Colonization dynamics of Klebsiella pneumoniae in the pet animals and human owners in a single household.

Klebsiella pneumoniae resides in the gastrointestinal (GI) microbiota of humans and animals. To characterize the population dynamics of GI-colonizing K. pneumoniae, we examined the clonality of K. pneumoniae isolates, which were longitudinally collected from the fecal samplings of a healthy married couple and their pet animals during Sep. 2015 to Oct. 2016. As revealed by XbaI-PFGE analysis, the K. pneumoniae populations detected in the male owner and in one of the dogs, consisted of clonally diverse K. pneumoniae isolates; whereas, a dominant clone persisted in the GI tract of the female owner who was prone to chronic diarrhea. Whole-genome sequencing analysis of a representative strain of this pathobiont clone revealed a sequence type (ST) 29 lineage with the carriage of KL54 cps locus and a 192,603 bp IncHIB-type virulence plasmid. After probiotics intervention, the pathobiont K. pneumoniae diminished. The vacant niche was transiently occupied by other clones of K. pneumoniae, one of which was also present in the male owner. Besides the dog, the fecal carriage of K. pneumoniae was also detected in a pet turtle. This turtle isolate was resistant to multiple antimicrobials, including carbapenems. Possible transmission of drug-resistant K. pneumoniae through human-pet bonds warrants our attention.

Serum Antibody Responses against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients.

Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR-Kp-infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp cultures. Isolate capsules were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, wzi154, and wzi50). High IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of nonconcordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains preopsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 antibodies (Abs) reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced poly-IgG efficacy against wzi29 CR-Kp We are the first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in vitro and in vivo models.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for Klebsiella pneumoniae pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host's capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both in vitro and in vivo models.

Immunomodulatory effects of colistin on host responses against carbapenem-resistant Klebsiella pneumoniae biofilms.

Colistin (CST) is a last-resort therapeutic option for carbapenem-resistant Klebsiella pneumoniae (CR-Kp) infections in critically ill patients. The effect of subinhibitory CST concentrations (sub-MICs) on biofilm formation is organism-dependent. We investigated the interactions between CST and innate immune cells against CR-Kp biofilms (CR-KpBF) by studying the effect of biofilm sub-MICs of CST on (i) damage induced by human polymorphonuclear neutrophils (PMNs) on CR-KpBF and (ii) the immunomodulatory potential on human mononuclear cells (MNCs) exposed to CR-KpBF. The impact of CST on PMN-induced biofilm damage was assessed by XTT reduction assay. Signal transduction and gene expression profiles in response to CST sub-MICs of MNCs exposed to CR-KpBF were studied by RT-PCR and multiplex ELISA. Pre-exposure of CR-Kp to 0.06 mg/L CST led to subsequent increased PMN-mediated biofilm damage against CR-KpBF in the presence of CST biofilm sub-MICs: there was an additive effect at 2, 4, 8 and 16 mg/L. However, the overall biofilm damage was not >52%. MNCs responded to CR-KpBF through Toll-like receptor 2 (TLR2) by 2.5-fold upregulation and NLRP3 inflammasome activation. CR-KpBF stimulated increased production of interleukin 1-beta (IL-1ß), tumour necrosis factor-alpha (TNFα), IL-8 and IL-6. In the combination treatment, 0.5 mg/L CST reduced IL-1ß, TNFα and IL-8 levels, whereas at 2 mg/L and 8 mg/L it increased the anti-inflammatory cytokine IL-10 (P < 0.05). Biofilm sub-MICs of CST enhance PMN killing capacity and attenuate production of inflammatory cytokines by MNCs exposed to CR-KpBF, playing a potentially important immunotherapeutic role especially for patients with cytokine deregulation.

Neutrophil extracellular traps (NETs)-mediated killing of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) are impaired in patients with diabetes mellitus.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) have been reported in recent years across Asian countries and pose a serious threat to public health. Neutrophils represent the first line of defense against numerous infectious pathogens, such as CR-hvKP. Neutrophil extracellular traps (NETs) constitute one of the major antimicrobial defense mechanisms in neutrophils against invading pathogens, especially against hvKP. Interestingly, previous studies have demonstrated that patients with type 2 diabetes mellitus (T2D) display elevated levels of NETosis but are vulnerable to infections caused by hvKP. The discrepancy propels us to investigate the role of NETs in hvKP infections in the context of T2D. By utilizing a clinical-derived CR-hvKP strain and a combination of NETs complex detection, phagocytosis testing, NETs killing assay and immunofluorescence, and scanning electron microscope assays, we identified defective NETs-mediated killing of CR-hvKP strain in patients with T2D. Specifically, we show that the impaired NETs-mediated killing in T2D is not due to the decreased NETs formation, as the neutrophils isolated from T2D patients exhibited enhanced NETs formation compared to healthy controls. Further, we demonstrate that the reduced NETs activity does not result from the trapping failure of CR-hvKP, but likely associated with the deficient surface damage conferred by the NETs of T2D patients. Our data provide a novel insight into the defective innate immune response against CR-hvKP in T2D.

The Role of IgG Subclass in Antibody-Mediated Protection against Carbapenem-Resistant Klebsiella pneumoniae.

Monoclonal antibodies (MAbs) have the potential to assist in the battle against multidrug-resistant bacteria such as carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, the characteristics by which these antibodies (Abs) function, such as the role of antibody subclass, must be determined before such modalities can be carried from the bench to the bedside. We performed a subclass switch on anticapsular monoclonal murine IgG3 (mIgG3) hybridomas and identified and purified a murine IgG1 (mIgG1) hybridoma line through sib selection. We then compared the ability of the mIgG1 and mIgG3 antibodies to control CR-Kp sequence type 258 (ST258) infection both in vitro and in vivo We found by enzyme-limited immunosorbent assay (ELISA) and flow cytometry that mIgG3 has superior binding to the CR-Kp capsular polysaccharide (CPS) and superior agglutinating ability compared to mIgG1 The mIgG3 also, predictably, had better complement-mediated serum bactericidal activity than the mIgG1 and also promoted neutrophil-mediated killing at concentrations lower than that of the mIgG1 In contrast, the mIgG1 had marginally better activity in improving macrophage-mediated phagocytosis. Comparing their activities in a pulmonary infection model with wild-type as well as neutropenic mice, both antibodies reduced organ burden in a nonlethal challenge, regardless of neutrophil status, with mIgG1 having the highest overall burden reduction in both scenarios. However, at a lethal inoculum, both antibodies showed reduced efficacy in neutropenic mice, with mIgG3 retaining the most activity. These findings suggest the viability of monoclonal Ab adjunctive therapy in neutropenic patients that cannot mount their own immune response, while also providing some insight into the relative contributions of immune mediators in CR-Kp protection.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an urgent public health threat that causes life-threatening infections in immunocompromised hosts. Its resistance to nearly all antibiotics necessitates novel strategies to treat it, including the use of monoclonal antibodies. Monoclonal antibodies are emerging as important adjuncts to traditional pharmaceuticals, and studying how they protect against specific bacteria such as Klebsiella pneumoniae is crucial to their development as effective therapies. Antibody subclass is often overlooked but is a major factor in how an antibody interacts with other mediators of immunity. This paper is the first to examine how the subclass of anticapsular monoclonal antibodies can affect efficacy against CR-Kp Additionally, this work sheds light on the viability of monoclonal antibody therapy in neutropenic patients, who are most vulnerable to CR-Kp infection.

Co-occurrence of a novel VIM-1 and FosA3-encoding multidrug-resistant plasmid and a KPC-2-encoding pKP048-like plasmid in a clinical isolate of Klebsiella pneumoniae sequence type 11.

The spread of carbapenem-resistant Enterobacteriaceae (CRE) worldwide remains a major threat to public health. Notably, carbapenemase-encoding genes are usually located in plasmids harboring other resistance determinants, and isolates having multiple plasmids are often highly resistant to carbapenems. In this study, we characterized the genetic context of coproduction of KPC-2, VIM-1, and FosA3 via two plasmids in the multidrug-resistant Klebsiella pneumoniae sequence type 11 (ST11) isolate JS187, recovered during an outbreak of KPC-2-producing K. pneumoniae in a Chinese teaching hospital in 2008. Plasmid p187-1, coharboring blaVIM-1 and fosA3, consisted of a pKOX-R1-like backbone and two multidrug resistant (MDR) regions separated by pKHS1-like backbone sequences involving plasmid replication and stability. The MDR region 1 was a chimera composed of the blaVIM-1-bearing In916-like integron and Tn1721-like transposon, and was disrupted by sequential insertion of an IS26-based transposition unit carrying blaCTX-M-3 and a ΔTn3-like transposon bearing blaTEM-1. MDR region 2 was an IS26-array structure with fosA3 and blaSHV-12. Plasmid p187-2 harboring blaKPC-2 was closely related to pKP048. blaKPC-2 in p187-2 was carried by a Tn1721 variant, which differed from the prototype Tn1721-blaKPC-2 transposon observed in pKP048 by disruption of an IS26 at Tn3 and deletion of a 31-kb MDR fragment. Co-existence of the novel VIM-1- and FosA3-encoding MDR plasmid p187-1 and the KPC-2-encoding pKP048-like plasmid p187-2 made K. pneumoniae JS187 highly resistant to carbapenems. Moreover, p187-1 still carried genes conferring resistance to cephalosporins, fosfomycin, chloramphenicol, and quaternary ammonium, posing substantial challenges for the treatment of Enterobacteriaceae infections. Thus, monitoring the prevalence and evolution of these plasmids and/or strains is critical.

Avibactam Sensitizes Carbapenem-Resistant NDM-1-Producing Klebsiella pneumoniae to Innate Immune Clearance.

Infections caused by New Delhi metallo-ß-lactamase (NDM)-producing strains of multidrug-resistant Klebsiella pneumoniae are a global public health threat lacking reliable therapies. NDM is impervious to all existing ß-lactamase inhibitor (BLI) drugs, including the non-ß-lactam BLI avibactam (AVI). Though lacking direct activity against NDMs, AVI can interact with penicillin-binding protein 2 in a manner that may influence cell wall dynamics. We found that exposure of NDM-1-producing K. pneumoniae to AVI led to striking bactericidal interactions with human cathelicidin antimicrobial peptide LL-37, a frontline component of host innate immunity. Moreover, AVI markedly sensitized NDM-1-producing K. pneumoniae to killing by freshly isolated human neutrophils, platelets, and serum when complement was active. Finally, AVI monotherapy reduced lung counts of NDM-1-producing K. pneumoniae in a murine pulmonary challenge model. AVI sensitizes NDM-1-producing K. pneumoniae to innate immune clearance in ways that are not appreciated by standard antibiotic testing and that merit further study.

Interleukin-10 Produced by Myeloid-Derived Suppressor Cells Provides Protection to Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 258 by Enhancing Its Clearance in the Airways.

Carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258) can cause chronic infections in lungs and airways, with repeated episodes of bacteremia. In this report we addressed whether the recruitment of myeloid cells producing the anti-inflammatory cytokine interleukin-10 (IL-10) modulates the clearance of CKRP-ST258 in the lungs and establishes bacterial persistence. Our data demonstrate that during pneumonia caused by a clinical isolate of CRKP-ST258 (KP35) there is an early recruitment of monocyte-myeloid-derived suppressor cells (M-MDSCs) and neutrophils that actively produce IL-10. However, M-MDSCs were the cells that sustained the production of IL-10 over the time of infection evaluated. Using mice unable to produce IL-10 (IL-10-/-), we observed that the production of this cytokine during the infection caused by KP35 is important to control bacterial burden, to prevent lung damage, to modulate cytokine production, and to improve host survival. Importantly, intranasal transfer of bone marrow-derived M-MDSCs from mice able to produce IL-10 at 1 day prior to infection improved the ability of IL-10-/- mice to clear KP35 in the lungs, decreasing their mortality. Altogether, our data demonstrate that IL-10 produced by M-MDSCs is required for bacterial clearance, reduction of lung tissue damage, and host survival during KP35 pneumonia.

Detection of Carbapenemase-Producing Enterobacteriaceae in Positive Blood Culture Using an Immunochromatographic RESIST-4 O.K.N.V. Assay.

We evaluated the performance of the RESIST-4 O.K.N.V. assay (Coris) with 98 isolates to detect OXA-48-like and KPC-, NDM-, and VIM-type carbapenemases directly on positive human blood cultures. OXA-48-like and KPC-type isolates were correctly detected, but the detection of NDM- and VIM-type carbapenemases was weak and variable. We show that repeating the test on a 4-h subculture improves the detection of NDM- and VIM-type carbapenemases to 100%.

Novel, Broadly Reactive Anticapsular Antibodies against Carbapenem-Resistant Klebsiella pneumoniae Protect from Infection.

Carbapenem-resistant (CR) sequence type 258 (ST258) Klebsiella pneumoniae has become an urgent health care threat, causing an increasing number of high-mortality infections. Its resistance to numerous antibiotics and threat to immunocompromised patients necessitate finding new therapies to combat these infections. Previous successes in the laboratory, as well as the conservation of capsular polysaccharide (CPS) among the members of the ST258 clone, suggest that monoclonal antibody (MAb) therapy targeting the outer polysaccharide capsule of K. pneumoniae could serve as a valuable treatment alternative for afflicted patients. Here, we isolated several IgG antibodies from mice inoculated with a mixture of CR K. pneumoniae CPS conjugated to anthrax protective antigen. Two of these MAbs, 17H12 and 8F12, bind whole and oligosaccharide epitopes of the CPS of clade 2 ST258 CR K. pneumoniae, which is responsible for the most virulent CR K. pneumoniae infections in the United States. These antibodies were shown to agglutinate all clade 2 strains and were also shown to promote extracellular processes killing these bacteria, including biofilm inhibition, complement deposition, and deployment of neutrophil extracellular traps. Additionally, they promoted opsonophagocytosis and intracellular killing of CR K. pneumoniae by human-derived neutrophils and cultured murine macrophages. Finally, when mice were intratracheally infected with preopsonized clade 2 CR K. pneumoniae, these MAbs reduced bacterial dissemination to organs. Our data suggest that broadly reactive anticapsular antibodies and vaccines against clade 2 ST258 CR K. pneumoniae are possible. Such MAbs and vaccines would benefit those susceptible populations at risk of infection with this group of multidrug-resistant bacteria.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an enteric bacterium that has been responsible for an increasing number of deadly outbreaks and hospital-acquired infections. The pathogen's resistance to numerous antibiotics, including new drugs, leaves few therapeutic options available for infected patients, who often are too sick to fight the infection themselves. Immunotherapy utilizing monoclonal antibodies has been successful in other medical fields, and antibodies targeting the outer polysaccharide capsule of these bacteria could be a valuable treatment alternative. This study presents two anticapsular antibodies, 17H12 and 8F12, that were found to be protective against the most virulent carbapenem-resistant K. pneumoniae clinical strains. These antibodies are shown to promote the killing of these strains through several extracellular and intracellular processes and prevent the spread of infection in mice from the lungs to distal organs. Thus, they could ultimately treat or protect patients infected or at risk of infection by this multidrug-resistant bacterium.

Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils.

Carbapenem-resistant Klebsiella pneumoniae is a problem worldwide. A carbapenem-resistant K. pneumoniae lineage classified as multilocus sequence type 258 (ST258) is prominent in the health care setting in many regions of the world, including the United States. ST258 strains can be resistant to virtually all clinically useful antibiotics; treatment of infections caused by these organisms is difficult, and mortality is high. As a step toward promoting development of new therapeutics for ST258 infections, we tested the ability of rabbit antibodies specific for ST258 capsule polysaccharide to enhance human serum bactericidal activity and promote phagocytosis and killing of these bacteria by human neutrophils. We first demonstrated that an isogenic wzy deletion strain is significantly more susceptible to killing by human heparinized blood, serum, and neutrophils than a wild-type ST258 strain. Consistent with the importance of capsule as an immune evasion molecule, rabbit immune serum and purified IgG specific for ST258 capsule polysaccharide type 2 (CPS2) enhanced killing by human blood and serum in vitro Moreover, antibodies specific for CPS2 promoted phagocytosis and killing of ST258 by human neutrophils. Collectively, our findings suggest that ST258 CPS2 is a viable target for immunoprophylactics and/or therapeutics.IMPORTANCE Infections caused by carbapenem-resistant K. pneumoniae are difficult to treat, and mortality is high. New prophylactic approaches and/or therapeutic measures are needed to prevent or treat infections caused by these multidrug-resistant bacteria. A strain of carbapenem-resistant K. pneumoniae, classified by multilocus sequence typing as ST258, is present in many regions of the world and is the most prominent carbapenem-resistant K. pneumoniae lineage in the United States. Here we show that rabbit antibodies specific for capsule polysaccharide of ST258 significantly enhance human serum bactericidal activity and promote phagocytosis and killing of this pathogen by human neutrophils. These studies have provided strong support for the idea that development of an immunotherapy (vaccine) for carbapenem-resistant K. pneumoniae infections is feasible and has merit.

Prospective evaluation of the OKN K-SeT assay, a new multiplex immunochromatographic test for the rapid detection of OXA-48-like, KPC and NDM carbapenemases.

Objectives: There is an urgent need for accurate and fast diagnostic tests capable of identifying carbapenemase producers. Here, we assessed the performance of a new multiplex lateral flow assay (OKN K -SeT) for the rapid detection of OXA-48-like, KPC and NDM carbapenemase-producing Enterobacteriaceae from culture colonies. Methods: Two hundred collection isolates with characterized ß-lactamase content and 183 non-duplicate consecutive isolates referred to two National Reference Centres over a 2 month period in 2016 were used to evaluate the OKN K -SeT assay. Results: The assay correctly detected all 42 OXA-48-like-, 27 KPC- and 30 NDM-producing isolates from the collection panel, including 7 isolates that co-produced NDM and OXA-181 carbapenemases. No cross-reactivity was observed with non-targeted carbapenemases ( n = 41) or with non-carbapenemase producers ( n = 60). Prospectively, all OXA-48-like ( n = 69), KPC ( n = 9) and NDM ( n = 19) carbapenemase-producing Enterobacteriaceae isolates were correctly detected, while 11 carbapenemase producers not targeted by the assay went undetected [VIM ( n = 8) and OXA-23/OXA-58-like ( n = 3)]. Overall, the sensitivity and specificity of the assay were 100%. Conclusions: The OKN assay is efficient, rapid and easy to implement in the workflow of a clinical microbiology laboratory for the confirmation of OXA-48, NDM and KPC carbapenemases. This test represents a powerful diagnostic tool as it enables the rapid detection of the most clinically important carbapenemases without the need for more costly and less frequently available molecular assays.