Strategic combination of bacteriophages with highly susceptible cells for enhanced intestinal settlement and resistant cell killing.

Avian pathogenic Escherichia coli (APEC) causes enormous economic losses and is a primary contributor to the emergence of multidrug resistance (MDR)-related problems in the poultry industry. Bacteriophage (phage) therapy has been successful in controlling MDR, but phage-resistant variants have rapidly emerged through the horizontal transmission of diverse phage defense systems carried on mobile genetic elements. Consequently, while multiple phage cocktails are recommended for phage therapy, there is a growing need to explore simpler and more cost-effective phage treatment alternatives. In this study, we characterized two novel O78-specific APEC phages, φWAO78-1 and φHAO78-1, in terms of their morphology, genome, physicochemical stability and growth kinetics. Additionally, we assessed the susceptibility of thirty-two O78 APEC strains to these phages. We analyzed the roles of highly susceptible cells in intestinal settlement and fecal shedding (susceptible cell-assisted intestinal settlement and shedding, SAIS) of phages in chickens via coinoculation with phages. Furthermore, we evaluated a new strategy, susceptible cell-assisted resistant cell killing (SARK), by comparing phage susceptibility between resistant cells alone and a mixture of resistant and highly susceptible cells in vitro. As expected, high proportions of O78 APEC strains had already acquired multiple phage defense systems, exhibiting considerable resistance to φWAO78-1 and φHAO78-1. Coinoculation of highly susceptible cells with phages prolonged phage shedding in feces, and the coexistence of susceptible cells markedly increased the phage susceptibility of resistant cells. Therefore, the SAIS and SARK strategies were demonstrated to be promising both in vivo and in vitro.

Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains.

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Optimized Detoxification of a Live Attenuated Vaccine Strain (SG9R) to Improve Vaccine Strategy against Fowl Typhoid.

The live attenuated vaccine strain, SG9R, has been used against fowl typhoid worldwide, but it can revert to the pathogenic smooth strain owing to single nucleotide changes such as nonsense mutations in the rfaJ gene. As SG9R possesses an intact Salmonella plasmid with virulence genes, it exhibits dormant pathogenicity and can cause fowl typhoid in young chicks and stressed or immunocompromised brown egg-laying hens. To tackle these issues, we knocked out the rfaJ gene of SG9R (named Safe-9R) to eliminate the reversion risk and generated detoxified strains of Safe-9R by knocking out lpxL, lpxM, pagP, and phoP/phoQ genes to attenuate the virulence. Among the knockout strains, live ΔlpxL- (Dtx-9RL) and ΔlpxM-9R (Dtx-9RM) strains induced remarkably less expression of inflammatory cytokines in chicken macrophage cells, and oil emulsion (OE) Dtx-9RL did not cause body weight loss in chicks. Live Dtx-9RM exhibited efficacy against field strain challenge in one week without any bacterial re-isolation, while the un-detoxified strains showed the development of severe liver lesions and re-isolation of challenged strains. Thus, SG9R was optimally detoxified by knockout of lpxL and lpxM, and Dtx-9RL and Dtx-9RM might be applicable as OE and live vaccines, respectively, to prevent fowl typhoid irrespective of the age of chickens.

Comparative genomics of bovine mastitis-origin Staphylococcus aureus strains classified into prevalent human genotypes.

Humans may serve as a reservoir host of Staphylococcus aureus, resulting in transmission to animals. Previously, we used RNA polymerase beta subunit gene (rpoB)-based genotyping and classified S. aureus strains into rpoB sequence types (RSTs). According to our previous work, the predominant genotypes of S. aureus in humans and cows differ in Korea, but some predominant genotypes (RST4-1 and RST2-1) in humans have been isolated from bovine mastitis. Therefore, it needs to be determined whether some strains of the predominant human genotypes have adapted to or caused occasional infections in cows. We determined the whole genome sequences of 2 bovine mastitis-origin strains, PMB179 (RST4-1) and PMB196 (RST2-1), and performed comparative genomics with the corresponding RST4-1 and RST2-1 S. aureus strains in the NCBI database. We identified 257 and 180 pseudogenes among 131 RST4-1 and 54 RST2-1 strains, respectively, for the comparison of pseudogene profiles. RST4-1 strains shared more common pseudogenes than RST2-1 strains, and some epidemiologically related strains shared common pseudogenes. However, most of the pseudogenes were strain-specific, and diverse pseudogene profiles were apparent in both the RST4-1 and RST2-1 strains. Furthermore, analysis of the mobile genetic elements, virulence genes, and antibiotic resistance genes revealed no molecular markers to differentiate PMB179 and PMB196 from human strains. Interestingly, the collective comparison of RST4-1 or RST2-1 strains revealed cumulative acquisition steps of genomic islands and antibiotic resistance genes. In conclusion, our data support PMB179 and PMB196 causing occasional infections that result in bovine mastitis.

Comparison of Humoral Immune Responses to Different Forms of Salmonella enterica Serovar Gallinarum Biovar Gallinarum.

Fowl typhoid is caused by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG), and live attenuated, rough vaccine strains have been used. Both humoral and cellular immune responses are involved in protection, but the humoral responses to different forms of SG antigens are unclear. In this study, we compared humoral responses to a killed oil-emulsion (OE) smooth vaccine (SG002) and its rough mutant vaccine (SR2-N6) strains using proteomics techniques. We identified two immunogenic outer membrane proteins (OmpA and OmpX), and the selected linear epitopes were successfully applied in peptide-ELISA. Our peptide- and total OMP-ELISAs were used to compare the temporal humoral responses to various SG antigens: OE SG002 and SR2-N6; live, killed [PBS-suspension (PS) and OE)] and mixed (live and PS) formulations of another rough vaccine strain (SG 9R); and orally challenge with a field strain. Serum antibodies to the linear epitopes of OmpA and OmpX lasted only for the first 2 weeks, but serum antibodies against OMPs increased over time. The rough strain (SR2-N6) and mixed SG 9R induced higher serum antibody titers than the smooth strain (SG002) and single SG 9R (OE, live and PS SG 9R), respectively. Infection with the field strain delayed the serum antibody response by ~2 weeks. Mucosal immunity was not induced by any formulation, except for infection with the field strain after SG 9R vaccination. Thus, our results may be useful to understand humoral immunity against various SG antigens and to improve vaccine programs and serological diagnosis in the field.

Evolution of a major bovine mastitic genotype (rpoB sequence type 10-2) of Staphylococcus aureus in cows.

Staphylococcus aureus is the major pathogen leading to bovine mastitis globally while livestock-associated methicillin resistant S. aureus (LA-MRSA) has become a potential threat to public health. MRSA from bovine mastitis is not common but a methicillin susceptible S. aureus (MSSA) genotype, rpoB sequence type (RST)10-2 (RST10-2), is prevalent in Korea. To date, many genomic sequences from S. aureus have been elucidated, but the complete genome sequences of RST10-2 MSSA from bovine mastitis has never been reported. In this study, we determined the complete genome sequence of two RST10-2 MSSA that differ from each other in staphylococcal protein A and molecular prophage types [PMB64-1 (t2489/ mPPT0) and PMB81-4 (t127/mPPT1-2-3)] and conducted a comparative genomics study. The genomic sequences of PMB64-1 and PMB81-4 were more homologous to the representative human RST10-2 strains (MSSA476, MW2 etc.) compared to other RSTs. Most of them shared five common pseudogenes, along with high amino acid identity of four variable virulence genes that were identified in this study. However, PMB64-1 and PMB81-4 acquired different strainspecific pseudogenes and mobile genetic elements than the human strains. The unique pseudogene profile and high identity of the virulence genes were verified in RST10-2 field strains from bovine mastitis. Thus, bovine mastitic RST10-2 MSSA may have an evolutionary relationship with the human RST10-2 community-associated (CA) MSSA and CA-MRSA strains but may have adapted to cows.

Molecular evolution of Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum in the field.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid (FT) and substantial economic loss in Korea due to egg drop syndrome and mortality. Despite the extensive use of vaccines, FT still occurs in the field. Therefore, the emergence of more pathogenic SG or the recovered pathogenicity of a vaccine strain has been suspected. SpvB, an ADP-ribosyl transferase, is a major pathogenesis determinant, and the length of the polyproline linker (PPL) of SpvB affects pathogenic potency. SG strains accumulate pseudogenes in their genomes during host adaptation, and pseudogene profiling may provide evolutionary information. In this study, we found that the PPL length of Korean SG isolates varied from 11 to 21 prolines and was longer than that of a live vaccine strain, SG 9R (9 prolines). According to growth competition in chickens, the growth of an SG isolate with a PPL length of 17 prolines exceeded that of an SG isolate with a PPL length of 15 prolines. We investigated the pseudogenes of the field isolates, SG 9R and reference strains in GenBank by resequencing and comparative genomics. The pseudogene profiles of the field isolates were notably different from those of the foreign SG strains, and they were subdivided into 7 pseudogene subgroups. Collectively, the field isolates had gradually evolved by changing PPL length and acquiring additional pseudogenes. Thus, the characterization of PPL length and pseudogene profiling may be useful to understand the molecular evolution of SG and the epidemiology of FT.

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.