Identification of a broad-spectrum lytic Myoviridae bacteriophage using multidrug resistant Salmonella isolates from pig slaughterhouses as the indicator and its application in combating Salmonella infections

Abstract

Background: Salmonella is a leading foodborne and zoonotic pathogen, and is widely distributed in different nodes of the pork supply chain. In recent years, the increasing prevalence of antimicrobial resistant Salmonella poses a threat to global public health. The purpose of this study is to the prevalence of antimicrobial resistant Salmonella in pig slaughterhouses in Hubei Province in China, and explore the effect of using lytic bacteriophages fighting against antimicrobial resistant Salmonella.

Results: We collected a total of 1289 samples including anal swabs of pigs (862/1289), environmental swabs (204/1289), carcass surface swabs (36/1289) and environmental agar plates (187/1289) from eleven slaughterhouses in seven cities in Hubei Province and recovered 106 Salmonella isolates. Antimicrobial susceptibility testing revealed that these isolates showed a high rate of antimicrobial resistance; over 99.06% (105/106) of them were multidrug resistant. To combat these drug resistant Salmonella, we isolated 37 lytic phages using 106 isolates as indicator bacteria. One of them, designated ph 2-2, which belonged to the Myoviridae family, displayed good capacity to kill Salmonella under different adverse conditions (exposure to different temperatures, pHs, UV, and/or 75% ethanol) and had a wide lytic spectrum. Evaluation in mouse models showed that ph 2-2 was safe and saved 80% (administrated by gavage) and 100% (administrated through intraperitoneal injection) mice from infections caused by Salmonella Typhimurium.

Conclusions: The data presented herein demonstrated that Salmonella contamination remains a problem in some pig slaughter houses in China and Salmonella isolates recovered in slaughter houses displayed a high rate of antimicrobial resistance. In addition, broad-spectrum lytic bacteriophages may represent a good candidate for the development of anti-antimicrobial resistant Salmonella agents.

Keywords: Antimicrobial resistance; Application; Lytic bacteriophage; Prevalence; Salmonella; Slaughterhouse.

Fig. 1

Isolation and antimicrobial resistant phenotypes of Salmonella from pig slaughter houses in Hubei Province in China. A A column chart showing the distribution of different types of samples collected for Salmonella isolation; B A column chart showing the isolation rates of Salmonella from different types of samples; C A column chart showing the distribution of different Salmonella serovars; D A column chart showing the percent isolates of Salmonella with different phenotypes against different antibiotics; E A column chart showing the numbers of Salmonella isolates with resistant phenotypes to different antimicrobial classes; F A column chart showing the numbers of Salmonella isolates resisting different numbers of antimicrobial classes

Fig. 2

Phenotypical characteristics of Salmonella phage ph 2–2. A Plaques of phage ph 2–2 on Salmonella Paratyphi 201,007; B Transmission electron micrograph of phage ph 2–2; C A column chart showing the titers of phage ph 2–2 at different multiplicity of infection (MOI) values; D One-step growth curve of phage ph 2–2; E A line chart showing the effect of phage ph 2–2 killing Salmonella Paratyphi strain 201,107 at different MOI values; F A line chart showing the effect of phage ph 2–2 killing Salmonella Typhimurium 1344 at different MOI values; G A line chart showing the changes of ph 2–2 titers at different temperatures; H A column chart showing the changes of ph 2–2 titers at different pHs; I A column chart showing the changes of ph 2–2 titers exposed to UV for different times; J A column chart showing the changes of ph 2–2 titers exposed to 75% ethanol for different times. Data represents mean ± SD. The significance level was set at P < 0.05 (*) or P < 0.001 (**); ns: No significance

Fig. 3

Genomic characteristics of Salmonella phage ph 2–2. A A circle map showing the complete genome sequence of phage ph 2–2; circles from inside to outside represent the numbers of coding sequences (CDS) and tRNAs (circle 1), depth of illumine sequencing (circle 2), GC skew (circle 3), G + C content (circle 4), and the genome circle (circle 5); B Phylogenetic relationships of bacterial phages belonging to the Myoviridae family; the tree was generated based on the nucleotide sequences of the large subunit of phage terminases; The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site; The optimal tree with the sum of branch length = 0.31527374 is shown; The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches; There were a total of 1605 positions in the final dataset. Evolutionary analyses were conducted in MEGA X; (C) A co-linearity comparison diagram of the genomic organization at the nucleotide level between Salmonella phages ph 2–2, SP2 SHa-2019 (GenBank accession number: MW362867) and SP4 SHa-2019 (GenBank accession number: MW321605); The figure was generated via Easyfig v.2.0. The color code refers to the BLASTn identity of those regions between genomes. Arrows represent putative CDSs encoded by different genomes

Fig. 4

Experimental scheme for the evaluation of ph 2–2 treatment efficacy in mice infected with Salmonella Typhimurium 1344. A Study design of the animal tests; dpc: days post challenge; hpc: hours post challenge; B A line chart showing changes of body weight of mice challenged with Salmonella Typhimurium 1344 by gavage and received a treatment of PBS by gavage (blue line); mice challenged with Salmonella Typhimurium 1344 by gavage and received a treatment of ph 2–2 by gavage (purple line); mice received an administration of PBS (green line) and/or ph 2–2 by gavage (orange line); Data represents mean ± SD. ns: No significance; C Mortality of mice challenged with Salmonella Typhimurium 1344 by gavage and received a treatment of PBS by gavage (blue line); mice challenged with Salmonella Typhimurium 1344 by gavage and received a treatment of ph 2–2 by gavage (purple line); mice received an administration of PBS (green line) and/or ph 2–2 by gavage (orange line); D Mortality of mice challenged with Salmonella Typhimurium 1344 through intraperitoneal injection and received a treatment of PBS through intraperitoneal injection (blue line); mice challenged with Salmonella Typhimurium 1344 through intraperitoneal injection and received a treatment of ph 2–2 through intraperitoneal injection (purple line); mice received an administration of PBS (green line) and/or ph 2–2 through intraperitoneal injection (orange line)