Isolation and Characterization of a Phage to Control Vancomycin Resistant Enterococcus faecium

2.1 Bacterial Strain Identification

The clinical isolates were collected from the microbial culture facility of Railway General Hospital, Rawalpindi. The isolates were validated using established microbiological methods. Analytical profile index testing kit [API 20E; Biomérieux Direct] was used for standardized identification and Kirby Baeur method was employed for antibiotic susceptibility testing [20]. Vancomycin resistance was checked via polymerase chain reaction (PCR) analysis for vanA gene using vanA specific primers. For molecular identification 16S rRNA gene sequencing was performed for the clinical isolate. The 16S rRNA gene amplification was carried out by PCR using primers: RS-1 5´-AAACTC-AAATGAATTGACGG-3´, RS3 5´-ACGGGCGGTGTGTAC-3´ [21]. Amplified PCR product of approximately 0.5 kb was analyzed on 1% agarose gel and purified using gel extraction kit [Invitrogen Cat. K210012]. The sequenced product was aligned using NCBI-BLAST for identification of the sequence (www.ncbi.nlm.nih.gov/BLAST).

2.2 Bacteriophage Isolation and Enrichment

Clinical strain of VREF was selected as host for bacteriophage isolation. Bacteriophages specific to clinical strain VREF was enriched by previously described method with few modifications [21]. Sewage water sample of a hospital at district Dera Ismail Khan, Pakistan was centrifuged at 1,300 rpm for 10 min for removal of impurities and cell debris. The supernatant was filter-sterilized through 0.2 μm filter. About 5mL of sample was then added to 30 mL log phase (OD₆₀₀ 0.4-0.6) E. faecium culture. The enriched culture was incubated at 37°C with shaking at 150 rpm. A drop of 1% chloroform was added to 1.5 mL of enriched culture for bacterial cell disruption and to release phages. Centrifugation at 14,000 rpm was then carried out for 15 min to settle down bacterial cell debris. Using 0.45 μm and 0.2 μm syringe filters sequentially, supernatant was filtered and transferred to a new Eppendorf tube.

Spot assay and plaque assay were used to isolate and detect phages on LB agar plates using soft agar. Overnight incubated bacterial culture and filtrate along with 0.7% soft agar were mixed and poured onto agar plates. Once solidified, plates were incubated overnight at 37°C to examine spots or plaques. For negative control only phage was added to soft agar. Amplification of phages was carried out by propagation of spots obtained after spot assay. Graph Pad Prism 5 was used to generate graphs of each test for phage characterization.

2.3 Host Range Determination

A wide range of clinical bacterial strains (Table 1) including E. faecium, Acinetobacter, Citrobacter, methicillin resistant Staphylococcus aureus, E. coli, Pseudomonas and Enterococcus were used to check the host specificity of bacteriophage STH1. To test the susceptibility of bacterial isolates, drop-on-lawn technique was used followed by plaque assay [22].

2.4 Morphology of the phage

The determination of phage morphology was done according to the previous reported methods [21]. A phage titer (≈10¹⁰ PFU/ml) was diluted 10 fold in 1× phosphate buffer saline in one liter distilled water having pH 7. On the surface of copper grids (formvar carbon), phage suspension was added. Uranyl acetate 2% was added for the negative staining of the samples. Through a filter paper the grids were instantly blotted and the grids were then air dried. The electron microscopy was done for the grid to analyze at 100 kV. For the classification of the phage STH1, morphological techniques were used according to the guidelines of international Committee on Taxonomy of Viruses.

2.5 Phage pH and Thermal Stability

Three consecutive experiments were conducted to determine appropriate phage pH stability using a range of values — 1, 3, 5, 7, 9, and 11 and thermal stability of phage STH1 according to the methods described previously [23, 24]. Phage filtrate (8.2x10⁷ PFU/mL) and LB media were mixed and incubated at 37°C for an hour (h). Phage viability was checked against the host for each treated sample by plaque assay. For thermal stability test, vials of phage filtrate were treated at a range of temperatures [37 (control), 45, 50, 55, 60, 65 and 70°C] for 1 h. Plaque assay was performed for each treated sample.

2.6 Effect of Calcium and Magnesium

E. faecium culture was equally poured into two sterile 50 mL tubes. Each of the two tubes were simultaneously inoculated with 0.25 mL (3.2×10⁸ PFU) phages and CaCl₂ or MgCl₂ (250 μL and 10 mmol/L each). The samples from the culture were collected from each flask at regular interval of times (0, 10, 20, and 30 min) to calculate titers of free phages in untreated and Ca2+ or Mg2+ supplemented supernatant. The effect of Ca2+ or Mg2+ ions on adsorption potential was investigated according to previously described method [24].

2.7 One Step Growth Experiment

One step growth experiment was carried out as described previously [25]. E. faecium culture was grown to mid exponential phase and cells were harvested by centrifugation (10,000 rpm for 5 min). About 0.5 mL LB media was used to re-suspend the pellet obtained and mixed with 0.5 mL phage (8.2×10⁷ PFU/mL). Phages were allowed to adsorb to bacteria for 2 min and free phages were later removed by centrifugation (13000 rpm for 30 s). About 100 mL fresh media was used to resuspend the pellet and the culture was incubated at 37°C. The incubated samples were taken at three minute interval and double layer agar assay was performed to determine the phage titer.

2.8 Bacterial Reduction Assay

Three consecutive bacterial reduction assays were conducted. Each experiment was performed by using E. faecium cultures in two flasks grown to mid exponential phase with absorbance (A₆₀₀) in the range of 0.4–0.6. One of the flasks had 1mL phage filtrate, while other flask was used as control without any phage. A₆₀₀ of the samples were taken after every 2 h for 24 h using a spectrophotometer.

2.9 Effect of Bacteriophage on VREF Grown in Milk Samples

The experiment was performed for milk samples according to already described methods with few modifications [26]. Bacterial culture incubated overnight at 37°C in tryptic soy broth with CFU of 3.9x10⁸ was used. About 1.5 mL culture was added to sterile Eppendorf tube and centrifuged for 10 min at 13,000 rpm. After discarding supernatant, double distilled autoclaved water was used to wash the pellet twice. To dilute the culture (2.0x10⁵ CFU), double distilled autoclaved water was used to dissolve the washed pellet. Diluted bacteria were added to 5 mL of each milk sample. The sample was transferred into two sterile flasks (sample and control) and incubated for 2 h. About of 500 μL phage filtrate (8.2×10⁷ PFU) was added to sample flask and was vigorously shaken. Both sample and control flasks were incubated at 37°C and CFU was calculated after 24 and 48 h for comparison.

3.1 Bacterial Strain Identification

Gram’s staining showed that the bacterium E. faecium is a gram positive cocci. Biochemical tests indicated that it a catalase negative bacterium able grow at alkaline pH and temperature range of 10-50 °C under anaerobic conditions. Using PCR an amplicon of approximately 470 bp was obtained and sequenced. The sequence was submitted to NCBI and is available under accession number [GeneBank: KF386009]. BLAST analysis indicated 98% sequence homology to E. faecium. Kirby Bauer method showed E. faecium resistance to vancomycin and teicoplanin. PCR amplification of right sized vanA gene confirmed vancomycin resistance.

3.2 Bacteriophage Isolation

Lytic phage that was isolated against E. faecium gave a clear zone. Soft agar method reconfirm the phage potential. The appearance of clear plaque on bacterial lawn showed that this phage was lytic and had plaques within a range of 1.5 to 3.5 mm having well defined boundaries. This phage was named STH1 (Figure 1.A& B).

Figure 1

(A), Spot assay for detection of phage and (B), shows plaque assay for detection and confirmation of phage clearly showing plaques ranging in size from 1.5 to 3.5 mm, (C) Transmission electron micrographic (TEM) view of the purified STH1 phage particle (negatively stained preparations). Scale bars, 100 nm.

3.3 Host Range Determination

During host-range determination of STH1 phage, where non-host bacteria were also considered, nineteen pathogenic bacterial isolates were tested. Phage showed

slightly lesser activity against two E. faecium strains U-520 and FT-549. However, no plaques were observed indicating narrow host range of the phage.

3.4 Morphology of Phage STH1

TEM analysis showed that phage head was isometric having contractile tail. The phage STH1 has a head of 132 nm with tail having a length of 120 nm and width of 20 nm. These morphological characteristics showed that phage STH1 belongs to family Myoviridae (Figure 1.C).

3.5 Characterization of Phage STH1

Reduced phage number was observed at extreme acidic conditions –pH 1, 3 and 5. Although with reduced growth, the phage exhibited plaques and that the phage was viable at the extreme pH. Optimum pH, however, was determined to be 7.0 with maximum number of plaques. The results suggest that extreme pH might be an obstacle to phage stability. Very few plaques were observed at pH 1. The number of plaques increased with increasing pH, reaching the highest number at pH 7.0. Gradual decrease in number of plaques was observed when pH elevated above 7.0 (Figure 2.A). To determine the heat resistant capability, phage thermal-stability test was performed at pH 7.0. The phage was incubated within a range of 35 to 70°C. Almost 100% infection activity was observed at 37°C. Although the phage was stable until 65°C, no plaque was observed at 70°C (Figure 2.B).

Figure 2

(A) pH stability test of phage at different pH values. At neutral pH maximum number of plaques were observed for STH1 (B) Thermal stability test of phage at different temperature. The optimal temperature for STH1 infection was found to be 37°C. (C)Test for adsorption rate of phage. The ability of divalent metal ions was observed by adding 10 mM CaCl₂ or MgCl₂ to the mixture of phage STH1 and host bacterium. A significant reduction in free phages in both Ca2+and Mg2+ ions treated samples was observed as compared with control (sample without Ca2+/Mg2+) by applying paired samples t-test (p< 0.05). All the values are mean with standard deviations.

3.6 Effect of Calcium and Magnesium

Calcium chloride or Magnesium chloride (either with concentration of 10 mmol/L) was added to the mixture of STH1 and the E. faecium to determine their effects on STH1 adsorption. The number of free floating phageswas determined by plaque assay method at 0, 10, 20, and 30 min of the time intervals. A significant difference was observed in calcium or magnesium ion-treated phage STH1 groups. We observed a significant reduction in free phages in both calcium/magnesium chloride treated samples as compared with control by applying paired samples t-test, p < 0.05 for both Ca²⁺ and Mg²⁺ ions). Furthermore, calcium and magnesium ions were found to stabilize the phage adsorption process. The frequency of free phages declined when treated with calcium and magnesium ions as compared to the control group as shown in Figure 2.C.

3.7 Latent Time Period and Burst Size

By using single-step growth experiment, a triphasic curve was obtained that showed the latent phase, log phase, and stationary phase. Latent time period was found to be 18 min having burst size of 334 virions per cell (Figure 3.A).

Figure 3

(A) One step growth experiment: Curve shows; latent period (18 min); log phase; stationary phase and burst size of 334 virions per cell. (B) Reduction in the growth of planktonic E. faecium by using phage STH1(8.0x107PFU/ml). Values are the means of 4 samples.

3.8 Bacterial Reduction Assay

Phage infected culture of E. faecium was monitored for 24 h along with the control to observe bacterial lysis. The bacterial culture infected with phage showed drastic decrease in turbidity. However a gradual increase in turbidity was observed after 11 h which could be either due to bacterial cell debris or growth of phage resistant cells (Figure 3.B). On the basis of these results, we have reasons to believe that bacterial reduction strategy if practiced for therapeutic or environmental control of VREF, will yield promising results.

3.9 Applications of STH1 Phage

The inhibition of E. faecium growth was observed after 24 to 48 h in sterile milk samples. The sterile milk sample treated with phage showed 2.5 fold reductions after 24 hours incubation whereas the 48 hour incubated sample when treated with the same amount of phages showed 3.2 fold reductions (Figure 4). Reductions in both cases were found to be significant by applying paired samples t-test (p <0.05) when compared with other control samples (sample without phages).

Figure 4

Effect of phage STH1 on reducing bacterial growth in milk samples when treated with 500 μL of phage filtrate (8.0x10⁷ PFU/ml) post 2 hours of bacterial inoculation. About 2.5 and 3.2 fold reductions were observed in 24 and 48 incubated milk samples, respectively. Reductions in both cases were found significant by applying by t-test (p<0.05) when compared with other control (without phages) samples.