Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice.

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Klebsiella pneumoniae-Induced Pneumonia-Derived Sepsis.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

Acyloxyacyl hydrolase promotes the resolution of lipopolysaccharide-induced acute lung injury.

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection.

Kaempferol-3-O-glucorhamnoside inhibits inflammatory responses via MAPK and NF-κB pathways in vitro and in vivo.

Klebsiella pneumoniae causes severe infections including pneumonia and sepsis and treatments are complicated by increased levels of antibiotic resistance. We have identified a flavonoid kaempferol-3-O-glucorhamnoside derived from the plant Thesium chinense Turcz that possessed potent anti-inflammatory effects in K. pneumoniae infected mice. Administration of kaempferol-3-O-glucorhamnoside before bacterial challenge effectively suppressed expression of the major inflammatory cytokines TNF-α, IL-6, IL-1ß and PGE2 and ameliorated lung edema. In addition, administration of this compound to cultured RAW macrophages or Balb/c mice resulted in the suppression of NFκB and MAP kinase phosphorylation indicating an inhibitory effect on inflammation in vitro and in vivo. Kaempferol-3-O-glucorhamnoside also decreased ROS levels and overall oxidative stress in lungs and in cultured cells generated by K. pneumoniae exposure. Taken together, kaempferol-3-O-glucorhamnoside is a potent anti-inflammatory in vitro and in vivo and is a promising therapeutic agent for treating K. pneumoniae infections in the clinic.

Phenotypic and Genotypic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli Isolates.

OBJECTIVES: Drug resistance among gram-negative bacteria is a worldwide challenge. Due to the importance of drug-resistant Klebsiella pneumoniae and Escherichia coli strains in hospital-acquired infections, we aimed to determine the phenotypic and genotypic characteristics of ESBL-, AmpC-, and carbapenemase-producing isolates obtained from hospitalized patients in Tehran and Ilam (Iran). MATERIALS AND METHODS: In total, 90 K. pneumoniae isolates and 65 E. coli isolates were collected from various infections. Phenotypic identification of bacterial isolates was performed using standard methods. Phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. RESULTS: Phenotypic detection tests showed that 36 (40%) K. pneumoniae and 23 (35.4%) E. coli isolates were ESBL producers. Moreover, 18 (20%) and 6 (9.2%) K. pneumoniae and E. coli isolates were AmpC producers, respectively. Modified Hodge test results indicated that 39 (43.3%) K. pneumoniae and 18 (27.7%) E. coli isolates produced carbapenemase. Molecular tests showed that 40% of K. pneumoniae and 36.9% of E. coli isolates were ESBL positive. AmpC was detected in 24.4 and 13.8% of K. pneumoniae and E. coli isolates. Carbapenemase was detected in 34 (37.8%) K. pneumoniae and 13 (20%) E. coli isolates. -Conclusion: In this study, 3 K. pneumoniae isolates simultaneously carried ESBL, AmpC, and carbapenemase genes. Up-to-date strategies such as combination therapy or utilization of new antimicrobial agents might help to combat such drug-resistant organisms.

Molecular epidemiology of ESBL-producing Klebsiella pneumoniae isolates in intensive care units of a tertiary care hospital, North of Iran.

The emergence of extended-spectrum beta-lactamases (ESBL) producing strains become a great concern, because few antimicrobial agents remain active against them. Due to the lack of data on the genotyping characteristics and antibiotics resistance of clinical isolates of Klebsiella pneumoniae in the north of Iran, this study aimed to determine the occurrence of ESBL-producing isolates and their molecular characteristics in order to analyses their epidemiological relationships. This cross-sectional study performed on 60 K. pneumoniae isolates which were recovered from different clinical specimens within May and November 2016. Isolates were identified by standard microbiologic tests and confirmed by API 20E strip. Antimicrobial susceptibility testing was carried out by disk diffusion method. The genetic relatedness among the isolates was assessed by RAPD-PCR. Totally, the lowest level of susceptibility was toward amoxicillin/clavulanat, and nalidixic acid. On the other hand, the highest level of susceptibility was toward imipenem (86.7%). The rate of ESBL-producing isolates was 45% (27/60). There was a significant association between production of ESBLs and higher antibiotic resistance in tested isolates. The RAPD-PCR dendrogram revealed 5 major clusters with a similarity of 80% which indicates the high relatedness of the studied isolates. Twenty-one isolates out of the 27 ESBL-producing isolates were clustered in cluster A. In summary, results showed the high prevalence of multiple-drug resistant and ESBL-producing K. pneumoniae isolates in our ICUs. Also, results revealed a significant similarity between ESBL-producing isolates that necessitate restricted infection control policies and rational prescription and use of antibiotics.

Previous Antibiotic Exposure Increases Risk of Infection with Extended-Spectrum-ß-Lactamase- and AmpC-Producing Escherichia coli and Klebsiella pneumoniae in Pediatric Patients.

The objective of this study was to determine whether antibiotic exposure is associated with extended-spectrum-beta-lactamase- or AmpC-producing Escherichia coli or Klebsiella pneumoniae infections in children. We collected extended-spectrum-beta-lactamase- or AmpC-producing E. coli or K. pneumoniae isolates and same-species susceptible controls from normally sterile sites of patients aged ≤21 years, along with associated clinical data, at four free-standing pediatric centers. After controlling for potential confounders, the relative risk of having an extended-spectrum-beta-lactamase-producing isolate rather than a susceptible isolate was 2.2 times higher (95% confidence interval [CI], 1.49 to 3.35) among those with antibiotic exposure in the 30 days prior to infection than in those with no antibiotic exposure. The results were similar when analyses were limited to exposure to third-generation cephalosporins, other broad-spectrum beta-lactams, or trimethoprim-sulfamethoxazole. Conversely, the relative risk of having an AmpC-producing versus a susceptible isolate was not significantly elevated with any antibiotic exposure in the 30 days prior to infection (adjusted relative risk ratio, 1.12; 95% CI, 0.65 to 1.91). However, when examining subgroups of antibiotics, the relative risk of having an AmpC-producing isolate was higher for patients with exposure to third-generation cephalosporins (adjusted relative risk ratio, 4.48; 95% CI, 1.75 to 11.43). Dose-response relationships between antibiotic exposure and extended-spectrum-beta-lactamase-producing or AmpC-producing isolates were not demonstrated. These results reinforce the need to study and implement pediatric antimicrobial stewardship strategies, and they indicate that epidemiological studies of third-generation cephalosporin-resistant E. coli and K. pneumoniae isolates should include resistance mechanisms when possible.

Transfer of CMY-2 Cephalosporinase from Escherichia coli to Virulent Klebsiella pneumoniae Causing a Recurrent Liver Abscess.

A CMY-2-producing capsular type K2 Klebsiella pneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the blaCMY-2-carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93.

Global dissemination of extensively drug-resistant carbapenemase-producing Enterobacteriaceae: clinical perspectives on detection, treatment and infection control.

The prevalence of carbapenem-resistant Gram-negative bacilli is on the rise worldwide, posing a major public health threat. Previously, this was mostly a problem in Pseudomonas and Acinetobacter, but during the last decade, carbapenem resistance has escalated in medically important species such as Klebsiella pneumoniae and Escherichia coli. In particular, the rising trend in E. coli is of concern, as this may lead to almost untreatable community-acquired infections. Resistance is conferred by carbapenemases, which are beta-lactamases that can breakdown essentially all beta-lactams. Moreover, bacteria carrying these resistance determinants are often resistant to other treatment options, due to the frequent co-acquisition of non-beta-lactam resistance genes located on the same mobile genetic elements. The detection of carbapenemase-producing Enterobacteriaceae (CPE) is a challenge, because some carbapenemases produce relatively discrete levels of carbapenem resistance. Current clinical evidence for treatment guidance is limited and based on retrospective observational studies and case reports. Existing data support the use of combination therapy for treatment of severe infections caused by CPE. Combination regimens including colistin, carbapenems, tigecycline, aminoglycosides and fosfomycin have been used. Randomized controlled studies of combination regimens are ongoing and may help to determine the optimal therapy. Novel beta-lactamase inhibitors may also have a role in future treatment of these infections. Strict infection control measures including isolation or cohort care of affected patients as well as contact tracing and active screening are needed to curb the spread of CPE. In this review, we provide a clinical perspective on the management of patients infected or colonized with CPE.

Effects of an anti-inflammatory VAP-1/SSAO inhibitor, PXS-4728A, on pulmonary neutrophil migration.

BACKGROUND AND PURPOSE: The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A. METHODS: Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A. RESULTS: Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

NLRC4 inflammasome-mediated production of IL-1ß modulates mucosal immunity in the lung against gram-negative bacterial infection.

Bacterial flagellin is critical to mediate NLRC4 inflammasome-dependent caspase-1 activation. However, Shigella flexneri, a nonflagellated bacterium, and a flagellin (fliC) knockout strain of Pseudomonas aeruginosa are known to activate NLRC4 in bone marrow-derived macrophages. Furthermore, the flagellin-deficient fliC strain of P. aeruginosa was used in a mouse model of peritonitis to show the requirement of NLRC4. In a model of pulmonary P. aeruginosa infection, flagellin was shown to be essential for the induction of NLRC4-dependent caspase-1 activation. Moreover, in all P. aeruginosa studies, IL-1ß production was attenuated in NLRC4(-/-) mice; however, the role of IL-1ß in NLRC4-mediated innate immunity in the lungs against a nonflagellated bacterium was not explored. In this article, we report that NLRC4 is important for host survival and bacterial clearance, as well as neutrophil-mediated inflammation in the lungs following Klebsiella pneumoniae infection. NLRC4 is essential for K. pneumoniae-induced production of IL-1ß, IL-17A, and neutrophil chemoattractants (keratinocyte cell-derived chemokines, MIP-2, and LPS-induced CXC chemokines) in the lungs. NLRC4 signaling in hematopoietic cells contributes to K. pneumoniae-induced lung inflammation. Furthermore, exogenous IL-1ß, but not IL-18 or IL-17A, partially rescued survival, neutrophil accumulation, and cytokine/chemokine expression in the lungs of NLRC4(-/-) mice following infectious challenge. Furthermore, IL-1R1(-/-) mice displayed a decrease in neutrophilic inflammation in the lungs postinfection. Taken together, these findings provide novel insights into the role of NLRC4 in host defense against K. pneumoniae infection.

Carbapenemase-producing Klebsiella pneumoniae in the Czech Republic in 2011.

Carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. are increasingly reported in many countries all over the world. Due to the resistance of those bacteria to almost all antibiotics (e.g.beta-lactams, aminoglycosides, fluoroquinolones),treatment options are seriously limited. In the Czech Republic, the incidence of carbapenemase-producing Enterobacteriaceae seems to be low, restricted to only three cases detected between 2009 and 2010.Here, we describe molecular typing of 15 carbapenemase-producing Klebsiella pneumoniae isolates identified in the Czech Republic during 2011. Five VIM-1-producing isolates belonging to sequence type (ST)11 and one VIM-4-producing isolate of ST1029 have been detected. blaVIM-1 and blaVIM-4 as a part of class 1 integrons were chromosomally located or carried by a plasmid belonging to A/C replicon type (blaVIM-4). KPC-3-producing isolates of ST512, recovered from six patients, caused an outbreak. Three more isolates producing KPC-2 enzyme belonged to ST258. Both blaKPCgenes were part of the Tn4401a transposon carried on plasmids of the pKpQIL type. The isolates were resistant to all antibiotics tested except colistin and/or gentamicin.Four of these 15 strains were recovered from patients repatriated to the Czech Republic from Greece and Italy. This is the first report of outbreaks caused by carbapenemase-producing Enterobacteriaceae in the Czech Republic.

Outcomes of transplantation using organs from a donor infected with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae.

Transmission of pathogens from donor to recipient is a potential complication of organ transplantation. Herein, we describe the clinical course and outcomes of 4 transplant recipients who received tissues from a donor with multi-organ infection with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae. Recipient 1 underwent simultaneous liver and kidney transplantation for alpha-1 antitrypsin deficiency and alcohol-related cirrhosis, and acute tubular necrosis, respectively. Soon after transplantation, he developed an infected hematoma and peritonitis due to KPC-producing K. pneumoniae despite receiving tigecycline prophylaxis. He was treated with a prolonged course of tigecycline, amikacin, and meropenem, in conjunction with surgical evacuation and percutaneous drainage of the infected fluid collections. Recipient 2 underwent living-donor liver transplantation for cholangiocarcinoma and primary sclerosing cholangitis using vein graft from the donor infected with KPC-producing K. pneumoniae. Culture of the preservation fluid containing the vein graft was positive for KPC-producing K. pneumoniae. The patient received preemptive amikacin and tigecycline, and he did not develop any infection (as evidenced by negative surveillance blood cultures). The isolates from the donor and Recipients 1 and 2 were indistinguishable by pulsed-field gel electrophoresis. Recipients 3 and 4 underwent kidney and heart transplantation, respectively; both patients received perioperative tigecycline prophylaxis and did not develop infections due to KPC-producing K. pneumoniae. All transplant recipients had good short-term outcomes. These cases highlight the importance of inter-institutional communication and collaboration to ensure the successful management of recipients of organs from donors infected with multidrug-resistant organisms.

Characterization of purified New Delhi metallo-ß-lactamase-1.

New Delhi metallo-ß-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used ß-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-ß-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of ß-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 µM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 µM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.

Kinase activity profiling of gram-negative pneumonia.

Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential to immune cells. The current study explored signal transduction events during murine Gram-negative pneumonia using a systems biology approach. Kinase activity arrays enable the analysis of 1,024 consensus sequences of protein kinase substrates. Using a kinase activity array on whole lung lysates, cellular kinase activities were determined in a mouse model of K. pneumoniae pneumonia. Notable kinase activities also were validated with phospho-specific Western blots. On the basis of the profiling data, mitogen-activated protein kinase (MAPK) signaling via p42 mitogen-activated protein kinase (p42) and p38 mitogen-activated protein kinase (p38) and transforming growth factor ß (TGFß) activity were reduced during infection, whereas v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) activity generally was enhanced. AKT signaling was represented in both metabolic and inflammatory (mitogen-activated protein kinase kinase 2 [MKK], apoptosis signal-regulating kinase/mitogen-activated protein kinase kinase kinase 5 [ASK] and v-raf murine sarcoma viral oncogene homolog B1 [b-RAF]) context. This study reaffirms the importance of classic inflammation pathways, such as MAPK and TGFß signaling and reveals less known involvement of glycogen synthase kinase 3ß (GSK-3ß), AKT and SRC signaling cassettes in pneumonia.

Epidemiology, Risk Factors, and Clinical Outcomes of Bloodstream Infection due to Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in Hematologic Malignancy: A Retrospective Study from Central South China.

Objective: To determine the epidemiology, risk factors, and prognosis of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae bloodstream infections (BSIs) among hematology malignancy (HM) patients in China. Method: From January 2010 to June 2018, we retrospectively collected and analyzed the 449 HM patients with E. coli or K. pneumoniae BSIs from three leading hospitals in Hunan Province, China. Results: Two hundred four (45.4%) patients harbored ESBL-producing bacteremia. The proportion of ESBL-producing bacteremia increased significantly with the growth of the year, with a ratio of 34.47% in 2010-2014 to 54.7% in 2015-2018. Comparing with non-ESBL groups in HM patients, central venous catheter (odds ratio [OR] 1.717, p = 0.009), previous antibiotic exposure (OR 1.559, p = 0.035), and E. coli (OR 2.561, p ≤ 0.001) among ESBL groups were independent risk factors. No significant differences in 30-day mortality were tested in patients with BSI caused by ESBL-producing or non-ESBL-producing E. coli and K. pneumoniae (17.1% vs. 16.7%; p = 0. 893). The proportion of carbapenem used within 72 hours after the onset of bacteremia in two groups was high, which was routinely used as "last-resort drugs" in Gram-negative bacterial infections. Risk factors associated with 30-day mortality in HM patients with E. coli or K. pneumoniae bacteremia were myelodysplastic syndrome, incomplete remission of the disease, Multinational Association of Supportive Care in Cancer score <21, Pitt bacteremia score ≥4, Charlson comorbidity score >3, catheter insertion, use of vasopressors, and inappropriate antibiotics within 72 hours of BSI onset. Conclusions: The results of this study may provide some references for the whole process management of HM patients with BSIs.

High-risk clones of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012).

The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection.

Klebsiella pneumoniae ST258 is a human pathogen associated with poor outcomes worldwide. We identify a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of these organisms in vivo. Comparison of a wild-type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by CRISPR-Cas9 targeting reveals greater NADH:ubiquinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. The acquisition of atf3 induces changes in the bacterial acetylome, promoting lysine acetylation of multiple proteins involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost leads to greater consumption of glucose in the host airway and increased bacterial burden in the lung, independent of cytokine levels and immune cell recruitment. Acquisition of this acyltransferase enhances fitness of a K. pneumoniae ST258 isolate and may contribute to the success of this clonal complex as a healthcare-associated pathogen.

Prevalence and patient related factors associated with Extended-Spectrum Beta-Lactamase producing Escherichia coli and Klebsiella pneumoniae carriage and infection among pediatric patients in Tanzania.

Extended-Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae (EPE) is increasing worldwide, though less documented in low-income settings. Here we determined the prevalence of EPE infection and carriage, and patient factors associated with EPE-carriage among pediatric patients in three health care levels in Tanzania. Between January and April 2016, 350 febrile children (median age 21 months) seeking care at a university or a regional referral hospital, or a health centre in Moshi municipality, Tanzania, were included. Socio-demographic characteristics were collected using a questionnaire. Rectal swabs and blood cultures were collected from all children (n = 350) and urinary samples from 259 children at admission. ESBL-phenotype and antimicrobial susceptibility were determined for Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolates. Only one EPE case (E. coli) in blood and four in urine (one E. coli and three K. pneumoniae) were found, whereas (n = 90, 26%) of the children were colonized in feces (ESBL-E. coli; n = 76, ESBL-K. pneumoniae, n = 14). High resistance rates were seen in fecal ESBL-E. coli (n = 76) against trimethoprim-sulfamethoxazole (n = 69, 91%), gentamicin (n = 51, 67%), ciprofloxacin (n = 39, 51%) and chloramphenicol (n = 27, 35%) whereas most isolates were sensitive to amikacin (n = 71, 93%). Similar rates were seen for fecal ESBL-K. pneumoniae. Resistance to first line antibiotics were also very high in fecal E. coli not producing ESBL. No sociodemographic factor was associated with EPE-carriage. Children colonized with EPE were younger than 12 months (n = 43, 48%) and often treated with antibiotics (n = 40, 44%) in the previous two months. After adjustment for age children admitted to the intensive care unit had higher odds of EPE fecal carriage compared with those in the general wards (OR = 3.9, 95%CI = 1.4-10.4). Despite comparatively high rates of fecal EPE-carriage and previous antibiotic treatment, clinical EPE cases were rare in the febrile children. The very high resistant rates for the EPE and the non-ESBL producing E. coli to commonly used antibiotics are worrying and demand implementation of antibiotic stewardship programs in all levels of health care in Tanzania.