Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

Ethical approval and patient information

Ethical approval to collect caecal effluent from patients was obtained from St Thomas’ Hospital Research Ethics Committee (06/Q0702/74) covering Guy’s and St Thomas’ Hospitals, and transferred by agreement to London Bridge Hospital. Patients provided written consent to provide samples. The caecal effluent was collected as described by Hoyles et al. (2014) from a 31-year-old female who showed no evidence of colonic abnormalities or disease as based on a routine colonoscopic examination.

Sample processing, and isolation of bacteria and phages from caecal effluent

The sample was transported to the University of Reading under anaerobic conditions (in a gas jar with an anaerobic gas-generating pack; Oxoid Ltd, Hampshire, UK) and on ice, where it was processed within 3 h of collection. The sample was transferred to an anaerobic cabinet (Whitley MG1000 anaerobic workstation; DW Scientific, West Yorkshire, UK; gas composition 80% N₂, 10% H₂, 10% CO₂) and mixed well by shaking. An aliquot (1 ml) of the sample was diluted with 9 ml sterile, anaerobic half-strength peptone water (Oxoid) in a sterile universal bottle containing 2-mm glass beads. A dilution series (10⁻¹ to 10⁻⁶) was prepared from the homogenate in sterile, anaerobic half-strength peptone water (Oxoid). Aliquots (20 µl) were plated in triplicate onto fastidious anaerobic agar (BIOTEC laboratories, Ipswich, UK) containing 5% laked horse blood. Bacteria were incubated anaerobically for 5 days at 37 °C, and then enumerated. Ten colonies were selected randomly, streaked to purity, and stored on Microbank cryogenic beads (Prolab Diagnostics, Merseyside, UK) at −70 °C.

The remaining neat caecal effluent (∼25 ml) was processed as described by Hoyles et al. (2014). Briefly, the sample was diluted 1:4 (v/v) with sterile TBT buffer (100 mM Tris/HCl, pH 8.0; 100 mM NaCl; 10 mM MgCl₂⋅6H₂O filtered through a 0.1 mm pore size filter prior to autoclaving). The sample was homogenized in a stomacher (Stomacher 400 Lab System; Seward, West Sussex, UK) for 2 min at ‘high’ speed, and then placed on ice for 2 h to allow detachment of phages from and settlement of particulate material. The homogenate was centrifuged at 11,180 g for 30 min at 4 °C, and the supernatant passed through a sterile 0.45 µm cellulose acetate filter (Millipore, Billerica, Massachusetts, USA). The filtrate was stored at 4 °C until used in spot assays.

Identification of isolated bacteria

DNA was isolated from bacteria using InstaGene Matrix (Bio-Rad, Deeside, UK) according to the manufacturer’s instructions. Partial (∼600 nt) 16S rRNA gene sequences were obtained for the isolates via the University of Reading’s Biocentre. Nearest relatives of isolates were determined using EzTaxon (Chun et al., 2007). The identity of strain L4-FAA5, tentatively identified as Klebsiella pneumoniae on the basis of its 16S rRNA gene sequence, was confirmed using a species-specific PCR that detects the 16S–23S internal transcribed spacer unit of K. pneumoniae as well as capsular-type-specific, and virulence gene targets (Turton et al., 2010). Malonate and Voges–Proskauer reactions were positive indicating that the isolate was subsp. pneumoniae (rather than subsp. ozaenae) (Turton et al., 2010). Typing was carried out by VNTR analysis at loci A, E, H, J, K and D (Turton et al., 2010) and an additional three loci (N1, N2 and N4). Primers, repeat sizes and flanking sequence sizes for the additional loci were: N1F 5’-CATCAGGTGCAAGATTCCA-3’ and N1R 5’- TGAGCGATTGCTGGCCTA-3’, 116 bp repeat with a 107 bp flanking sequence; N2F 5’-GATGCGGCAAGCACCAC-3’ and N2R 5’-ACGCCCTGACCATTATGC-3’, 57 bp repeat with a 109 bp flanking sequence; and N4F 5’-GTGCGGTGATTGTGATGG-3’ and N4R 5’-CTGACAACGTCGATGTGG-3’, 67 bp repeat with a 119 bp flanking sequence.

Screening of bacteria against filtered caecal effluent

K. pneumoniae strains isolated from the caecum were grown to OD₆₆₀ ∼ 0.4 in tryptone soya broth (Oxoid Ltd), and used in spot assays as follows. An aliquot of culture (200 µl) was inoculated into 3 ml tryptone soya broth containing 0.3% (w/v) agarose (SeaKem LE agarose; Lonza Rockland) that had been heat-treated by microwaving and dispensed aseptically from a larger volume before cooling to 48 °C. The overlays containing bacteria were poured over 20 ml solid agar plates of autoclaved tryptone soya agar (Oxoid Ltd). Once the agar had solidified, a 10 µl spot of filtered caecal effluent was applied to the overlays, and the plates were incubated overnight at 37 °C in the anaerobic cabinet. Identical plaques were observed on all K. pneumoniae isolates; strain L4-FAA5 was used to propagate and purify an isolated phage in tryptone soya broth or reinforced clostridial and nutrient media (Oxoid Ltd). Neither calcium nor magnesium was added to media during propagations.

The ability of the purified phage to infect a panel of K. pneumoniae subsp. pneumoniae clinical isolates (Table 1) was determined using the spot assay as described above, except that nutrient agar plates were used for the base. Strain L4-FAA5 was used as a positive control.

Purification of phage particles

A 100-ml culture of strain L4-FAA5 was grown to mid-exponential phase, inoculated with 100 µl of phage stock (∼10¹⁰ pfu/ml) and incubated until the medium became clear (∼2 h after infection). The lysate was centrifuged at 4,500 g for 10 min, then passed through a 0.45 µm cellulose acetate filter. PEG 8000 (10%, w/v) and NaCl (6%, w/v) were added to the filtrate, and mixed until all particulates had dissolved. The sample was left at 4 °C for 16 h, then centrifuged at 4,500 g for 30 min. The supernatant was removed and the pellet resuspended in 4 ml TBT buffer. A CsCl block gradient was formed with 5 M and 3 M CsCl, both prepared in TBT buffer and samples were placed on top of this gradient and subjected to centrifugation at 100,000 g for 2 h at 4 °C. The band containing ∼2 ml of TBT buffer and the phages was drawn out of the tube and the purified sample was transferred to dialysis tubing (4,000–6,000 Da cut-off), and dialysed against TBT buffer overnight. The TBT buffer was replaced with fresh TBT buffer, and the sample dialysed for a further 4 h. The purified phage particles were removed from the dialysis tubing and stored at 4 °C.

Transmission electron microscopy

This was done as described by Hoyles et al. (2014).

Extraction of DNA from phage KLPN1 and restriction enzyme profiles

An aliquot (250 µl) of the purified phage particle stock was treated with DNAse (2 U) for 30 min at 37 °C, then heated for 10 min at 80 °C. Two phenol/chloroform extractions were performed, before the DNA was precipitated using 1/10 volume of 3 M sodium acetate (pH 4.8) and 2 volumes of ice-cold ethanol. After air-drying, DNA was resuspended in 100 µl TE buffer. Restriction profiles were obtained for phage KLPN1 using the enzymes EcoRV and HaeIII (SuRE/Cut; Roche Applied Science, Basel, Switzerland): restriction digests contained 3 µl H₂O, 1 µl enzyme, 1 µl enzyme buffer and 5 µl DNA, and were incubated for 3 h at 37 °C before being run on a 0.8% (w/v) agarose gel (1 h, 75 V) and stained with ethidium bromide.

Whole-genome sequencing and annotation of the phage genome

DNA (5 µg) was extracted and concentration verified by Nanodrop quantification. Confirmatory molecular identification tests were also conducted on the DNA extract prior to shipment to the contract sequencing facility (Macrogen Inc., Seoul, Korea). Eighty-fold sequencing coverage was obtained using pyrosequencing technology on a 454 FLX instrument. The individual sequence files generated by the 454 FLX instrument were assembled with GSassembler (454 Lifesciences, Branford, Connecticut, USA) to generate a consensus sequence. To ensure correct assembly and resolve any remaining base-conflicts, short segments of the genome were amplified by PCR and the generated amplicons were then subjected to Sanger sequencing (MWG, Ebersberg, Germany). Open reading frames (ORFs) were automatically predicted using Genemark (Besemer & Borodovsky, 1999). General feature format (gff) files were generated for the predicted phage proteome (retaining proteins with a minimum size of 30 amino acids) and visualized using the annotation software Artemis v10.0 (Rutherford et al., 2000). ORF boundaries were verified and, where required, adjusted by manual inspection of Shine–Delgarno sequences. BLASTP (Altschul et al., 1990) was used to provide preliminary functional annotation data, and to carry out a comparative analysis of KLPN1 with previously sequenced Klebsiella phages at the protein level (Altschul et al., 1990). To improve genome annotations/predictions, all proteins encoded by ORFs in the genome sequence of KLPN1 were searched against InterProScan (http://www.ebi.ac.uk/interpro/). HHpred (http://toolkit.tuebingen.mpg.de/hhpred) searches were done to identify domains associated with depolymerase activity.

Screening virome datasets for phage sequences related to KLPN1

Fasta files associated with public virome datasets were downloaded from the METAVIR web server (http://metavir-meb.univ-bpclermont.fr; Roux et al., 2011) on 31 March 2015 (Table S1). Each fasta file was used to create a BLAST database, against which the genome sequence of phage KLPN1 was searched using BLASTN.

Isolation and characterization of bacteria isolated from caecal effluent

On fastidious anaerobe agar, 2.22 × 10⁸ ± 5.30 × 10⁷ (n = 3 replica plating) bacteria were isolated per millilitre of caecal effluent sample (log₁₀ 8.35 ± 7.72 bacteria/ml caecal effluent). Ten colonies were randomly selected from one of the triplicate plates used to enumerate the bacteria, and streaked to purity. On the basis of 16S rRNA gene sequence analysis (EzTaxon), isolates represented Klebsiella pneumoniae (n = 5), Bacteroides vulgatus (n = 2), Bacteroides massiliensis (n = 1), a novel member of the order Erysipelotrichales distantly related to [Clostridium] innocuum (n = 1), and Haemophilus parainfluenzae (n = 1).

It is widely accepted that identification of members of the family Enterobacteriaceae is difficult on the basis of 16S rRNA gene sequence data alone; therefore, one of the strains identified as K. pneumoniae, L4-FAA5, was characterized using the PCR-based methods of Turton et al. (2010). The isolate was found to be a strain of K. pneumoniae subsp. pneumoniae, capsular type K2, rmpA⁺. Its VNTR profile was unique among the clinical isolates tested and on the wider database of clinical isolates held by Public Health England–Colindale.

Screening of filter-sterilized caecal effluent against bacteria to isolate phages

The K. pneumoniae isolates were used in spot assays with the caecum filtrate, which was free of bacteria (as verified by Gram-stained smear and plating on nutrient agar; not shown). Phages infecting these isolates were initially identified in the caecal effluent by spotting 10 µl aliquots of filtered caecal effluent onto TSA overlays containing 200 µl of a given culture in the exponential phase of growth. Plaques identical to those shown in Fig. 1A were observed with all K. pneumoniae isolates in the spot assays, and phages were present at 2 × 10⁵ ± 2.65 × 10³ (n = 3) pfu/ml caecal effluent. Clear plaques of 2 mm diameter were visible within 3 h of spotting onto an agar overlay. After prolonged incubation, the area around the plaques developed opaque haloes presumably caused by depolymerase activity, which increased in size over the course of 4 days, although the central clear area of the plaques remained 2 mm in diameter (Fig. 1B). This phenomenon has been observed previously for lytic phages of the families Siphoviridae, Podoviridae and Myoviridae against K. pneumoniae, and is associated with degradation of capsular polysaccharides (Geyer et al., 1983; Verma, Harjai & Chhibber, 2009; Hsu et al., 2013; Lin et al., 2014a; Shang et al., 2015). The non-Klebsiella isolates were not screened against the caecum filtrate for phages.

Strain L4-FAA5 was used as the host bacterium on which to isolate and propagate one phage (which we named KLPN1) to purity. KLPN1 infected all K. pneumoniae isolates recovered from the filter-sterilized caecal effluent.

The ability of phage KLPN1 to infect a panel of K. pneumoniae subsp. pneumoniae clinical isolates was determined (Table 1), which revealed that all K2 strains tested (including the type strain) were sensitive to this phage, though KLPN1 did not infect any of the other tested strains belonging to other capsular types. VNTR analysis showed that the six K2 isolates in the panel represented five, distinct strains, suggesting a wide susceptibility to the phage among isolates of this capsular type. In addition, it did not exhibit depolymerase activity with any of the clinical K2 isolates.

Characterization of phage KLPN1

Phage KLPN1 is chloroform-resistant. In addition, it displayed stability to prolonged storage in TSB at 4 °C: after 6 and 18 months’ storage, titres for the phage were still 10¹⁰ pfu/ml, comparable with the original stock. Transmission electron microscopy revealed the isometric-headed phage to possess a capsid of ∼62.7 ± 2.3 nm (n = 30) in diameter with a long non-contractile tail of ∼164.4 ± 3.0 nm (n = 29), thus indicating that this phage is a member of the family Siphoviridae (Fig. 2). Notably, the base-plate structure was unusual with a distinct central tail fibre [length 33.4 ± 1.7 nm (n = 43)] with apparently three elongated spherical structures [length, 13.2 ± 1.3 nm (n = 43); width, 6.9 ± 0.7 nm (n = 48)] loosely associated with the central regions of the tail fibre. This unique base-plate structures resembles a rosette with three leaves, and can be observed in all five micrographs shown in Fig. 2. A similar structure has been described for Rtp, a lytic phage of Escherichia coli (Wietzorrek et al., 2006), with which KLPN1 shares some sequence identity (discussed below). It is also visible in electron micrographs of phage ϕ28, which infects K. pneumoniae capsular type K28 (Geyer et al., 1983).

It was straightforward to isolate high-quality DNA from phage KLPN1. Heating an aliquot of CsCl-purified, DNAse-/RNAse-treated sample at 80 °C for 10 min followed by phenol/chloroform/isoamyl alcohol extractions and ethanol precipitation gave large quantities of high-quality DNA that was used for restriction digests (not shown) and genome sequencing. The estimated genome size of KLPN1 based on restriction digests with EcoRV or HaeIII was ∼45 kbp, which was an underestimation of the genome size of 49,037 bp as determined by sequencing (see below).

Sequence analysis of KLPN1

The genome of KLPN1 was 49,037 bp with a G + C content of 50.53%, similar to previously sequenced Klebsiella Siphoviridae phages such as KP36 (Table 2). Initial genome annotation was performed using Genemark (Besemer & Borodovsky, 1999), from which a gff file was generated to allow visualisation of the predicted ORFs in Artemis v10.0 (Rutherford et al., 2000). Each ORF with a minimum amino acid content of 30, a start and stop codon as well as a ribosomal-binding site was retained (Table 3). The genome of KLPN1 was predicted to encompass 73 ORFs divided into four clusters, two rightward and two leftward (Fig. 3, Table 3). Of the 73 predicted ORFs, 23 were assigned a function with the remainder representing hypothetical proteins with no assignable function (Table 3). BLAST analysis of the complete nucleotide sequence indicated that KLPN1 is closely related to the Klebsiella phages KP36 (95% identity across 86% of the genome; Kęsik-Szeloch et al., 2013), F20 (84% identity across 82% of the genome, GenBank accession no. JN672684; Mishra, Choi & Kang, 2012) and phage 1513 (95% identity across 85% of the genome, GenBank accession no. KP658157; Cao et al., 2015). Furthermore, partial identity was observed against Shigella phage Shfl1 as well as enterobacterial phages T1 (Roberts, Martin & Kropinski, 2004) and RTP (Wietzorrek et al., 2006) (GenBank accession numbers HM035024, AY216660 and AM156909, respectively). A comparative analysis of KLPN1 was performed against both known members (KP36, F20) of the genus “Kp36likevirus” (Niu et al., 2014; Table 3, Fig. 3). For completion, KLPN1 was also compared with previously sequenced Klebsiella phages (Table 2). KLPN1 was shown not to share detectable homology with Klebsiella phages belonging to the family Podoviridae or Myoviridae. Based on the amino acid percentage identities presented in Table 3, the genomes of KLPN1 and related phages were divided into functional conserved modules: packaging, phage particle morphogenesis and DNA replication (Fig. 3, Table 3). Sections of the KLPN1 genome were shown to exhibit homology to the Siphoviridae phage phiKO2 genome, although such homology was confined to the tail morphogenesis region and one hypothetical protein (Table 3). Of the 73 predicted ORFs, only three appeared unique to KLPN1: two genes encoding hypothetical proteins located at the 5’ end of the genome and a putative homing endonuclease-gene positioned in the capsid morphogenesis module (Table 3).

Based on sequence homology to ORF33 (encoding the host-specificity J protein; InterPro IPR021034) of phage T1, the phage receptor-binding protein of KLPN1 was predicted to be encoded by ORF43. The deduced ORF43 protein was found to exhibit similarity to fibronectin type III domain-containing protein, and potentially encodes the depolymerase whose effect was observed on the phage assay plates (Fig. 1). However, a HHpred search with the protein sequence encoded by this ORF failed to detect any domains associated with enzymes (such as glycanases, deacetylases and lyases) associated with depolymerase activity of Klebsiella lytic phages (Geyer et al., 1983). The protein encoded by ORF34 of phage NTUH-K2044-K1-1 has proven depolymerase activity associated with pectate lyase (Lin et al., 2014a); a HHpred search confirmed the protein encoded by this ORF has a lyase/polygalacturonase domain (1ru4_A pectate lyase, 94.3% probability of true positive, E-value 0.055, P-value 1.6 × 10⁻⁶; 1bhe_A PEHA, polygalacturonase, 90.1% probability of true positive, E-value 13, P-value 0.00038). ORF96 of phage 0507-KN2-1 also encodes proven depolymerase activity (Hsu et al., 2013). A HHpred search demonstrated the protein encoded by this ORF has an acetylneuraminidase domain (3gw6_A endo-N-acetylneuraminidase, 99.8% probability of true positive, E-value 1 × 10⁻¹⁹, P-value 2.9 × 10⁻²⁴). The whole-genome sequence of phage P13 cannot be retrieved from GenBank; however, on the basis of a PSI-BLAST search conducted by Shang et al. (2015), genes 49 and 50 of phage P13 are predicted to encode the exopolysaccharide depolymerase, but no further substrate/functional information is available. HHpred searches of all proteins encoded by KLPN1 showed that ORF34 and ORF35 encode an endo-N-acetylneuraminidase/endosialidase domain (Table 4). Consequently, we predict ORF34 and/or ORF35 encode the depolymerase activity of phage KLPN1. Further work will be required to confirm our hypothesis.

The proteins encoded by ORF36 and ORF36.1 are predicted to be two chaperones, analogous to gpG and gpGT required for phage λ tail assembly (Xu, Hendrix & Duda, 2013). These ORFs are believed to keep the tail tape measure protein (TMP) soluble and to recruit the major tail protein subunits, both essential processes for tail assembly. ORF36 encodes the short chaperone (gpG), while a programmed -1 translational frameshift facilitates a translational fusion between the products of ORF36 and ORF36.1 so as to form the long chaperone (gpGT) (hence the lack of a ribosome-binding site for ORF36.1) (Fig. 4). A BLASTP search of the predicted 213 aa fusion protein showed it shares 47% identity (E-value 3 × 10⁻⁵⁶) with the tape measure chaperone of the Citrobacter Siphoviridae phage Stevie, reported to have a conserved translational frameshift (Shaw et al., 2015; Fig. 4).

The genome sequence of KLPN1 was subjected to BLASTN searches against all publicly available virome sequence data held by METAVIR (51,992,208 sequence reads associated with 70 projects from a range of habitats, including human faeces (Reyes et al., 2010; Kim et al., 2011; Minot et al., 2011; Minot et al., 2013); Table S1). No hits corresponding to KLPN1 were found among these publicly available datasets. However, using InterProScan, the protein sequence encoded by ORF64 was found to belong to the family ‘Protein of unknown function DUF3987’, representing uncharacterized human-gut-microbiome-specific proteins (Ellrott et al., 2010; Table 3).