The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 080015-1329341-028710US_ST25.txt, was created on Jan. 6, 2023 and is 187,901 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
Highly drug-resistant enteric bacteria, including carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE), are emerging worldwide1. In the United States, CRE infections have been confirmed in 48 states and are estimated at 2.93 per 100,000 persons with almost 10,000 infections per year2. Enterobacteriaceae causes 30% of healthcare-associated infections. Although they are susceptible to carbapenems3,4,5, the emergence of bacteria with new β-lactamase enzymes with direct carbapenem-hydrolyzing activity has contributed to an increased prevalence of CRE in the past decade6. Some CRE bacteria have become resistant to most available antibiotics and patients carrying these bacteria are at a high risk of severe infections and a high mortality rate5. Currently, decolonization strategies are lacking, and targeted selective digestive decontamination resulted in short-term benefits and increased risks of resistance to the antibiotics used7.
Fecal microbiota transplantation (FMT) is highly effective in the treatment of recurrent Clostridioides difficile infections (CDI)8, and has recently emerged as a promising therapy for decolonization of intestinal multi-drug resistant microorganisms9. In four case series with varying study protocols, FMT resulted in 33-50% of decolonization in CRE infections10-13. However, the fate of native and introduced microbes and which species are enriched or cleared after FMT in CRE recipients remain unclear11. Apart from the bacterial community, accumulating evidence showed that gut fungal (fungome) and viral microbiome (virome) which consists of eukaryotic RNA and DNA viruses and bacteriophage are also associated with FMT treatment outcome in CDI14. To date, there are limited data on how FMT affects CRE carriage in relation to the gut microbiome after FMT.
FMT can restore the gut microbial ecology, and has proven to be a breakthrough for the treatment of recurrent CDI. Furthermore, clinical trials are being conducted to evaluate its use for other conditions including treating multi-drug resistant microorganisms. There is accumulating evidence showing that the gut microbiota plays an important role in the control of intestinal colonization and infection by pathogenic bacteria. In addition, as bacteriophages propagate via exclusively lytic or lysogenic infection of bacteria, bacteriophage has the potential for eradicating multi-drug resistant microorganisms.
In one aspect, the disclosure features a method for identifying a donor subject for fecal microbiota trans-plantation (FMT), comprising: (a) analyzing a fecal sample obtained from a candidate subject to detect the presence of one or more predetermined species of bacteriophages in the fecal sample; and (b) determining the candidate subject as a donor subject when the presence of the one or more predetermined species of bacteriophages is detected in the fecal sample. In some embodiments, the method further comprises step (c) administering a fecal material obtained from the donor subject to a subject in need of FMT.
In some embodiments of this aspect, the subject in need of FMT has a bacterial infection, for example, a recurring or an antibiotic-resistant bacterial infection.
In another aspect, the disclosure features a method for treating or preventing a bacterial infection in a subject in need of FMT, comprising: (a) analyzing a fecal sample obtained from an individual as a proposed donor to detect the presence of one or more predetermined species of bacteriophages in the fecal sample, upon confirmation of the presence, especially at a desirable level (e.g., above an average level), of the one or more predetermined species of bacteriophages in the fecal sample the individual is chosen as FMT donor; and (b) administering to the subject in need of FMT a processed fecal sample from the donor containing the predetermined species of bacteriophages in an effective amount.
In some embodiments, the bacterial infection is an antibiotic resistant bacterial infection. In some embodiments, the bacterial infection is caused by bacteria in the family Enterobacteriaceae. In some embodiments, the bacterial infec-tion is caused by bacteria in the genus Enterococcus, Klebsiella, or Escherichia. In some embodiments, the bacterial infection is caused by carbapenem-resistant Enterobacteriaceae (CRE). In some embodiments, the bacterial infection is caused by vancomycin-resistant Enterococci (VRE). In some embodiments, the bacteria infection is caused by Klebsiella pneumonia, Klebsiella variicola, or Escherichia coli.
In some embodiments, the bacteriophage is selected from the group consisting of Klebsiella phage KP34 (NCBI:txid674081), genus KP32virus (NCBI: txid1985720), genus Kp36virus (NCBI: txid1920860), Klebsiella virus Kp15 (NCBI:txid1985328), and Klebsiella phage KP27 (NCBI:txid1129147), or from the group consisting of Klebsiella phage vB_Kpn_IME260 (NCBI: taxid 1912318), Klebsiella phage vB_KpnM_KB57 (NCBI: taxid 1719140), Klebsiella phage vB_KpnM_KpV52 (NCBI: taxid 1912321), Klebsiella virus 0507KN21 (NCBI: taxid 2169687), Klebsiella phage F19 (NCBI: taxid 1416011), Klebsiella phage K5 (NCBI: taxid 1647374), Klebsiella phage Matisse (NCBI: taxid 1675607), Klebsiella phage Sugarland (NCBI: taxid 2053603), Klebsiella phage PKP126 (NCBI: taxid 1654927), Klebsiella phage K64-1 (NCBI: taxid 1439894), Klebsiella phage KpV71 (NCBI: taxid 1796998), and Klebsiella phage Matisse (NCBI: taxid 1912318).
In some embodiments, the bacteriophage in the genus KP32virus (NCBI: txid1985720) is selected from the group consisting of Klebsiella phage K5 (NCBI:txid1647374), Klebsiella phage K11 (NCBI:txid532077), Klebsiella phage vB Kp1 (NCBI:txid1701804), Klebsiella phage KP32 (NCBI:txid674082), and Klebsiella phage vB KpnP_KpV289(NCBI:txid1671396).
In some embodiments, the bacterial infection is caused by carbapenem-resistant Klebsiella pneumonia, and the bacteriophage is selected from the group consisting of Klebsiella phage KP34 (NCBI:txid674081), genus KP32virus (NCBI: txid1985720), and genus Kp36virus (NCBI:txid1920860).
In some embodiments, the bacteriophage comprises a genome comprising a nucleic acid sequence of any one of SEQ ID NOS: 1-324 and 333-335, or any one in List 6 or 7.
In some embodiments, the bacterial infection is caused by carbapenem-resistant Klebsiella variicola, and the bacteriophage is selected from the group consisting of Klebsiella virus Kp15 (NCBI:txid1985328) and Klebsiella phage KP27 (NCBI:txid1129147).
In some embodiments, the bacteriophage comprises a genome comprising a nucleic acid sequence of any one of SEQ ID NOS:325-332.
In some embodiments, the bacterial infection is caused by carbapenem-resistant Escherichia coli, and the bacteriophage comprises a genome comprising a nucleic acid sequence of any one of SEQ ID NOS:336-384.
In some embodiments, the methods further comprise, prior to step (a), the step of obtaining the fecal sample from a candidate subject. In some embodiments, the candidate subject previously had the same bacterial infection as the subject in need of FMT and is now cured. In some embodiments, the donor subject is cured by fecal microbiota transplantation (FMT). In some embodiments, the fecal sample comprises a bacteriophage selected from the group consisting of Klebsiella phage KP34 (NCBI:txid674081), genus KP32virus (NCBI: txid1985720), and genus Kp36virus (NCBI: txid1920860). In some embodiments, the fecal sample comprises a bacteriophage comprising a sequence of any one of SEQ ID NOS: 1-324 and 333-335.
In some embodiments, the fecal sample comprises a bacteriophage selected from the group consisting of Klebsiella virus Kp15 (NCBI:txid1985328) and Klebsiella phage KP27 (NCBI:txid1129147). In some embodiments, the fecal sample comprises a bacteriophage comprising a sequence of any one of SEQ ID NOS:325-332.
In some embodiments of the methods described herein, the fecal sample is obtained from a stool bank.
In some embodiments of the methods described herein, the methods further comprise identifying the bacteria causing the bacterial infection in the subject in need of FMT.
In some embodiments of the methods described herein, the fecal material or the processed fecal sample is administered to the small intestine, the ileum, and/or the large intestine of the subject in need of FMT. In other embodiments, the fecal material or the processed fecal sample is administered via direct transfer to the GI track. In other embodiments, the fecal material or the processed fecal sample is formulated for oral administration. In other embodiments, the fecal material or the processed fecal sample is administered before food intake or together with food intake. In further embodiments, the subject in need of FMT is further administered an antibiotic.
The invention provides methods for treating or preventing a bacterial infection in a subject in need of FMT by administering to the subject a processed fecal sample that contains the bacteriophages that inhibit the bacteria causing the bacterial infection in the subject. The processed fecal sample can first be obtained from a donor subject, analyzed for its bacteriophage content, and then processed to be ready for administration. During their studies, the present inventors performed a longitudinally and in-depth metagenomics analysis of the gut bacteriome, fungome, and virome in CRE-positive patients who successfully decolonized CRE after FMT. As described herein, the bacteria-bacteriophage correlation before and after FMT and its association with treatment outcome were explored. The inventors discovered that the bacteriophage used in the treatment showed a negative correlations between the bacteriophage and bacteria that caused infection. The determination and analysis of the species of bacteriophages in a potential donor’s fecal sample thus can be used to guide donor selection.
As used herein, the term “fecal microbiota transplantation (FMT)” refers to a medical procedure during which fecal matter containing live fecal microorganisms (bacteria, fungi, and the like) obtained from a healthy individual is transferred into the gastrointestinal tract of a recipient to restore healthy gut microflora that has been disrupted or destroyed by a variety of medical conditions. Typically, the fecal matter from a healthy donor is first processed into an appropriate form for the transplantation, which can be made through direct deposit into the lower gastrointestinal tract such as by colonoscopy, or by nasal intubation, or through oral ingestion of an encapsulated material containing dried and frozen fecal matter. Clostridium difficile infection (CDI) is the condition most commonly treated by FMT, although a number of other diseases and disorders including in the digestive system and in the nervous system have been reported to be successfully treated by FMT.
As used herein, the term “antibacterial” refers to a molecule or agent that is destructive to or inhibits the growth of bacteria.
As used herein, the term “bacteriophage” or “phage” refers to a bacteriophage isolate in which members of the isolate has substantially the same genetic makeup, such as sharing at least about any of 90%, 95%, 99%, 99.9% or more sequence identity in the genome. “Bacteriophage” or “phage” refers to the parent bacteriophage as well as the progeny or derivatives (such as genetically engineered versions) thereof. The bacteriophage can be a naturally occurring phage isolate, or a synthetic or engineered phage, including vectors, or nucleic acids that encode at least all essential genes, or the full genome of a phage to carry out the life cycle of the phage inside a host bacterium.
As used herein, a bacteriophage “targeting” or “targets” a bacterium means that the bacteriophage can infect the bacterium, and inhibit the growth of the bacterium. The bacteriophage can be either a lysogenic bacteriophage of the bacterium, or a lytic bacteriophage of the bacterium.
As used herein, the term “inhibiting” or “inhibition” refers to any detectable negative effect on a target biological process, such as RNA/protein expression of a target gene, the biological activity of a target protein, cellular signal transduction, cell proliferation, and the like. Typically, an inhibition is reflected in a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater in the target process (e.g., growth or proliferation of bacterial cells), or any one of the downstream parameters mentioned above, when compared to a control. “Inhibition” further includes a 100% reduction, i.e., a complete elimination, prevention, or abolition of a target biological process or signal. The other relative terms such as “suppressing,” “suppression,” “reducing,” and “reduction” are used in a similar fashion in this disclosure to refer to decreases to different levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater decrease compared to a control level) up to complete elimination of a target biological process or signal. On the other hand, terms such as “activate,” “activating,” “activation,” “increase,” “increasing,” “promote,” “promoting,” “enhance,” “enhancing,” or “enhancement” are used in this disclosure to encompass positive changes at different levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or greater such as 3, 5, 8, 10, 20-fold increase compared to a control level) in a target process or signal.
As used herein, the term “treatment” or “treating” refers to an approach for obtaining beneficial or desired results including clinical results. The beneficial or desired clinical results can include, but are not limited to, alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, and decreasing the dose of one or more other medications required to treat the disease.
As used herein, the term “prevent” or “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
The term “effective amount,” as used herein, refers to an amount that is sufficient to produces an intended effect for which a substance is administered. The effect may include a desirable change in a biological process as well as the prevention, correction, or inhibition of progression of the symptoms of a disease/condition and related complications (e.g., suppressed or prevented bacterial infection) to any detectable extent. The exact amount “effective” for achieving a desired effect will depend on the nature of the therapeutic agent, the manner of administration, and the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
As used herein, the term “subject” refers to an animal, including, but not limited to, a cow, a goat, a sheep, a buffalo, a camel, a donkey, a llama, a horse, a pig, a human, a primate, an avian, a fish, a mule, a cat and a dog. In some embodiments, the subject is a human.
As used herein, the term “about” denotes a range of value that is +/- 10% of a specified value. For instance, “about 10” denotes the value range of 10 +/- 10 x 10%, i.e., 9 to 11.
A fecal matter containing bacteriophages can be administered to a subject having a bacterial infection or at risk of having a bacterial infection. The fecal matters can be obtained from a donor subject or from a stool bank. The fecal matter can be processed into appropriate forms for the intended means of delivery in an FMT procedure. An FMT donor can be a healthy individual without any known diseases or disorders especially in the digestive tract, although some preference is often given to the members of the same household as the recipient. In some embodiments, a fecal matter can comprise one type of bacteriophages or can comprise two or more (e.g., three, four, five, six, seven, eight, nine, or ten) different types of bacteriophages.
Examples of bacteriophages include, but are not limited to, Przondovirus (NCBI:txid1985720), Webervirus (NCBI:txid1920860), Slopekvirus (NCBI:txid1985328), Klebsiella phage KP27 (NCBI:txid1129147), Klebsiella phage K11 (NCBI:txid532077), Klebsiella phage K5 (NCBI:txid1647374), Klebsiella phage vB Kp1 (NCBI:txid1701804), Klebsiella phage KP32 (NCBI:txid674082), Klebsiella phage vB KpnP KpV289 (NCBI:txid1671396), Klebsiella phage F19 (NCBI:txid1416011), Klebsiella phage NTUH-K2044-K1-1 (NCBI:txid1194091), Klebsiella phage Kp2 (NCBI:txid1701805), Klebsiella phage KP34 (NCBI:txid674081), Klebsiella phage KpV41 (NCBI:txid1747282), Klebsiella phage KpV475 (NCBI:txid1852657), Klebsiella phage KpV71 (NCBI:txid1796998), Klebsiella phage vB KpnP SU503 (NCBI:txid1610834), Klebsiella phage vB_KpnP_SU552A (NCBI:txid1610835), Klebsiella phage KLPN1 (NCBI:txid1647408), Klebsiella phage Kp36 (NCBI:txid1129191), Escherichia virus 186 (NCBI:txid29252), Escherichia virus HK97 (NCBI:txid37554), Escherichia phage HK633 (NCBI:txid1147147), Escherichia virus P1 (NCBI:txid10678), Escherichia phage mEpX2 (NCBI:txid1147154), Escherichia phage TL-2011b (NCBI:txid1124654), Escherichia phage HK75 (NCBI:txid906668), Escherichia phage K30 (NCBI:txid1041524), Escherichia phage HK446 (NCBI:txid1147145), Escherichia virus HK022 (NCBI:txid10742), Escherichia phage HK629 (NCBI:txid1147148), Enterobacteria phage HK106 (NCBI:txid432198), Escherichia phage phiV10 (NCBI:txid343516), Escherichia virus P2 (NCBI:txid10679), Escherichia phage mEp234 (NCBI:txid1147157), Escherichia phage HK544 (NCBI:txid432201), Escherichia phage pro483 (NCBI:txid1649240), Escherichia phage HK542 (NCBI:txid432200), Escherichia phage Pollock (NCBI:txid1540097), Escherichia virus Lambda (NCBI:txid10710), Escherichia phage pro147 (NCBI:txid1649239), Escherichia phage Av-05 (NCBI:txid1527519), Escherichia virus Wphi (NCBI:txid103216), Escherichia phage HK639 (NCBI:txid906669), Escherichia virus Mu (NCBI:txid10677), Escherichia phage mEpX1 (NCBI:txid1147153), Escherichia phage 64795 ec1 (NCBI:txid1837842), Enterobacteria phage If1 (NCBI:txid10868), Enterobacteria phage Bp7 (NCBI:txid1052121), Enterobacteria phage RB69 (NCBI:txid12353), Escherichia virus N4 (NCBI:txid10752), Escherichia virus N15 (NCBI:txid40631), Escherichia phage FFH2 (NCBI:txid1446490), Escherichia phage phAPEC8 (NCBI:txid1229753), Escherichia phage ECBP5 (NCBI:txid1498172), Escherichia phage SUSP2 (NCBI:txid1718669), Escherichia phage 121Q (NCBI:txid1555202), Escherichia phage wV8 (NCBI:txid576791), Escherichia phage QL01 (NCBI:txid1673871), Escherichia phage V5 (NCBI:txid399183), Escherichia Stxl converting phage (NCBI:txid194948), Escherichia phage AR1 (NCBI:txid66711), Escherichia phage JSE (NCBI:txid576789), Enterobacter phage CC31 (NCBI:txid709484), Escherichia virus VR20 (NCBI:txid1913684), Escherichia virus VR26 (NCBI:txid1913686), Escherichia virus SU10 (NCBI:txid1987942), Escherichia virus K1H (NCBI:txid1911010), Escherichia virus ECB2 (NCBI:txid1987939) and Escherichia phage WG01 (NCBI:txid1837931).
In some embodiments, the bacteriophages target bacteria in the genus Klebsiella, such as Klebsiellapneumonia (e.g., carbapenem-resistant Klebsiellapneumonia). Examples of such bacteriophages include, but are not limited to, Webervirus (NCBI:txid1920860), Drulisvirus (NCBI:txid1920774), Przondovirus (NCBI:txid1985720), Klebsiella phage KLPN1 (NCBI:txid1647408), Klebsiella phage KpV71 (NCBI:txid1796998), Klebsiella phage vB_KpnP_SU552A (NCBI:txid1610835), Klebsiella phage NTUH-K2044-K1-1 (NCBI:txid1194091), Klebsiella phage KpV41 (NCBI:txid1747282), Klebsiella phage KP34 (NCBI:txid674081), Klebsiella phage F19 (NCBI:txid1416011), Klebsiella phage Kp2 (NCBI:txid1701805), Klebsiella phage KpV475 (NCBI:txid1852657) and Klebsiella phage vB_Kp1 (NCBI:txid1701804). The bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella pneumonia (e.g., carbapenem-resistant Klebsiella pneumonia) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS:1-324 and 333-335. In other embodiments, the bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella pneumonia can be any of the species listed in Table 1 below.
Klebsiella phage KLPN1
Klebsiella pneumoniae
Klebsiella phage vB Kp1
Klebsiella pneumoniae
Klebsiella phage K11
Klebsiella pneumoniae
Klebsiella phage KP32
Klebsiella pneumoniae
Klebsiella phage vB KpnP KpV289
Klebsiella pneumoniae
Klebsiella phage K5
Klebsiella pneumoniae
Klebsiella phage KpV71
Klebsiella pneumoniae
Klebsiella phage vB KpnP SU552A
Klebsiella pneumoniae
Klebsiella phage NTUH-K2044-K1-1
Klebsiella pneumoniae
Klebsiella phage KpV41
Klebsiella pneumoniae
Klebsiella phage KP34
Klebsiella pneumoniae
Klebsiella phage F19
Klebsiella pneumoniae
Klebsiella phage Kp2
Klebsiella pneumoniae
Klebsiella phage KpV475
Klebsiella pneumoniae
Klebsiella phage vB KpnP SU503
Klebsiella pneumoniae
In other embodiments, the bacteriophages target bacteria in the genus Klebsiella, such as Klebsiella variicola (e.g., carbapenem-resistant Klebsiella variicola). Examples of such bacteriophage include, but are not limited to, Slopekvirus (NCBI:txid1985328), Klebsiella phage KP27 (NCBI:txid1129147). The bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella variicola (e.g., carbapenem-resistant Klebsiella variicola) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS:325-332. In other embodiments, the bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella variicola can be species Klebsiella phage KP27 (NCBI:txid1129147).
In yet other embodiments, the bacteriophages target bacteria in the genus Escherichia, such as Escherichia coli (e.g., carbapenem-resistant Escherichia coli). Examples of such bacteriophage include, but are not limited to, Escherichia virus 186 (NCBI:txid29252), Escherichia virus HK97 (NCBI:txid37554), Escherichia phage HK633 (NCBI:txid1147147), Escherichia virus P1 (NCBI:txid10678), Escherichia phage mEpX2 (NCBI:txid1147154), Escherichia phage TL-2011b (NCBI:txid1124654), Escherichia phage HK75 (NCBI:txid906668), Escherichia phage K30 (NCBI:txid1041524), Escherichia phage HK446 (NCBI:txid1147145), Escherichia virus HK022 (NCBI:txid10742), Escherichia phage HK629 (NCBI:txid1147148), Enterobacteria phage HK106 (NCBI:txid432198), Escherichia phage phiV10 (NCBI:txid343516), Escherichia virus P2 (NCBI:txid10679), Escherichia phage mEp234 (NCBI:txid1147157), Escherichia phage HK544 (NCBI:txid432201), Escherichia phage pro483 (NCBI:txid1649240), Escherichia phage HK542 (NCBI:txid432200), Escherichia phage Pollock (NCBI:txid1540097), Escherichia virus Lambda (NCBI:txid10710), Escherichia phage pro147 (NCBI:txid1649239), Escherichia phage Av-05 (NCBI:txid1527519), Escherichia virus Wphi (NCBI:txid103216), Escherichia phage HK639 (NCBI:txid906669), Escherichia virus Mu (NCBI:txid10677), Escherichia phage mEpX1 (NCBI:txid1147153), Escherichia phage 64795_ ecl (NCBI:txid1837842), Enterobacteria phage If1 (NCBI:txid10868), Enterobacteria phage Bp7 (NCBI:txid1052121), Enterobacteria phage RB69 (NCBI:txid12353), Escherichia virus N4 (NCBI:txid10752), Escherichia virus N15 (NCBI:txid40631), Escherichia phage FFH2 (NCBI:txid1446490), Escherichia phage phAPEC8 (NCBI:txid1229753), Escherichia phage ECBP5 (NCBI:txid1498172), Escherichia phage SUSP2 (NCBI:txid1718669), Escherichia phage 121Q (NCBI:txid1555202), Escherichia phage wV8 (NCBI:txid576791), Escherichia phage QL01 (NCBI:txidI673871), Escherichia phage V5 (NCBI:txid399183), Escherichia Stxl converting phage (NCBI:txid194948), Escherichia phage AR1 (NCBI:txid66711), Escherichia phage JSE (NCBI:txid576789), Enterobacter phage CC31 (NCBI:txid709484), Escherichia virus VR20 (NCBI:txid1913684), Escherichia virus VR26 (NCBI:txid1913686), Escherichia virus SU10 (NCBI:txid1987942), Escherichia virus K1H (NCBI:txid1911010), Escherichia virus ECB2 (NCBI:txid1987939), and Escherichia phage WG01 (NCBI:txid1837931). The bacteriophages that target bacteria in the genus Escherichia, such as Escherichia coli (e.g., carbapenem-resistant Escherichia coli) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS:336-384. In other embodiments, the bacteriophages that target bacteria in the genus Escherichia, such as Escherichia coli can be any of the species listed in Table 2 below.
Escherichia virus 186
Escherichia coli
Escherichia virus HK97
Escherichia coli
Escherichia phage HK633
Escherichia coli
Escherichia virus P1
Escherichia coli
Escherichia phage mEpX2
Escherichia coli
Escherichia phage TL-2011b
Escherichia coli
Escherichia phage HK75
Escherichia coli
Escherichia phage K30
Escherichia coli
Escherichia phage HK446
Escherichia coli
Escherichia virus HK022
Escherichia coli
Escherichia phage HK629
Escherichia coli
Enterobacteria phage HK106
Escherichia coli
Escherichia phage phiV10
Escherichia coli
Escherichia virus P2
Escherichia coli
Escherichia phage mEp234
Escherichia coli
Escherichia phage HK544
Escherichia coli
Escherichia phage pro483
Escherichia coli
Escherichia phage HK542
Escherichia coli
Escherichia phage Pollock
Escherichia coli
Escherichia virus Lambda
Escherichia coli
Escherichia phage pro147
Escherichia coli
Escherichia phage Av-05
Escherichia coli
Escherichia virus Wphi
Escherichia coli
Escherichia phage HK639
Escherichia coli
Escherichia virus Mu
Escherichia coli
Escherichia phage mEpX1
Escherichia coli
Escherichia phage 64795 ec1
Escherichia coli
Enterobacteria phage If1
Escherichia coli
Enterobacteria phage Bp7
Escherichia coli
Enterobacteria phage RB69
Escherichia coli
Escherichia virus N4
Escherichia coli
Escherichia virus N15
Escherichia coli
Escherichia phage FFH2
Escherichia coli
Escherichia phage phAPEC8
Escherichia coli
Escherichia phage ECBP5
Escherichia coli
Escherichia phage SUSP2
Escherichia coli
Escherichia phage 121Q
Escherichia coli
Escherichia phage wV8
Escherichia coli
Escherichia phage QL01
Escherichia coli
Escherichia phage V5
Escherichia coli
Escherichia Stx1 converting phage
Escherichia coli
Escherichia phage AR1
Escherichia coli
Escherichia phage JSE
Escherichia coli
Enterobacter phage CC31
Escherichia coli
Escherichia virus VR20
Escherichia coli
Escherichia virus VR26
Escherichia coli
Escherichia virus SU10
Escherichia coli
Escherichia virus K1H
Escherichia coli
Escherichia virus ECB2
Escherichia coli
Escherichia phage WG01
Escherichia coli
As described in the Examples section, the bacteriophage used in the treatment should show a negative correlations (r value less than 0) between the bacteriophage and bacteria that caused infection (see, e.g.,
The bacteriophages described herein can be lytic or lysogenic. A lytic phage has the ability to lyse out of the bacterial host cell following phage replication, and the phage progeny is able to infect new bacterial host cells. A lysogenic phage, in contrast, integrates its viral genome with the host DNA, replicating along with the host’s DNA. The lysogenic phage then undergoes replication resulting in lysis of the host cell releasing phage.
A fecal sample obtained from a donor subject can be processed to obtain a processed fecal sample that is in an appropriate form for the intended means of delivery in an FMT procedure. For example, a fecal sample for treating a Klepsiella infection can be processed as described below. Fecal samples are incubated overnight at 37° C. in LB Broth and then centrifuged at 5,500 g. The supernatant is filtered through 0.22 µm. The solution is then mixed with 2.5 mL of the host bacteria, Klebsiella (at exponential phage) and added to 10 mL LB broth for overnight incubation at 37° C. The mixture is then screened for the presence of phage by the Double-Layer Agar (DLA) method. Supernatants with positive phages are purified by picking a single plaque with a sterile pasteur pipette tip, resuspending the plaque in 1 mL LB broth, incubating for 1 h at 37° C., tittering, and plating by the DLA method.
In other examples, a composition to administer to a recipient can contain synthetic bacteriophages. Synthetic bacteriophages can be made, for example by creating functional phage particles from phage genomes modified in vitro, with transformation as the means of getting phage genomic DNA back into the host bacterium, where phage particles are produced from the genomic DNA. Recombinant DNA (rDNA) technology refers to the process of joining DNA molecules from two different sources and inserting them into a host organism, to generate products for human use. Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice, the process often involves combining the DNA of different organisms (e.g., bacteria and phages). Though molecular cloning technologies, a promoter can also be operatively linked to the nucleic acid of a bacteriophage. Further, other components that can promote the expression and/or activity of the bacteriophage can also be linked to the nucleic acid of the bacteriophage, e.g., a nucleic acid encoding an antimicrobial polypeptide.
For example, the following steps can be followed when generating artificial bacteriophages: isolation of genetic material via restriction enzyme digestion; amplification using PCR; ligation of DNA molecules to create recombinant DNA that is within a plasmid vector; and insertion of recombinant DNA into host cell by transformation of competent bacterial cells. The plasmid vector is now able to replicate because plasmids normally have a replication origin. However, now that the DNA insert is part of the vector’s length, the DNA is automatically replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell.
In some embodiments, the amount of the beneficial bacteriophages in the processed fecal sample to be administered to the subject in need is expressed as a percentage over the total level of all bacteriophage species in the sample. In some embodiments, the amount of the beneficial bacteriophages is determined as greater than 10% (e.g., greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of total bacteriophages in the processed fecal sample. In some cases, the potential recipient is then immediately given FMT, without any further treatment or preparation such as administration of an antibiotic in the effective amount. In some embodiments, when the amount of beneficial bacteriophage is no greater than 5% (e.g., 4%, 3%, 2%, or 1%) of total bacteriophages in the processed sample, FMT is assessed as unlikely to be effective for the potential recipient. In some embodiments, amount of bacteriophages is determined by quantitative polymerase chain reaction (PCR). In some embodiments, the levels of all bacteriophage species present in the sample is determined by the Internal transcribed spacer 2 (ITS2) sequencing.
In some embodiments, the processed fecal sample comprises at least about any one of 104, 105, 106, 107, 108, 109, 1010, 1011, or 1012 PFU/mL of each bacteriophage. The concentration of bacteriophage can be determined using known phage titration protocols. In some embodiments, the processed fecal sample comprises an effective amount of the bacteriophages. The concentration of bacteriophage varies depending upon the carrier and method of administration. For a processed fecal sample that comprises two or more different types of bacteriophages, the relative ratio by PFU between different bacteriophages in the processed fecal sample can be chosen to optimize the efficacy of the processed fecal sample or to enhance synergy among the different bacteriophages. In some embodiments, each bacteriophage is present at about equal PFU in the processed fecal sample. In some embodiments, one bacteriophage is present at about any one of 1.5, 2, 3, 4, 5, 10 or more PFU than another bacteriophage in the processed fecal sample.
One or more antibiotics can be added to the processed fecal sample. Examples of antibiotics include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, torezolid, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temocillin, ticarcillin, amoxicillin clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (tmp-smx), sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol(bs), ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, and trimethoprim.
The fecal sample obtained from the donor subject can be processed, formulated, and packaged to be in an appropriate form in accordance with the delivery means in the FMT procedure, which may be by direct deposit in the recipient’s lower gastrointestinal track (e.g., wet or semi-wet form) or by oral ingestion (e.g., frozen dried encapsulated). In some embodiments, the processed fecal sample can be formulated for FMT by direct transfer to the GI tract (e.g., via colonoscopy or via nasal intubation). In some embodiments, the processed fecal sample can be formulated for FMT by rectal deposit.
In some embodiments, the processed fecal sample comprising bacteriophages can be stored as an aqueous solution or lyophilized powder preparation. A delivery vehicle is suitable for the route of delivery or administration. In some embodiments, the delivery vehicle is suitable for oral administration. In some embodiments, the delivery vehicle is suitable for direct transfer to the GI track. In some embodiments, the delivery vehicle further stabilizes the bacteriophages, and/or enhances the efficacy of the bacteriophages on inhibiting bacterial infection.
In some embodiments, the delivery vehicle is a buffer, such as phosphate buffered saline (PBS), Luria-Bertani Broth, phage buffer (100 mM NaCl, 100 mM Tris-HCl, 0.01% (w/v) Gelatin), or Tryptic Soy broth (TSB). In some embodiments, the delivery vehicle comprises food grade oils, and inorganic salts useful for adjusting the viscosity of the bacteriophage composition. Examples of pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985). Suitable pharmaceutical carriers include, but are not limited to, sterile water; saline, dextrose; dextrose in water or saline; condensation products of castor oil and ethylene oxide combining about 30 to about 35 moles of ethylene oxide per mole of castor oil; liquid acid; lower alkanols; oils such as corn oil; peanut oil, sesame oil and the like, with emulsifiers such as mono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin, and the like; glycols; polyalkylene glycols; aqueous media in the presence of a suspending agent, for example, sodium carboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and the like, alone, or with suitable dispensing agents such as lecithin; polyoxyethylene stearate; and the like. The carrier may also contain adjuvants such as preserving stabilizing, wetting, emulsifying agents and the like together with the penetration enhancer. The final form may be sterile and may also be able to pass readily through an injection device such as a hollow needle. The proper viscosity may be achieved and maintained by the proper choice of solvents or excipients.
In some embodiments, the delivery vehicle comprises other agents, excipients, or stabilizers to improve properties of the composition, which do not reduce the effectiveness of the bacteriophage. Examples of suitable excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. Examples of emulsifying agents include tocopherol esters such as tocopheryl polyethylene glycol succinate and the like, PLURONIC®, emulsifiers based on polyoxy ethylene compounds, Span 80 and related compounds and other emulsifiers known in the art and approved for use in animals or human dosage forms. The compositions (such as pharmaceutical compositions) can be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to an individual by employing procedures well known in the art.
In some embodiments, the processed fecal sample comprises a delivery vehicle suitable for oral administration. In some embodiments, the delivery vehicle is an aqueous medium, such as deionized water, mineral water, 5% sucrose solution, glycerol, dextran, polyethylene glycol, sorbitol, or such other formulations that maintain phage viability, and are non-toxic to animals, including lactating mammals and humans. In some embodiments, the composition is prepared by resuspending purified phage preparation in the aqueous medium.
The disclosure provides methods of treating or preventing a bacterial infection in a subject in need of FMT, comprising: (a) analyzing the fecal sample obtained from a potential donor to determine the presence and/or relative quantity of one or more species of the pertinent bacteriophages in the fecal sample, thereby determining whether potential donor can properly serve as a donor to provide fecal material advantageous in FMT; (b) processing the fecal sample that has been deemed suitable for FMT into a processed fecal sample; and (c) administering to the subject in need of FMT the processed fecal sample. A fecal sample from the subject in need of FMT can be analyzed to find the species of bacteria causing the infection, which can help to determine the species of bacteriophage needed in a fecal sample obtained from a donor subject. A fecal sample from a donor subject can be analyzed to find if the sample contains the predetermined species of bacteriophage.
As described herein, the bacteriophages in the processed fecal sample should target the bacteria that caused the bacterial infection in the subject in need. One or more methods available in the art can be used to analyze and determine the species of bacteriophage present in the fecal sample. For example, as described herein, megagenomics sequencing using PCR can be applied to determine the species of bacteriophage present in the fecal sample. The methods described herein can further comprise the step of determining the bacteria that caused the infection in the subject. For example, before the subject undergoes FMT, a stool sample can be obtained from the subject and analyzed for the bacteria that caused the infection. Once the bacteria is determined, the appropriate bacteriophage that targets the bacteria can be chosen, and a fecal sample from a donor subject containing the appropriate bacteriophage can be selected. In some embodiments, the processed fecal sample can be administered via direct transfer to the GI track. In other embodiments, the processed fecal sample can be administered orally, i.e., before food intake or together with food intake.
In one example, the bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella pneumonia (e.g., carbapenem-resistant Klebsiella pneumonia) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS: 1-324 and 333-335. In another example, the bacteriophages that target bacteria in the genus Klebsiella, such as Klebsiella variicola (e.g., carbapenem-resistant Klebsiella variicola) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS:325-332. In yet another example, the bacteriophages that target bacteria in the genus Escherichia, such as Escherichia coli (e.g., carbapenem-resistant Escherichia coli) can comprise a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a sequence of any one of SEQ ID NOS:336-384. In other embodiments, the bacteriophage that targets bacteria is selected from the group consisting of Klebsiella phage vB_Kpn_IME260 (NCBI: taxid 1912318), Klebsiella phage vB_KpnM_KB57 (NCBI: taxid 1719140), Klebsiella phage vB_KpnM_KpV52 (NCBI: taxid 1912321), Klebsiella virus 0507KN21 (NCBI: taxid 2169687), Klebsiella phage F19 (NCBI: taxid 1416011), Klebsiella phage K5 (NCBI: taxid 1647374), Klebsiella phage Matisse (NCBI: taxid 1675607), Klebsiella phage Sugarland (NCBI: taxid 2053603), Klebsiella phage PKP126 (NCBI: taxid 1654927), Klebsiella phage K64-1 (NCBI: taxid 1439894), Klebsiella phage KpV71 (NCBI: taxid 1796998), and Klebsiella phage Matisse (NCBI: taxid 1912318) and can comprise a polynucleotide sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences set forth in List 6 or 7.
In the methods described herein, the fecal sample can be obtained from a donor subject. For example, the donor subject can be someone who previously had the same bacterial infection (i.e., caused by the same bacteria) and who is now cured. For example, the donor subject can be cured by FMT using a fecal sample that was obtained from another donor subject. Thus, the donor subject is likely to have the appropriate bacteriophage that targets the infection-causing bacteria in the subject in need of the bacteriophage. In other embodiments, a donor subject can simply be a healthy individual without any known diseases or disorders especially in the digestive tract. In another example, the fecal sample used in the methods can be obtained from a stool bank. A stool bank can have a variety of fecal samples obtained from donor subjects who previously had a bacterial infection and is now cured.
The methods described herein can be used to treat or prevent bacterial infections that are antibiotic resistant, for example, carbapenem-resistant Enterobacteriaceae (CRE) infections and vancomycin-resistant Enterococci (VRE) infections. A bacterial infection can be caused by bacteria in the family Enterobacteriaceae, such as bacteria in the genus Enterococcus, Klebsiella (e.g., Klebsiella pneumonia, Klebsiella variicola), or Escherichia (e.g., Escherichia coli). A bacteriophage used in methods described herein can comprise a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS: 1-384. A bacteriophage used in methods described herein can be any of the species listed in Tables 1 and 2 and species Klebsiella phage KP27 (NCBI:txid1129147) or any of those indicated in List 6 or 7. The bacteriophage can be administered to the small intestine, the ileum, and/or the large intestine of the subject in need of FMT. In some embodiments, the bacteriophage can be administered in combination with an antibiotic.
A fecal sample containing bacteriophages obtained from a donor subject can be processed and administered to a subject in need to prevent or treat a bacterial infection in the subject. In some embodiments, the fecal sample containing the bacteriophages can be processed and formulated for oral administration. For example, the subject can ingest the processed fecal sample before food intake or together with food intake. In other examples, the processed fecal sample containing the bacteriophages can be administered by direct transfer to the GI track. For example, the subject can undergo FMT where the processed fecal sample is delivered to the small intestine, the ileum, and/or the large intestine of the subject. In other embodiments, the processed fecal sample containing the bacteriophages can also be formulated for rectal administration.
The donor subject can be someone who previously had the same bacterial infection as the subject and is now cured. For example, frozen or fresh stool can be freshly prepared on the day of administration using stool from a single donor subject or using stools from a mixture of multiple donor subjects. Fecal samples can be diluted with sterile saline (0.9%). This solution can then be blended and strained with filter. The resulting supernatant can then be used directly as fresh FMT solution or stored as frozen FMT solution to be used on another day.
The processed fecal sample containing the bacteriophages can be formulated for oral delivery. The following is an example of capsulized, freeze-dried fecal microbiota. Processing is carried out under aerobic conditions. A fecal suspension is generated in normal saline without preservatives using a commercial blender. The slurry is centrifuged at 200 g for 10 minutes to remove debris. The separate fraction was centrifuged at 6,000 × g for 15 min and re-suspended to one-half (0.5 mL) the original volume in trehalose (at 5% and 10% concentrations) in saline. The supernatant is lyophilized and stored at -80° C. Commercially available acid-resistant hypromellose capsules (DRCaps, Capsugel) are used. Double-encapsulated capsules are prepared by using a filled size 0 capsule packaged inside a size 00 capsule. Capsules are manually filled using a 24-hole filler (Capsugel) to a final concentration of about 1011 cells/capsule. The capsules are stored at -80° C. in 50 mL conical tubes until needed. Once removed from the freezer, a 1 g silica gel canister (Dry Pak Industries, Encino, CA) is added to the container. Another example is a capsulized preparation of bacteriophages. The isolated phage is grown in host to make high-titer stocks by standard procedures. The high-titer phage preparations are filtered through a 0.22 µm filter. These filterates are stored at 4° C. until use. Double-encapsulated capsules are prepared by using a filled size 0 capsule packaged inside a size 00 capsule. Capsules are manually filled using a 24-hole filler (Capsugel) to a final concentration of about 3×1011 PFU/capsule.
As outlined in
Microbiome analysis was performed on the stool samples from three subjects having a CRE infection pre-FMT and after FMT. Microbiome analysis was also performed on samples collected from the FMT donor. In addition, stool samples from four healthy subjects, and three controls with spontaneous clearance of their CRE infection status were included. Approximately 100 mg fecal sample was prewashed with 1 mL ddH2O and pelleted by centrifugation at 13,000×g for 1 min. The fecal pellet was resuspended in 800 µL TE buffer (pH 7.5), supplemented with 1.6 µL 2-mercaptoethanol and 500 U lyticase (Sigma), and incubated at 37° C. for 60 min. The sample was then centrifuged at 13,000×g for 2 min and the supernatant was discarded. After pretreatment, fecal DNA was subsequently extracted from the pellet using Maxwell® RSC PureFood GMO and Authentication Kit (Promega) following manufacturer’s instructions. Briefly, 1 mL of CTAB buffer was added to the fecal pellet and vortexed for 30 seconds. Then the sample was heated at 95° C. for 5 minutes. After that, the samples were vortexed thoroughly with beads at maximum speed for 15 min. Then 40 µL of proteinase K and 20 µL of RNase A was added into the sample and the mixture was incubated at 70° C. for 10 minutes. The supernatant was then obtained by centrifuging at 13,000×g for 5 min and was added in Maxwell® RSC machine for DNA extraction. The extracted fecal DNA was subject to metagenomics sequencing.
Virus-like particles (VLPs) were enriched using protocol described in previous study14. 200 mg of stool sample was added in 400 µL saline-magnesium buffer (0.1 M NaCl, 0.002% gelatin, 0.008 M MgSO4H2O, 0.05 M Tris pH 7.5) and vortexed for 10 min. The sample then was centrifuged at 2,000xg and suspension was obtained. To remove the bacterial cells and residual host, the suspension was further filtered by one 0.45 mm and two 0.22 mm filters. The cleared suspension was incubated with lysozyme (1 mg/mL at 37° C. for 30 min) and chloroform (0.2x volume at RT for 10 min) in turn to degrade any remaining bacterial and host cell membranes. A DNase cocktail including 1 U Baseline zero DNase (Epicenter) and 10 U TubroDNaseI (Ambion) was added into the sample and the mixture was incubated at 65° C. for 10 min to eliminate non-virus protected DNA. VLPs were lysed (4% SDS plus 38 mg/mL Proteinase K at 56° C. for 20 min), treated with CTAB (2.5% CTAB plus 0.5 M NaCl at 65° C. for 10 min), and the nucleic acid was extracted with phenol:chloroform pH 8.0 (Invitrogen). The aqueous fraction was washed once with an equal volume of chloroform, purified, and concentrated on a column (DNA Clean & Concentrator™ 89-5, Zymo Research). VLP DNA was amplified for 2 h using Phi29 polymerase (GenomiPhi V2 kit, GE Healthcare) prior to sequencing. Four independent reactions were performed for each sample and pooled together to reduce amplification bias.
Qualified fecal DNA and VLP DNA was cut into fragments, the sequencing libraries were prepared through the processes of end repairing, adding A to tails, purification, and PCR amplification. The fecal DNA libraries were sequenced on an Illumina Novaseq 6000 with PE150 sequencing strategy by Novogene and yielded an average of 48 ± 5.3 million reads (12G data) per sample. The VLPs libraries were sequenced by Illumina Novaseq 6000 with PE150 sequencing strategy by Novogene and an average of 25 ± 3.3 million reads (6G data) per sample were obtained.
Raw sequence reads were filtered and quality-trimmed using Trimmomatic v0.3615 as follows: 1) trimming with a quality sliding window of 4:8; 2) cropping sequences to remove 20 bases from the start and bases beyond 220 from the end; 3) removing sequences less than 150 bp long. Then the human host contaminate reads were filtering out by Kneaddata (web site: bitbucket.org/biobakery/kneaddata/wiki/Home, reference database: GRCh38 p12) with default argument to generate clean reads.
Taxonomic profile of fungi and viruses were determined from the fecal DNA metagenomic dataset and VLP DNA metagenomic dataset respectively, using Kraken2 v2.0.7-beta. The full NCBI fungal and viral RefSeq database16 was built from NCBI using Jellyfish by counting distinct 31-mers in the reference libraries, with each k-mer in a read mapped to the lowest common ancestor of all reference genomes with exact k-mer matches. Each query was thereafter classified to a taxon with the highest total hits of k-mer matched by pruning the general taxonomic trees affiliated with mapped genomes.
Other methods as follows for taxonomy assignment that compare a sequence in the sample to a database of known sequences are also applicable. First, sequence alignment can be conducted by BLASTN similarity searches against customized databases (with cut-off e-value <0.0001). Secondly, Bowtie2 will step deep sequence obtained quickly mapped into the database. Reads per sample are mapped against this dataset using Bowtie2 v.2.2.8 with the following parameters:-local-maxins 800 -k = 3. Genome coverage per base was calculated considering only reads with a mapping quality above 20 using view and depth Samtools commands.
The bacteriophage should show a negative correlations (r value less than 0) between the bacteriophage and bacteria that caused infection in any one of the fecal samples collected from subject 2 or subject 3 in
To isolate CRE bacteria, colonies were picked from dilution cultures (stool samples from recipient 1) by specific selective media (chromID CARBA SMART by bioMerieux, France) and streaked onto fresh agar to ensure purity. Isolated clones were resuspended in PBS plus glycerol (20%) and stored at -80° C. For animal experiments, the bacterial inoculum administered to mice was normalized to total 109 CFU. Bacterial were administered to mice by oral gavage in 100 µL daily for 2 days. Genomic DNA of the strains were extracted using QIAamp DNA Mini Kit (Qiagen, Germany) and were sent to BGI Genomics (Shenzhen, China) immediately on dry ice for WGS. Sequencing was carried out using the Illumina Hiseq Xten PE150 sequencer (Illumina, United States) with a high-throughput 2 × 100 bp pair end sequencing strategy. Reads were filtered as described previously and the resulting clean reads were assembled using SPAdes software (Bankevich et al., 2012). The assemblies were further examined for characteristics.
C57BL/6J male mice were used at 6-8 weeks of age and were randomly assigned to experimental and control groups. In all experiments, age- and gender-matched mice were used. All mice were kept at a strict 24 hr light-dark cycle, with lights on from 6am to 6pm. For antibiotic treatment, mice were given a combination vancomycin (0.125 g)-neomycin (0.25 g)-metronidazole (0.25 g)-ampicillin (0.25 g, combined in 250 ml water) in their drinking water for two weeks as previously described 18 . On the day of FMT, a fresh FMT was prepared by harvesting the stool from normal healthy mice. The stool pellets were then suspended in 100 µL sterile PBS and mice were subsequently orally gavaged with 100 µL suspension.
Viral microbial fraction transplantation (VMT) was obtained by VLP preparation. A stool pellet from the untreated healthy mice were suspended in 300 µL sterile PBS and centrifuged at 2500 g for 10 min. Then bacteria were removed in the VLP-containing supernatant using a 0.45 µm filter, which follow by a 0.22 µm filter. Using a 100 kDa centrifugal filter at 3220 g for 5 min to capture VLPs in the fecal filtrated and then washed 3 times with PBS under the same conditions. Afterwards, VLPs on the filter were suspended in 100 µL PBS and mice were orally gavaged with 100 µL suspension. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.
The abundance data of bacteria, viruses, and fungi were imported into R 3.3.5. Richness, diversity, and rarefaction calculations were performed using the phyloseq package. Data visualization was done in R (package ggplot2). Pearson’s correlation test was applied to determine correlations between the bacteriophage and bacteria that caused infection. For Pearson correlations, we used the cor.test function in R to conduct significance test and obtain the P values (two-sided). An r value less than 0 indicates a negative association.
Patients who were 18 years old or older, had two or more stool or rectal swab positive for CRE at least one week apart, and did not receive antimicrobial therapy for at least 48 hours prior to infusion of FMT were recruited to a clinical trial (NCT03479710). Patients who had an active infection of CRE or VRE requiring antimicrobial therapy, pregnancy, active gastrointestinal tract infections, or inflammatory disorders, had recent intro-abdominal surgery, had short gut syndrome, or use of medications which alter gastrointestinal motility were excluded. CRE infection was defined as the presence of any Enterobacteriaceae with resistance to any of the carbapenems. In this study, patients received 2 FMTs using frozen donor stool samples. 100 mL of FMT solution (raw stool 50 g) in 0.9% sterile saline were infused over 2-3 minutes into the distal duodenum or jejunum via oesophago-gastro-duodenoscopy (OGD). Stool samples were collected from patients before and after FMT prospectively. Recipients received FMT from the same donor for the 2 FMTs. Stools for FMT infusions were obtained from donors recruited to Stool Biobank for the Faculty of Medicine, The Chinese University of Hong Kong. Donors were volunteers from the general population, including spouses or partners, first-degree relatives, other relatives, friends, and others who were known or unknown to the potential patients. Donors need to fulfil a set of eligibility criteria and passed screening laboratory tests for infectious diseases, including CRE and VRE infections.
Three CRE-positive patients, detected on two consecutive rectal swabs, with CRE isolates clinically identified as Klebsiella pneumonia, Klepsiella variicola, and Escherichia coli (Table 3), were successfully cleared from CRE after receiving two FMTs each (
Klebsiella pneumoniae
Klebsiella pneumoniae
Escherichia coli
Klebsiella variicola
The bacteria profile of patients with CRE infections was significantly different from healthy controls. We first determined the differences in fecal microbiome between patients with CRE infections and healthy controls via shotgun metagenomic profiling. Stool of patients with CRE infections was characterized by a lower bacterial and fungal α-diversity (Shannon index P < 0.05;
To explore alterations in the patient’s gut virome after multiple FMTs, we enriched virus like particles (VLPs) in stools and subsequently sequenced DNA extracted from VLP-enriched stool preparations. On average, we obtained 13,886,857 ± 4,552,632 (mean ± s.d.) clean paired-end reads per sample. The alpha diversity of virome demonstrated substantial variations among individuals (
Bacteriophages are known natural predators that control the bacterial population and have a large impact on bacteria ecosystems. Strikingly, following an FMT, a substantial decrease of Klebsiella spp. abundance (clinical identified CRE species prior to FMT) was seen with a concomitant marked increase of Klebsiella phages (
Since approach based on metagenomic analysis of VLPs focuses primarily on the free phages at the time of sampling, to test bacteriophage in full metagenome, we then further identified bacteriophages by comparing the whole metagenome sequences to viral database using the Kraken classification and alignment programs. In FMT recipients, we observed a marked increased level of Klebsiella bacteriophage (Drulisvirus (NCBI:txid1920774) and Slopekvirus (NCBI:txid1985328)) from full metagenomes and VLP in post-FMT samples (
A striking increase in the relative abundance of Escherichia virus was only found in recipient 2, the only patient who carry CRE Escherichia coli (
We validated the results in a parallel in vivo study of mice specifically gavaged with carbapenem-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae was introduced to antibiotics-treated mice and treated with healthy fecal microbiota/virome fraction twice by oral gavage (
From human clinical trial, longitudinal metagenomic analysis of stool samples from CRE patients have demonstrated the bloom of Klebsiella phages after FMT. Herein, we found ten Klebsiella virus (List 6) exhibited higher relative abundance level in FMT treated mice than control animals (
All patents, patent applications, and other publications, including GenBank Accession Numbers or equivalents, cited in this application are incorporated by reference in the entirety for all purposes.
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP34 (NCBI:txid674081)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage KP27 (NCBI:txid1129147)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage KP27 (NCBI:txid1129147)
Klebsiella phage KP27 (NCBI:txid129147)
Klebsiella phage vB_KpnP_KpV289 (NCBI:txid1671396)
Klebsiella phage vB_KpnP_KpV289 (NCBI:txid1671396)
Klebsiella phage KLPN1 (NCBI:txid1647408)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Escherichia virus 186 (NCBI:txid29252)
Klebsiella virus (target bacteria Klebsiella pneumoniae) sequences in whole-community metagenomes in post-FMT samples
1 Ferri, M., Ranucci, E., Romagnoli, P. & Giaccone, V. Antimicrobial resistance: a global emerging threat to public health systems. Critical reviews in food science and nutrition 57, 2857-2876 (2017).
2 Guh, A. Y. et al. Epidemiology of carbapenem-resistant Enterobacteriaceae in 7 US communities, 2012-2013. JAMA 314, 1479-1487 (2015).
3 Sievert, D. M. et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection Control & Hospital Epidemiology 34, 1-14 (2013).
4 Satlin, M. J. et al. Multicenter clinical and molecular epidemiological analysis of bacteremia due to carbapenem-resistant Enterobacteriaceae (CRE) in the CRE epicenter of the United States. Antimicrobial agents and chemotherapy 61, e02349-02316 (2017).
5 Gupta, N., Kallen, A. J., Limbago, B. M. & Patel, J. B. Carbapenem-Resistant Enterobacteriaceae: Epidemiology and Prevention. Clinical Infectious Diseases 53, 60-67, doi:10.1093/cid/cir202 (2011).
6 Urban, C. et al. Carbapenem-resistant Escherichia coli harboring Klebsiella pneumoniae carbapenemase β-lactamases associated with long-term care facilities. Clinical Infectious Diseases 46, e127-e130 (2008).
7 Oostdijk, E. A., de Smet, A. M. G., Kesecioglu, J. & Bonten, M. J. J. J. o. A. C. Decontamination of cephalosporin-resistant Enterobacteriaceae during selective digestive tract decontamination in intensive care units. 67, 2250-2253 (2012).
8 Van Nood, E. et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. 368, 407-415 (2013).
9 Manges, A. R., Steiner, T. S. & Wright, A. J. J. I. D. Fecal microbiota transplantation for the intestinal decolonization of extensively antimicrobial-resistant opportunistic pathogens: a review. 48, 587-592 (2016).
10 Manges, A. R., Steiner, T. S. & Wright, A. J. Fecal microbiota transplantation for the intestinal decolonization of extensively antimicrobial-resistant opportunistic pathogens: a review. Infectious Diseases 48, 587-592 (2016).
11 Davido, B. et al. Is faecal microbiota transplantation an option to eradicate highly drug-resistant enteric bacteria carriage? Journal of Hospital Infection 95, 433-437 (2017).
12 Gopalsamy, S. N., Sherman, A., Woodworth, M. H., Lutgring, J. D. & Kraft, C. S. Fecal Microbiota Transplant for Multidrug-Resistant Organism Decolonization Administered During Septic Shock. Infection Control & Hospital Epidemiology 39, 490-492 (2018).
13 Dinh, A. et al. Clearance of carbapenem-resistant Enterobacteriaceae vs vancomycin-resistant enterococci carriage after faecal microbiota transplant: a prospective comparative study. Journal of Hospital Infection 99, 481-486 (2018).
14 Davido, B. et al. Is faecal microbiota transplantation an option to eradicate highly drug-resistant enteric bacteria carriage? 95, 433-437 (2017).
15 Zuo, T. et al. Bacteriophage transfer during faecal microbiota transplantation in Clostridium difficile infection is associated with treatment outcome. 67, 634-643 (2018).
16 Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014).
17 Wood, D. E. & Salzberg, S. L. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome biology 15, R46 (2014).
18 Croswell, A., Amir, E., Teggatz, P., Barman, M. & Salzman, N. H. Prolonged impact of antibiotics on intestinal microbial ecology and susceptibility to enteric Salmonella infection. Infection and immunity 77, 2741-2753 (2009).
19 Minot, S. et al. The human gut virome: inter-individual variation and dynamic response to diet. Genome research 21, 1616-1625 (2011).
20 Reyes, A. et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334 (2010).
21 Minot, S. et al. Rapid evolution of the human gut virome. Proceedings of the National Academy of Sciences 110, 12450-12455 (2013).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
This application is a 371 U.S. National Phase Application of PCT/CN2020/135614, international filing date Dec. 11, 2020, which claims priority to U.S. Provisional Pat. Application No. 62/946,781, filed Dec. 11, 2019, the contents of which are hereby incorporated by reference in the entirety for all purposes.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/CN2020/135614 | 12/11/2020 | WO |
Number | Date | Country | |
---|---|---|---|
62946781 | Dec 2019 | US |