Assessment of phage-mediated inhibition of Salmonella Typhimurium treated with sublethal concentrations of ceftriaxone and ciprofloxacin.

This study was designed to evaluate the synergistic effect of phage (P22) and antibiotic on the inhibition of Salmonella Typhimurium exposed to ceftriaxone (CEF) and ciprofloxacin (CIP). The effect of phage and antibiotic treatments was evaluated by plaque size, disk diffusion, antibiotic susceptibility and phage multiplication assays. The sequential treatment effect of phage and antibiotic was carried out in different treatment order and time for 12 h at 37°C. P22 plaque sizes were increased by 28 and 71%, respectively, in the presence of CEF and CIP. The clear zone sizes in disk diffusion assay were significantly increased to >37 mm in the presence of CEF and CIP compared to the control (28-31 mm). Pre-treatment with P22 enhanced the antimicrobial effect of CIP, showing >2 log reduction after a 12 h incubation. Phage P22 combined with antibiotics (CEF and CIP) effectively inhibited the growth of S. Typhimurium depending on the treatment order and time. These results provide useful information for understanding the synergistic effect of phage and antibiotic treatment which can be an effective option to control antibiotic resistant pathogens.

Assessment of the alteration in phage adsorption rates of antibiotic-resistant Salmonella typhimurium.

This study was designed to evaluate the phage-binding receptors on the surface of antibiotic-sensitive Salmonella typhimurium (ASST) and antibiotic-resistant S. typhimurium (ARST). The antibiotic susceptibilities of plasmid-cured ASST and ARST were evaluated against ampicillin, cephalothin, ciprofloxacin, kanamycin, penicillin, and tetracycline. The capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) were quantified using carbazole assay and HPLC, respectively. The amounts of CPSs and LPSs in ARST were decreased from 108 to 62 µg/ml and 284-111 ng/ml, respectively, after plasmid curing. The adsorption rates of P22, PBST10, and PBST13 to plasmid-uncured and plasmid-cured ASST and ARST were decreased after proteinase K and periodate treatments. The highest reduction in phage adsorption rate was observed for P22 to the plasmid-cured ARST treated with periodate (71%). The relative expression levels of btuB, fhuA, and rfaL were decreased by more than twofold in the plasmid-cured ASST, corresponding to the decrease in the adsorption rates of P22 and PBST10. The plasmid-cured ARST lost the ability to express the ß-lactamase gene, which was related to the loss of resistance to ampicillin, cephalothin, kanamycin, penicillin, and tetracycline. The results provide valuable insights into understanding the interaction between phage and antibiotic-resistant bacteria.

Treatment of Salmonella-Contaminated Eggs and Pork with a Broad-Spectrum, Single Bacteriophage: Assessment of Efficacy and Resistance Development.

Numerous studies have assessed the efficacy of phage-based methods to inhibit Salmonella contamination in food products. As with most antibacterials, bacteria can develop resistance to phage in vitro. Here, we applied a single broad-spectrum Salmonella phage, vB_SalS_SJ_2 (SJ2; 108 PFU; MOI = 10), to Salmonella-contaminated meat and eggs to quantify the development of resistance in actual food matrices. Treatment with a single phage significantly reduced Salmonella Typhimurium contamination in both ground pork and liquid egg at various time points. Similarly, the same phage significantly reduced Salmonella Enteritidis in both food matrices. Efficacy was temperature dependent as larger reductions were seen at higher temperatures (21°C) versus lower temperatures (4°C) at 24 h. Following phage treatment, over 10,000 Salmonella isolates were examined for resistance to the treatment phage. The percentages of phage-resistant Salmonella (either serovar) recovered from phage-treated versus untreated pork did not differ. Conversely, significantly (p < 0.05) higher percentages of phage-resistant Salmonella Typhimurium (92.50% vs. 0.56% of control) and Salmonella Enteritidis (50.83% vs. 0.56% of control) isolates were observed in phage-treated versus untreated egg samples after incubation at room temperature for 48 h. Taken together, these data indicate that the food matrix may influence the emergence of phage resistance with resistance developing more rapidly in foods with less complex microbial communities. Future studies will focus on the impact the development of resistance in production and processing settings may have on the efficacy of phage treatments for longer term biocontrol of pathogens.

Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, ß-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to > 512, 16, 16, and 256 µg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85 µmol/min/ml) and ARST(CIP) (48.83 µmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95 % for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52 % for ARST(CIP) at 15 min of infection. The least lytic activity of phage was 20 % against the ARST(CIP), followed by ASST(CIP) (30 %). The adsorption rate of phage against ARST(CIP) was 52 % at 15 min of infection, which resulted in the decrease in lytic activity (12 %). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens.

In vitro assessment of the susceptibility of planktonic and attached cells of foodborne pathogens to bacteriophage p22-mediated salmonella lysates.

This study was designed to evaluate the lytic activity of bacteriophage P22 against Salmonella Typhimurium ATCC 19585 (Salmonella Typhimurium P22(-)) at various multiplicities of infections (MOIs), the susceptibility of preattached Salmonella cells against bacteriophage P22, and the effect of P22-mediated bacterial lysates (extracellular DNA) on the attachment ability of Listeria monocytogenes ATCC 7644 and enterohemorrhagic Escherichia coli ATCC 700927 to surfaces. The numbers of attached Salmonella Typhimurium P22(-) cells were effectively reduced to below the detection limit (1 log CFU/ml) at the fixed inoculum levels of 3 × 10(-) CFU/ml (MOI = 3.12) and 3 × 10(3) CFU/ml (MOI = 4.12) by bacteriophage P22. The attached Salmonella Typhimurium P22(-) cells remained more than 2 log CFU/ml, with increasing inoculum levels from 3 × 10(4) to 3 × 10(7) CFU/ml infected with 4 × 10(8) PFU/ml of P22. The number of preattached Salmonella Typhimurium P22(-) cells was noticeably reduced by 2.72 log in the presence of P22. The highest specific attachment ability values for Salmonella Typhimurium P22(-), Salmonella Typhimurium ATCC 23555 carrying P22 prophage (Salmonella Typhimurium P22(+)), L. monocytogenes, and enterohemorrhagic E. coli were 2.09, 1.06, 1.86, and 1.08, respectively, in the bacteriophage-mediated cell-free supernatants (CFS) containing high amounts of extracellular DNA. These results suggest that bacteriophages could potentially be used to effectively eliminate planktonic and preattached Salmonella Typhimurium P22(-) cells with increasing MOI. However, further research is needed to understand the role of bacteriophage-induced lysates in bacterial attachment, which can provide useful information for the therapeutic use of bacteriophage in the food system.

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.

Increased burden of illness associated with antimicrobial-resistant Salmonella enterica serotype typhimurium infections.

This study investigated the burden of illness associated with 440 cases of Salmonella enterica serotype Typhimurium infection identified in Canada between December 1999 and November 2000. We categorized case subjects' infections by definitive phage type 104 (DT104) and antimicrobial-resistance patterns. These variables were then investigated as risk factors for hospitalization. Hospitalization was more likely to occur among case subjects whose infections were resistant to at least ampicillin, chloramphenicol and/or kanamycin, streptomycin, sulphamethoxazole, and tetracycline (R-type AK/CSSuT; odds ratio [OR], 2.3; P=.003), compared with case subjects with AK/CSSuT-susceptible infections, and among case subjects with non-DT104 R-type AKSSuT infections (OR, 3.6; P=.005), compared with case subjects with non-DT104 AKSSuT-susceptible infections. In contrast, hospitalization rates did not differ between case subjects with DT104 infections and case subjects with non-DT104 infections or between case subjects with DT104 R-type ACSSuT infections and case subjects with DT104 ACSSuT-susceptible infections. We estimated that 57% of the hospitalizations among AK/CSSuT case subjects and 72% of the hospitalizations among non-DT104 AKSSuT case subjects were attributable to the resistance patterns of the infections.