Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by progressive biliary inflammation and fibrosis. Although gut commensals are associated with PSC, their causative roles and therapeutic strategies remain elusive. Here we detect abundant Klebsiella pneumoniae (Kp) and Enterococcus gallinarum in fecal samples from 45 PSC patients, regardless of intestinal complications. Carriers of both pathogens exhibit high disease activity and poor clinical outcomes. Colonization of PSC-derived Kp in specific pathogen-free (SPF) hepatobiliary injury-prone mice enhances hepatic Th17 cell responses and exacerbates liver injury through bacterial translocation to mesenteric lymph nodes. We developed a lytic phage cocktail that targets PSC-derived Kp with a sustained suppressive effect in vitro. Oral administration of the phage cocktail lowers Kp levels in Kp-colonized germ-free mice and SPF mice, without off-target dysbiosis. Furthermore, we demonstrate that oral and intravenous phage administration successfully suppresses Kp levels and attenuates liver inflammation and disease severity in hepatobiliary injury-prone SPF mice. These results collectively suggest that using a lytic phage cocktail shows promise for targeting Kp in PSC.

Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation.

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.

Evolution and dissemination of the Klebsiella pneumoniae clonal group 258 throughout Israeli post-acute care hospitals, 2008-13.

Objectives: The KPC-producing Klebsiella pneumoniae (KPC-KP) clonal group (CG) 258 has disseminated throughout Israeli post-acute care hospitals (PACHs). The objectives of the study were (i) to describe the evolution and (ii) to understand the dissemination modes of CG 258 in the PACH system in Israel. Methods: KPC-KP surveillance cultures isolates were collected in Israeli PACHs in three national point-prevalence surveys: 2008, 2011 and 2013. CG 258 was identified by pilv-l PCR. WGS was performed for CG 258 isolates from 9 of 14 PACHs and data extracted for core-genome MLST (cgMLST) and for capsule polysaccharide gene cluster analysis. Results: The proportional representation of CG 258 among the KPC-KP isolates increased from 72 of 104 isolates (69.2%) in 2008 to 113 of 133 isolates (85%) in 2011 ( P = 0.004 for 2008 versus 2011) and remained high in 2013 [56 of 67 isolates (83.6%)]. All isolates were related to CG 258 clade 2. cgMLST phylogenetic analysis showed relative convergence in the 2008 survey, with increasing diversification in the subsequent surveys. A predominantly institutional dissemination pattern was observed only in centre F from southern Israel. A predominantly regional dissemination pattern was observed in the two PACHs in Jerusalem. The other PACHs were characterized by a combined institutional and generalized pattern, with the majority of isolates clustering within the same PACH and survey. Conclusions: CG 258 clade 2 has retained its predominance despite increased diversification. Although interchanging of CG 258 strains occurred between most PACHs, local spread is the leading cause of its dissemination.

A prospective survey of Pseudomonas aeruginosa colonization and infection in the intensive care unit.

BACKGROUND: Pseudomonas aeruginosa (PA) surveillance may improve empiric antimicrobial therapy, since colonizing strains frequently cause infections. This colonization may be 'endogenous' or 'exogenous', and the source determines infection control measures. We prospectively investigated the sources of PA, the clinical impact of PA colonization upon admission and the dynamics of colonization at different body sites throughout the intensive care unit stay. METHODS: Intensive care patients were screened on admission and weekly from the pharynx, endotracheal aspirate, rectum and urine. Molecular typing was performed using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain reaction (ERIC-PCR). RESULTS: Between November 2014 and January 2015, 34 patients were included. Thirteen (38%) were colonized on admission, and were at a higher risk for PA-related clinical infection (Hazard Ratio = 14.6, p = 0.0002). Strains were often patient-specific, site-specific and site-persistent. Sixteen out of 17 (94%) clinical isolates were identical to strains found concurrently or previously on screening cultures from the same patient, and none were unique. Ventilator associated pneumonia-related strains were identical to endotracheal aspirates and pharynx screening (87-75% of cases). No clinical case was found among patients with repeated negative screening. CONCLUSION: PA origin in this non-outbreak setting was mainly 'endogenous' and PA-strains were generally patient- and site-specific, especially in the gastrointestinal tract. While prediction of ventilator associated pneumonia-related PA-strain by screening was fair, the negative predictive value of screening was very high.

Peripartum maternal transmission of extended-spectrum ß-lactamase organism to newborn infants.

The aim of this study was to determine whether the route of extended-spectrum ß-lactamase (ESBL) transmission to hospitalized newborns was from their mothers during delivery. Neonatal intensive care unit (NICU) hospitalized newborns were sampled for ESBL presence by stool cultures on the first and fourth days of life. Mothers of ESBL-positive newborns were sampled for possible correlation detection. Bacteria isolates were molecularly identified and susceptibility tests for antibiotic agents were performed. Of the 225 newborns, 14 (6.2%) were ESBL positive, 10 (4.4%) were Escherichia coli positive, and 4 (1.7%) were Klebsiella pneumoniae positive. Among the 14 mothers of positive newborns, 13 (92.8%) were found ESBL positive and one mother of a newborn with E. coli carriage was found ESBL negative. Genes encoding for ESBL resistance were identified. Antibiotic sensitivity and resistance were tested. This study demonstrated that ESBL bacteria carrier neonates hospitalized in NICU may be a result of transmission from mother to baby during delivery.

Dissemination of the blaKPC gene by clonal spread and horizontal gene transfer: comparative study of incidence and molecular mechanisms.

OBJECTIVES: In addition to the global spread of the KPC-producing Klebsiella pneumoniae (KPC-KP) clonal complex (CC)-258 clone, the blaKPC gene may also spread by horizontal gene transfer (HGT), as suspected when more than one KPC-producing Enterobacteriaceae (KPC-Ent) species are isolated in a single patient. We aimed to characterize the incidence and molecular features of KPC-KP that were isolated alone (singular KPC-KP) versus KPC-KP that were isolated together with another KPC-Ent species (joint KPC-KP). METHODS: Isolates were collected from April 2011 to August 2012 at the Laniado Medical Center. Typing was done by CC-258 multiplex PCR and MLST. Plasmids were characterized by plasmid MLST (pMLST). The genetic environment of the blaKPC gene was studied by sequencing. RESULTS: During the 17 month period, there were 281 cases of singular KPC-KP and 8 cases of joint KPC-KP (P < 0.0001). Among the patients with joint KPC-KP, the additional KPC-Ent species were Escherichia coli (n = 6), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 1) and Citrobacter freundii (n = 1). All singular KPC-KP isolates tested (n = 27) belonged to the CC-258 clone and carried the blaKPC-3 allele, located inside a Tn4401a transposon. In contrast, joint KPC-KP/KPC-Ent isolates belonged to different STs and all but one carried the blaKPC-2 allele. The blaKPC-2 gene was located inside ΔTn4401c transposons that were harboured by IncN/pMLST ST-15-type plasmids possessing high conjugation efficiency. CONCLUSIONS: This study highlights two dissemination modes of the blaKPC gene: clonal spread of the CC-258 clone and, far less commonly, HGT-related spread, mediated by ST-15 plasmids that shuttle between a variety of species and clones.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone.

The Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non-ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.