Comparative analysis of phage cocktail activity against uropathogenic E. coli strains using spot overlay assay

Abstract

Spot overlay assays are a common method for measuring phage activity against target microorganisms and determining % host range activity. Dilutions of phage demonstrate activity by creating a zone of clearing indicative of bacterial killing; however, this yields only semi-quantitative results for each target strain tested. In addition, zones can be completely clear or incomplete cloudy zones of varying intensity. These factors make it difficult to compare the overall strength of activity between monophages or phage cocktails when numerous bacterial strains are involved. Here we demonstrate a method to analyze spot overlay results to provide insight beyond just host range activity; three phage cocktails with host range activity of 92–96 % had different overall activities. While the spot overlay is a qualitative assay, consolidation of activity results can derive semi-quantitative information. This method converts activity of phage dilutions from a spot overlay assay into an overall activity of cocktails for comparison. Our method:

• •Uses standard laboratory protocol for analysis
• •Expands information beyond host range activity
• •Consolidates results of all strains tested to assess overall cocktail strength of activity for comparison

Graphical abstract

Graphical Abstract.

Specifications table

Subject area	Immunology and Microbiology
More specific subject area	Phage cocktail activity comparison
Name of your protocol	Comparative analysis of phage cocktail activity using spot overlay assay
Reagents/tools	•Uropathogenic Escherichia coli (UPEC) (ATCC, BEI, IHMA) •Luria broth (LB) (Difco) •Phage cocktails (Intralytix, Inc.) •Bacto-Agar (Difco) •Petri dishes (100 mm x 15 mm) (Fisher Scientific) •SM buffer (100 mM NaCl, 8 mM MgSO4 × 7H2O, 50 mM Tris–HCl pH 7.4) •Spectrophotometer (UV-1600, VWR) •Microcentrifuge (Eppendorf 5415C, Eppendorf North America) •Vortexer (Vortex Genie 2, Scientific Industries) •Laminar flow biohood (NuAire Type A/B3) •Incubator (VWR) •Shaker incubator (Environ Shaker, LabLine) •Micropipets (P10, P100, P1000)
Experimental design	Data from spot overlay traditionally used for screening phage activity can also be used to assess and compare overall strength of activity. Phage cocktails of similar concentration were tested against a matched set of E. coli strains. For each strain, zones of clearing from each phage 10-fold dilution were categorized: completely clear = no cells; cloudy or hazy = partially active; and no zone = not active. Zones are then converted to an overall cocktail strength of activity (highly, fully, partially or not active) for each strain of E. coli. Strain results were consolidated for comparative analysis and the outcome determined strength of activity for each cocktail.
Trial registration	Not applicable
Ethics	Not applicable
Value of the Protocol	Uses standard laboratory protocol for analysis Expands information beyond host range activity Consolidates results of all strains tested to assess overall cocktail strength of activity for comparison

Background

There is great interest in using phage as an alternative to antibiotics for therapeutic applications [1]. Phage have been used to target numerous antibiotic-resistant bacteria such as Acinetobacter baumannii, Staphylococcus aureus and E. coli [[2], [3], [4]]. Phage have also been investigated for numerous other applications including: food safety, disinfection of food prep surfaces [5], wound healing items [6], use in the agri-food sector [7] and control of pathogens associated with hospital acquired infections [8]. Phage cocktails are generally employed to prevent emergence of microbial resistance, and cocktails with wide host range activities are utilized for various applications [9].

There are numerous methods for characterizing phage to determine host range activity against individual target strains and identify phage for inclusion to a cocktail; these include spot overlay, efficiency of plating (EOP), culture lysis assays, and others [9]. However, there is no standard approach for utilizing these methods for establishing host range to determine composition of phage cocktails [10]. While the spot overlay assay is a commonly used method for characterizing activity and screening phage for use in cocktails [7,11,12], there are few reports of it or other methods being used to characterize or compare cocktail activities. Interpretation and comparison of cocktail activities using quantitative methods may be difficult due to presence of multiple phage; for example, it is unclear how quantitative data such as EOP would be assessed for comparative purposes, with multiple isolation strains potentially needed.

Here we report expansion of the spot overlay assay for comparative analysis of phage cocktail activities. This method converts activity of phage dilutions into an overall activity of cocktails for comparison. This approach does not require specialized equipment and takes advantage of the simplicity of the spot overlay protocol. Additional information is generated about the properties of phage cocktails beyond host range. It is particularly useful to compare and differentiate cocktails of similar host range to identify the one most suitable for the microbial panel of interest. While the spot overlay is qualitative in nature, semi-quantitative information can be derived from the consolidated results. It is not intended as a substitute for more quantitative methods, but rather a screening tool for down selection of cocktails for further investigation.

Description of protocol

Fig. 1 is the workflow schematic of the notional determination and comparison of the overall activities of four phage cocktails against ten E. coli strains. Zones of clearing of each dilution were categorized and converted to an overall cocktail activity determined against each strain. The results were then consolidated in table form and represented graphically to determine and compare the overall activity against the E. coli collection. The method is demonstrated below by comparing the activity for four cocktails of phage that target uropathogenic E. coli (UPEC) as determined by spot assay against a collection of 26 strains.

Fig. 1.

Schematic of cocktail activity comparison.

Phage cocktails

Cocktails designed to target uropathogenic E. coli (UPEC) strains were kindly provided by Intralytix, Inc. and stored at 4 °C; see Table 1 for details. Each cocktail contained three viruses that were purified in buffer. Working stocks of the solution formulations were made by 1:10 dilution to 10⁹ PFU/mL in SM buffer (100 mM NaCl, 8 mM MgSO₄ × 7H₂O, 50 mM Tris–HCl pH 7.4). Spray dried formulations were reconstituted to 10⁹ PFU/mL in SM.

Table 1.

Composition of phage cocktails used in this study.

Cocktail	Titer	Phage constituents (genera)
UPP-302 (solution)	1010 PFU/mL	Mosigvirus, Gaprivevirus95, Gaprivevirus27
05EP (dry formulation)	1010 PFU/g	Mosigvirus, Gaprivevirus18, Felixounavirus
59EP (solution)	1010 PFU/mL	Gaprivevirus95, Gaprivevirus27, Phapecoctavirus
05EP (dry formulation)	1010 PFU/g	Gaprivevirus95, Gaprivevirus27, Phapecoctavirus

UPEC strains

Bacteria were purchased from ATCC (Manassas, VA), BEI (managed by ATCC) and IHMA (Schaumburg, IL). Strains from ATCC and BEI were supplied lyophilized and reconstituted as per manufacturer’s instructions. IHMA strains were supplied as frozen stocks. Single use frozen stocks in 15 % glycerol were made of each strain from mid-log cultures in LB broth (OD₆₀₀ = 0.5 to 1 as determined on UV-1600 PC spectrophotometer, previously determined by spread plating to be approximately 10⁸ CFU/mL). Inoculation of 3 mL LB working cultures with 100 uL frozen stock grown at 37 °C with 100–200 rpm agitation typically took about 3 h to reach the desired OD₆₀₀. Overnight cultures were not used as they are a potential source of variability.

Bottom agar plates

Plates were made by adding agar to 1.5 % (w/v) to LB broth in glass bottle and sterilized by autoclaving. Solutions were swirled to mix to ensure complete mixing of agar. Temperature was brought to approximately 50 °C in an incubator (alternatively, a water bath could be used) and approximately 20 mL poured into 100 mm diameter petri dishes and allowed to solidify. Plates sat at room temperature overnight before use.

Top agar

LB with 0.6 % (w/v) agar was prepared in glass bottle, sterilized and brought to 50 °C in an incubator for immediate or short term (2–3 days) use. If longer, agar was allowed to solidify and reheated as needed (LB 0.6 % agar can be reheated up to 10 times; number of heating cycles for other media would need to be determined). Bottle was equilibrated to 50 °C before use.

Spot overlay assay

Cocktails were tested against a matched set of 26 E. coli strains. For each strain, 100 µL mid-log E. coli was pipetted into 16 × 100 mm borosilicate glass tubes, to which 3 mL top agar was added using sterile technique. Tubes were gently vortexed to mix, avoiding formation of bubbles and poured onto LB plates (minimizing any bubbles) while swirling plate to evenly distribute molten agar and cells. Plates solidified on the bench at least 15 min. Cocktails were 10-fold serially diluted to 10^–5 using 10 µL phage + 90 µL SM buffer in 0.5 mL microtubes. After vortexing briefly to mix, 5 µL of undilute and serially diluted cocktails were added to the top agar layer. When making the dilutions and applying to top agar, tubes were arranged so that each was moved to an adjacent row to avoid losing track of the samples. Plates were left in the biohood to let samples dry, then incubated overnight at 30 °C (or other desired temperature) and monitored for formation of zones of clearing.

Assay analysis

For each strain, the zones of clearing were categorized as degrees of activity for each phage dilution on the E. coli lawn by the extent clearing: complete, partial or no clearing (Fig. 2). Based on the dilution zones of clearing, the overall cocktail strength of activity for each strain was determined as follows: 1) highly active = complete clearing in 2 or more dilutions; 2) fully active = complete clearing in 1 dilution; 3) partially active: partial clearing in 1 or more dilutions; and 4) not active = no clearing in any dilution (Fig. 2).

Fig. 2.

Spot overlay results and interpretation. Overall strength of phage cocktail against each E. coli strain was assessed by zones of clearing for phage dilutions.

Results for each cocktail activity were consolidated to determine host range and strength of activity against the matched set of E. coli strains (Fig 3A). Activity was represented graphically to distinguish differences in strength for cocktails with similar % host range coverage (Fig. 3B). Here, host range is defined as any level of activity against each E. coli strain.

Fig. 3.

Strength of phage cocktail activity vs UPEC strains. A. Overall activity summarized for each strain based on zones of clearing of 10-fold dilutions using spot overlay. B. Comparison showing overall strength of activity (high, full, partial, none) of each cocktail. Graphical representation allows additional assessment beyond host range activity. Here, cocktail UPP-302 is considered the most active.

Protocol validation

While the level of activity of the phage cocktails against the individual E. coli strains varied, three of the four cocktails had similar overall host range activities of 92–96 % against the collection. Strains tested were from a variety of geographic locations (number of isolates in parenthesis), including North America (11) Central America (2), Europe (5), Middle East (2) and Asia (4); the source of one isolate is unknown. Within each specific cocktail, the activity against individual E. coli strains was variable, which was to be expected (Suppl. Fig 1). In addition, activities across the four cocktails against a given E. coli strain were somewhat variable, but general trends were observed.

Using a spot overlay assay with 10-fold phage dilutions, zones of clearing were classified based on degree of lysis of E. coli. Consolidated dilution results were assembled into a heat map table to visualize the activity for each cocktail. Organizing the results graphically further demonstrates activity differences. Of the cocktails tested, overall strength of activity against each strain indicated that UPP-302 was considered the most active.

Spot overlay assay using an array of phage dilutions has been recognized as providing useful information compared to determining activity by a single concentration. Classifying zones of clearing from serial dilutions has been employed to identify individual phage infectivity and establish host range for an E. coli collection [11]. Ranking was very detailed but due to the many types of zones with subtle differences, interpretation can sometimes be difficult and lead to bias. The method presented here minimizes the classification categories and therefore potential source of bias. In addition, spot assay information was revealed beyond host range, demonstrating details of the types of activity observed. Compared to the simplicity of the spot overlay assay, other methods are more cumbersome (plaque and EOP assays) or require specialized equipment (culture lysis studies). This method could also be applied to other microorgansims, using the appropriate collection for cocktail comparison.

Limitations

The method presented here is qualitative, broadly classifying activity of phage dilutions, particulary partial activity. It also does not characterize activity in detail (e.g., lytic vs lysogenic lysis, lysis without, etc.), information that would be revealed by quantitative method such as EOP. In addition, variations in reagents and prototols may affect reproducability, which can be more of an issue when comparing results between laboratories. Reagent sources should be standardized and protocols conducted consistently to reduce variability. There are likely small differences in how laboratories conduct a particular protocol; therefore, details should be tracked carefully to enable interpretation when comparing results from different labs.

CRediT author statement

Steven Arcidiacono: Conceptualization, Validity tests, Writing (drafts including final draft), Reviewing and Editing. Robert Stote: Validity tests, Reviewing and Editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.