Lytic phage typing against Salmonella enterica.

Stuart Torres1, Teresa Guerrero1, Sonia Zapata Mena1

1
Instituto de Microbiología, Colegio de Ciencias Biológicas y Ambientales, Universidad San
Francisco de Quito, Quito, Ecuador.

INTRODUCTION

Foodborne infections caused by infectious Salmonella enterica serotypes are of great

healthcare importance worldwide (Majowicz, 2010). In Ecuador, 3373 cases of salmonellosis
have been reported in 2019, 1099 cases in 2020 and 587 cases until September of 2021. This
shows a decrease in infections but not a complete stop, also most infections occur in warm-
weather cities like Guayaquil and Morona Santiago (MSP, 2021).

Figure 1 Salmonellosis cases until week 35 of 2021. Most cases come from Guayas and Morona
Santiago province (MSP, 2021).

Generally, poultry is the main animal reservoir of Salmonella spp and in Ecuador, the most
common serotypes isolated are Infantis, Typhimurium and Enteritidis (Vinueza, 2017).
Currently, bacteriophages are being used as an alternative against Salmonella colonization in
poultry, but for a bacteriophage to be useful, it must present a wide host range, it must not
have a lysogenic cycle and must not have virulence or antibiotic-resistances genes that could
be horizontally transmitted to bacteria.

METHODS
 RESULTS

From a phage cocktail we were able to isolate 5 new phages, we named them as ph115-1,
ph115-2, ph115-3, ph115-4, and ph115-5. Host range of these phages against a panel of 18
Salmonella strains are shown on Figure 2.

Figure 2: The host range of 5 Salmonella phages against 18 Salmonella strains were measured
by Spot Test Assay. Phages were tested and scored on the following criteria: 4 - Presence of a
clear inhibition zone around the phage spot, 3 – Presences of a clear inhibition zone with small
bacterial colonies in the middle of the phage spot, 2 – Presence of an unclear or turbid
inhibition zone around the phage spot, 0 – Absence of an inhibition zone.

Certain isolated phages shown similar lytic patterns against the 18 Salmonella strains. Thus, we
classified the phages that had similar lytic activity in a same group, therefore phages ph115-1
and ph115-3 were grouped under the name Ph115-1 and phages ph115-4 and ph115-5 were
grouped under the name Ph115-3. Phage Ph115-2 had a different lytic pattern, so it was not
grouped.

Serum Neutralization Assay

We obtain 3 types of antisera from chicken immunization, the different type of sera was
named according to its corresponding phage: antiph115-1, antiph115-2, and antiph115-3.
Serum-neutralization assay showed that the antisera could block phage infection and lysis
against its Salmonella host (Figure 3).

Figure 3: Serum neutralization assay, 50 μL of each antiserum was pitted against 50 μL of: A)
Ph115-1, B) Ph115-2, and C) Ph115-3
Cross sera-neutralization assay showed that some phages were able to produce plaques after
being incubated with a non-corresponding serum (Figure 4), these results showed that each
isolated phage induced the production of different antibodies that differ in their neutralization
capabilities and could help in the isolation of different types of phages.

Figure 4: Cross sera-neutralization assay of each isolated phage against the 3 types of antisera:
In clockwise order: Phage ph115-1, Phage ph115-2 and Ph115-3.

Isolation of 2 new phages

We were able to isolate 2 new phages from the cross sera-neutralization assay, these phages
were named as F3S1 Delta and F3S2 Alfa. Cross sera-neutralization assay showed that these
new phages could not be neutralized by sera used in previous assays. The morphologies of the
two new phages were observed under SEM, F3S2 Alfa belongs to Siphoviridae Family due to its
isometric head and long non contractile tail, F3S1 Delta belongs to Podoviridae Family due to
its short non contractile tail and isometric head (Figure 5).

Figure 5: Scanning Electron Microscope analysis of phages F3S2 Alfa and F3S1 Delta, head and
tails measurements of each phage are represented below each micrograph, scale = 500 nm.
 CONCLUSION.

Overall, our results demonstrate that phage typing using serum neutralization is a feasible
option in the isolation and characterization of environmental phages for their potential use as
alternative Salmonella control in farms.

References:

Gauger, P., & Vincent, A. (2020). Serum Virus Neutralization Assay for Detection and
Quantitation of Serum Neutralizing Antibodies to Influenza A Virus in Swine. Animal Influenza
Virus, 321-333.

Loc-Carrillo, C., & Abedon, S. (2011). Pros and cons of phage therapy. Bacteriophage, doi:
10.4161/bact.1.2.14590.

Majowicz, H. (2010). The Global Burden of Nontyphoidal Salmonella Gastroenteritis. Clinical

Infectious Diseases, 882–889.

MSP. (2019). Gaceta Epidemiológica Semanal No. 52. Subsistema de vigilancia SIVE- ALERTA.

Vinueza, C. (2017). Salmonella and Campylobacter in broilers at slaughter age: a possible

source for carcasses contamination in Ecuador. Ghent University.