to allow a rapid influx of β-lactams through its wide porin channels.ImportanceIn Gram-negative bacteria, drugs must first enter the outer membrane, usually through porin channels. Thus, the quantitative examination of influx rates is essential for the assessment of resistance mechanisms, yet no such studies exist for a very important nosocomial pathogen, Klebsiella pneumoniae We found that the larger channel porin of this organism, OmpK35, produces a significantly larger channel than its Escherichia coli homolog, OmpF. This makes unmodified K. pneumoniae strains more susceptible to relatively large antibiotics, such as the third- and fourth-generation cephalosporins. Also, even the acquisition of powerful β-lactamases is not likely to make them fully resistant in the presence of such an effective influx process, explaining why so many clinical isolates of this organism lack porins.

Carbapenemases are enzymes that are capable of inactivating all or almost all beta-lactam antimicrobial agents. These enzymes are frequently coexpressed with other resistance mechanisms to non-beta-lactams, leading to extremely drug-resistant pathogens. Once a curiosity, these enzymes have spread into organisms that are among the most common causes of infection, such as Klebsiella pneumoniae and Escherichia coli. Identification of these organisms has proved challenging for clinical microbiology laboratories, leading to revisions in susceptibility standards for carbapenems. Although currently a rare cause of infection in children, these carbapenem-resistant Enterobacteriaceae (CRE) are becoming endemic in a variety of healthcare settings. Management of infections due to CRE is complicated by a lack of effective treatment options and clinical data on their effectiveness. Treatment of CRE infections in children is particularly challenging because therapeutic options for CRE lack adequate data on dosing and safety in children. Use of unconventional combination treatment regimens, including agents to which the organism is resistant in vitro, may provide some benefit in the treatment of severe CRE infection. Fortunately, several agents with the potential for treatment of CRE infections have been recently approved or are in late clinical development, although few data will be available in the short term to inform use in children.

BackgroundRapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians.MethodsWe compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making.ResultsIn PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in 95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem.ConclusionsRMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.

COVID‐19 patients are prone to coinfections during their hospitalization. These coinfections are challenging as they involve longer hospital stays, high costs, and higher risk of mortality. Here, we present a case of a patient with multi‐infection by resistant parasites, fungi, and bacteria during his hospitalization in a hospital in Lima, Peru. [ABSTRACT FROM AUTHOR]

The human innate immune system is equipped with multiple mechanisms to detect microbe-associated molecular patterns (MAMPs) to fight bacterial infections. The metabolite short-chain fatty acids (SCFAs) acetate, propionate and butyrate are released by multiple bacteria or are food ingredients. SCFA production, especially acetate production, is usually essential for bacteria, and knockout of pathways involved in acetate production strongly impairs bacterial fitness. Because host organisms use SCFAs as MAMPs and alter immune reactions in response to SCFAs, interventions that modulate SCFA levels can be a new strategy for infection control. The interaction between SCFAs and host cells has been primarily investigated in the intestinal lumen because of the high local levels of SCFAs released by bacterial microbiome members. However, members of not only the intestinal microbiome but also the skin microbiome produce SCFAs, which are known ligands of the seven-transmembrane G-protein-coupled receptor FFAR2. In addition to enterocytes, FFAR2 is expressed on other human cell types, including leukocytes, especially neutrophils. This finding is in line with other research that determined that targeted activation of FFAR2 diminishes susceptibility toward various types of infection by bacteria such as Klebsiella pneumonia, Citrobacter rodentium , and Staphylococcus aureus but also by viruses such as respiratory syncytial and influenza viruses. Thus, our immune system appears to be able to use FFAR2-dependent detection of SCFAs for perceiving and even averting severe infections. We summarize recent advances in understanding the role of SCFAs and FFAR2 in various infection types and propose the manipulation of this receptor as an additional therapeutic strategy to fight infections. [ABSTRACT FROM AUTHOR]

Abstract COVID‐19 patients are prone to coinfections during their hospitalization. These coinfections are challenging as they involve longer hospital stays, high costs, and higher risk of mortality. Here, we present a case of a patient with multi‐infection by resistant parasites, fungi, and bacteria during his hospitalization in a hospital in Lima, Peru.

Lasianthus stipularis Blume is a medicinal plant found in the tropical rainforests of peninsular Thailand and Malaysia. Its leaves are used in traditional medicine to treat a condition called "noise in the head" (i.e., ear infections). As such infections are often caused by Gram-negative bacteria; this species of Lasianthus may have antibacterial effects. In this study, we examined the non-polar fractions from the leaves and stem bark of L. stipularis using thin-layer chromatography and highperformance liquid chromatography (HPLC). The non-polar fractions exhibited a high coumarin content, while stem bark extracts showed a high concentration of 7-hydroxy-6-methoxycoumarin when isolated by column chromatography. The antibacterial activity of these extracts was evaluated by disk diffusion and broth microdilution against seven Gram-negative bacteria include Escherichia coli ATCC 25922, Pseudomonas aeruginosa DMSC 37166, P. aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC-BAA 1705, K. pneumoniae ATCC-BAA 1706, Acinetobacter baumannii ATCC 19606, and A. baumannii DMSC 3487. The DNA of L. stipularis was sequenced to support species identification and authentication. The minimum inhibitory concentration of fractions and pure compounds against pathogenic bacteria ranged from 100 to 200 µg/ml. Both strains of P. aeruginosa revealed the highest antibacterial activity at 100 µg/ml and 400 µg/ml concentration effect on Acinetobacter baumannii. In conclusion, leaf and stem bark non-polar extracts of L. stipularis have clear antibacterial activities. [ABSTRACT FROM AUTHOR]

ObjectiveTo assess the time-dependent exposure of California healthcare facilities to patients harboring methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, and Clostridium difficile infection (CDI) upon discharge from 1 hospital.MethodsRetrospective multiple-cohort study of adults discharged from 1 hospital in 2005-2009, counting hospitals, nursing homes, cities, and counties in which carriers were readmitted, and comparing the number and length of stay of readmissions and the number of distinct readmission facilities among carriers versus noncarriers.ResultsWe evaluated 45,772 inpatients including those with MRSA (N=1,198), VRE (N=547), ESBL (N=121), and CDI (N=300). Within 1 year of discharge, MRSA, VRE, and ESBL carriers exposed 137, 117, and 45 hospitals and 103, 83, and 37 nursing homes, generating 58,804, 33,486, and 15,508 total exposure-days, respectively. Within 90 days of discharge, CDI patients exposed 36 hospitals and 35 nursing homes, generating 7,318 total exposure-days. Compared with noncarriers, carriers had more readmissions to hospitals (MRSA:1.8 vs 0.9/patient; VRE: 2.6 vs 0.9; ESBL: 2.3 vs 0.9; CDI: 0.8 vs 0.4; all P

Ceftazidime-avibactam is the first antimicrobial approved by the U.S. FDA for the treatment of carbapenem-resistant Enterobacteriaceae. Avibactam, a non-β-lactam β-lactamase inhibitor, inactivates class A serine carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC). We report a KPC-producing K. pneumoniae isolate resistant to ceftazidime-avibactam (MIC, 32/4 μg/ml) from a patient with no prior treatment with ceftazidime-avibactam.

ObjectiveThe emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria is now a critical concern. The ESBL-producing Klebsiella pneumoniae constitutes one of the most common multidrug-resistant (MDR) groups of gram-negative bacteria involved in nosocomial infections worldwide. In this study we report on the molecular characterization through whole genome sequencing of an ESBL-producing K. pneumoniae strain, LAU-KP1, isolated from a stool sample from a patient admitted for a gastrointestinal procedure/surgery at the Lebanese Amrican University Medical Center-Rizk Hospital (LAUMCRH) in Lebanon.MethodsIllumina paired-end libraries were prepared and sequenced, which resulted in 4,220,969 high-quality reads. All sequence processing and assembly were performed using the A5 assembly pipeline.ResultsThe initial assembly produced 86 contigs, for which no scaffolding was obtained. The final collection of contigs was submitted to GenBank. The final draft genome sequence consists of a combined 5,632,663 bases with 57% G+C content. Automated annotation was performed using the RAST annotation server. Sequencing analysis revealed that the isolate harbored different β-lactamase genes, including bla oxa-1, bla CTX-M-15, bla SHV-11, and bla TEM-1b. The isolate was also characterized by the concomitant presence of other resistance determinants most notably acc(6')-lb-cr and qnrb1. The entire plasmid content was also investigated and revealed homology with four major plasmids pKPN-IT, pBS512_2, pRSF1010_SL1344, and pKPN3.ConclusionsThe potential role of K. pneumonia as a reservoir for ESBL genes and other resistance determinants is along with the presence of key factors that favor the spread of antimicrobial resistance a clear cause of concern and the problem that Carbapenem-non-susceptible ESBL isolates are posing in hospitals should be reconsidered through systematic exploration and molecular characterization.

Mathematical models of metabolism from bacterial systems biology have proven their utility across multiple fields, for example metabolic engineering, growth phenotype simulation, and biological discovery. The usefulness of the models stems from their ability to compute a link between genotype and phenotype, but their ability to accurately simulate gene-gene interactions has not been investigated extensively. Here we assess how accurately a metabolic model for Escherichia coli computes one particular type of gene-gene interaction, synthetic lethality, and find that the accuracy rate is between 25% and 43%. The most common failure modes were incorrect computation of single gene essentiality and biological information that was missing from the model. Moreover, we performed virtual and biological screening against several synthetic lethal pairs to explore whether two-compound formulations could be found that inhibit the growth of Gram-negative bacteria. One set of molecules was identified that, depending on the concentrations, inhibits E. coli and S. enterica serovar Typhimurium in an additive or antagonistic manner. These findings pinpoint specific ways in which to improve the predictive ability of metabolic models, and highlight one potential application of systems biology to drug discovery and translational medicine.

BackgroundKlebsiella pneumoniae is a leading cause of hospital-acquired urinary tract infections and pneumonia worldwide, and is responsible for many cases of pyogenic liver abscess among diabetic patients in Asia. A defining characteristic of this pathogen is the presence of a thick, exterior capsule that has been reported to play a role in biofilm formation and to protect the organism from threats such antibiotics and host immune challenge.FindingsWe constructed two knockout mutants of K. pneumoniae to investigate how perturbations to capsule biosynthesis alter the cellular phenotype. In the first mutant, we deleted the entire gene cluster responsible for biosynthesis of the extracellular polysaccharide capsule. In the second mutant, we deleted the capsule export subsystem within this cluster. We find that both knockout mutants have lower amounts of capsule but produce greater amounts of biofilm. Moreover, one of the two mutants abolishes fimbriae expression as well.ConclusionsThese results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease that affects about a quarter of the world population. MAFLD encompasses different disease stadia ranging from isolated liver steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Although MAFLD is considered as the hepatic manifestation of the metabolic syndrome, multiple concomitant disease-potentiating factors can accelerate disease progression. Among these risk factors are diet, lifestyle, genetic traits, intake of steatogenic drugs, male gender and particular infections. Although infections often outweigh the development of fatty liver disease, pre-existing MAFLD could be triggered to progress towards more severe disease stadia. These combined disease cases might be underreported because of the high prevalence of both MAFLD and infectious diseases that can promote or exacerbate fatty liver disease development. In this review, we portray the molecular and cellular mechanisms by which the most relevant viral, bacterial and parasitic infections influence the progression of fatty liver disease and steatohepatitis. We focus in particular on how infectious diseases, including coronavirus disease-19, hepatitis C, acquired immunodeficiency syndrome, peptic ulcer and periodontitis, exacerbate MAFLD. We specifically underscore the synergistic effects of these infections with other MAFLD-promoting factors. [ABSTRACT FROM AUTHOR]

Prior work established that a deficiency in the cysteine protease dipeptidyl peptidase I (DPPI) improves survival following polymicrobial septic peritonitis. To test whether DPPI regulates survival from severe lung infections, DPPI(-/-) mice were studied in a Klebsiella pneumoniae lung infection model, finding that survival in DPPI(-/-) mice is significantly better than in DPPI(+/+) mice 8d after infection. DPPI(-/-) mice have significantly fewer bacteria in the lung than infected DPPI(+/+) mice, but no difference in lung histopathology, lung injury, or cytokine levels. To explore mechanisms of enhanced bacterial clearance in DPPI(-/-) mice, we examined the status of pulmonary collectins, finding that levels of surfactant protein D, but not of surfactant protein A, are higher in DPPI(-/-) than in DPPI(+/+) BAL fluid, and that DPPI(-/-) BAL fluid aggregate bacteria more effectively than control BAL fluid. Sequencing of the amino terminus of surfactant protein D revealed two or eight additional amino acids in surfactant protein D isolated from DPPI(-/-) mice, suggesting processing by DPPI. These results establish that DPPI is a major determinant of survival following Klebsiella pneumoniae lung infection and suggest that the survival disadvantage in DPPI(+/+) mice is in part due to processing of surfactant protein D by DPPI.

The production of β-lactamase is the most common mechanism of resistance to β-lactam antibiotics among gram-negative bacteria. Extended-spectrum β-lactamases (ESBLs) are capable of hydrolyzing most penicillins, extended-spectrum cephalosporins, and aztreonam, but their activity is suppressed in the presence of a β-lactamase inhibitor. Serious infections with ESBL-producing isolates are associated with high rates of mortality, making early detection and adequate medical management essential to ensure optimal patient outcomes. Much controversy has centered on the recommendations for testing and reporting of antibiotic susceptibility of potential ESBL-producing organisms. The latest version of the Clinical Laboratory Standards Institute (CLSI) susceptibility reporting guidelines, published in 2010, no longer advocates for phenotypic testing of ESBL-producing isolates. From newer studies demonstrating a correlation between organism minimum inhibitory concentration (MIC) and clinical outcome, along with pharmacokinetic/pharmacodynamic (PK/PD) modeling demonstrating the importance of the MIC to achieving therapeutic targets, the CLSI has assigned lower susceptibility breakpoints for aztreonam and most cephalosporins. The new guidelines recommend using the lower MIC breakpoints to direct antibiotic selection. This article reviews the microbiology and epidemiology of ESBLs, the recent change in CLSI susceptibility reporting guidelines for ESBLs, and the clinical and PK/PD data supporting the relationship between in vitro susceptibility and clinical outcome. Finally, considerations for antimicrobial selection when treating patients with infections caused by ESBL-producing organisms from various sources are discussed.

S-nitrosoglutathione reductase (GSNOR) is a key denitrosylase and critically important for protecting immune and other cells from nitrosative stress. Pharmacological inhibition of GSNOR is being actively pursued as a therapeutic approach to increase S-nitrosoglutathione levels for the treatment of asthma and cystic fibrosis. In the present study, we employed GSNOR-deficient (GSNOR(-/-)) mice to investigate whether inactivation of GSNOR may increase susceptibility to pulmonary infection by Klebsiella pneumoniae, a common cause of nosocomial pneumonia. We found that compared to wild-type mice, bacterial colony forming units 48 h after intranasal infection with K. pneumoniae were increased over 4-folds in lung and spleen and strikingly, over a 1000-folds in blood of GSNOR(-/-) mice. Lung injury was comparable between infected wild-type and GSNOR(-/-) mice, but inflammation and injury was significantly elevated in spleen of GSNOR(-/-) mice. Whereas all wild-type mice survived 48 h after infection, 10 of 23 GSNOR(-/-) mice died. Thus, GSNOR appears to play a crucial role in controlling pulmonary and systemic infection by K. pneumoniae. Our results suggest that patients treated in clinical trials with inhibitors of GSNOR should be carefully monitored for signs of infection.

Effective host defense requires a robust, yet self-limited response to pathogens. A poorly calibrated response can lead to either bacterial dissemination due to insufficient inflammation or organ injury due to excessive inflammation. Recent evidence suggests that the cholinergic anti-inflammatory reflex helps calibrate the immune response. However, the influence of peripheral noradrenergic neurons, which are primarily sympathetic neurons, in regulating immunity remains incompletely characterized. Using a model of 6-hydroxydopamine-mediated noradrenergic nerve ablation, we show that elimination of noradrenergic neurons improves survival during Klebsiella pneumoniae peritonitis (67 versus 23%, p < 0.005) in mice. The survival benefit results from enhanced MCP-1-dependent monocyte recruitment and a subsequent decrease in bacterial loads. Splenectomy eliminated both the survival benefit of 6-hydroxydopamine and monocyte recruitment, suggesting that monocytes recruited to the peritoneum originate in the spleen. These results suggest that noradrenergic neurons regulate the immune response through two pathways. First, sympathetic nerve-derived norepinephrine directly restrains MCP-1 production by peritoneal macrophages during infection. Second, norepinephrine derived from the vagally innervated splenic nerve regulates splenic monocyte egress. Removal of these two modulators of the immune response enhances antibacterial immunity and improves survival. These results may have implications for how states of catecholamine excess influence the host response to bacterial infections.

BackgroundHuman exposure to nanoparticles (NPs) and environmental bacteria can occur simultaneously. NPs induce inflammatory responses and oxidative stress but may also have immune-suppressive effects, impairing macrophage function and altering epithelial barrier functions. The purpose of this study was to assess the potential pulmonary effects of inhalation and instillation exposure to copper (Cu) NPs using a model of lung inflammation and host defense.MethodsWe used Klebsiella pneumoniae (K.p.) in a murine lung infection model to determine if pulmonary bacterial clearance is enhanced or impaired by Cu NP exposure. Two different exposure modes were tested: sub-acute inhalation (4 hr/day, 5 d/week for 2 weeks, 3.5 mg/m(3)) and intratracheal instillation (24 hr post-exposure, 3, 35, and 100 μg/mouse). Pulmonary responses were evaluated by lung histopathology plus measurement of differential cell counts, total protein, lactate dehydrogenase (LDH) activity, and inflammatory cytokines in bronchoalveolar lavage (BAL) fluid.ResultsCu NP exposure induced inflammatory responses with increased recruitment of total cells and neutrophils to the lungs as well as increased total protein and LDH activity in BAL fluid. Both inhalation and instillation exposure to Cu NPs significantly decreased the pulmonary clearance of K.p.-exposed mice measured 24 hr after bacterial infection following Cu NP exposure versus sham-exposed mice also challenged with K.p (1.4 × 10(5) bacteria/mouse).ConclusionsCu NP exposure impaired host defense against bacterial lung infections and induced a dose-dependent decrease in bacterial clearance in which even our lowest dose demonstrated significantly lower clearance than observed in sham-exposed mice. Thus, exposure to Cu NPs may increase the risk of pulmonary infection.

Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2-4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient Kit(W-sh)/Kit(W-sh) mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to Kit(W-sh)/Kit(W-sh) mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.

KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.