Assessment of the potential of phage-antibiotic synergy to induce collateral sensitivity in Salmonella Typhimurium.

This study was designed to evaluate the synergistic effect of phage and antibiotic on the induction of collateral sensitivity in Salmonella Typhimurium. The synergistic effects of Salmonella phage PBST32 combined with ciprofloxacin (CIP) against S. Typhimurium KCCM 40253 (STKCCM) were evaluated using a fractional inhibitory concentration (FIC) assay. The CIP susceptibility of STKCCM was increased when combined with PBST32, showing 16-fold decrease at 7 log PFU/mL. The combination of 1/2 × MIC of CIP and PBST32 (CIP[1/2]+PBST32) effectively inhibited the growth of STKCCM up to below the detection limit (1.3 log CFU/mL) after 12 h of incubation at 37 °C. The significant reduction in bacterial swimming motility was observed for PBST32 and CIP[1/4]+PBST32. The CIP[1/4]+PBST32 increased the fitness cost (relative fitness = 0.57) and decreased the cross-resistance to different classes of antibiotics. STKCCM treated with PBST32 alone treatment exhibited the highest coefficient of variation (90%), followed by CIP[1/4]+PBST32 (75%). These results suggest that the combination of PBST32 and CIP can be used to control bacterial pathogens.

Assessment of phage-mediated control of antibiotic-resistant Salmonella Typhimurium during the transition from planktonic to biofilm cells.

This study was designed to evaluate the abilities of phage P22 to lyse, eradiate, and disperse the biofilm cells of Salmonella enterica serovar Typhimurium ATCC 19585 (STWT), ciprofloxacin-induced Typhimurium ATCC 19585 (STCIP), S. Typhimurium KCCM 40253 (STKCCM), and multidrug-resistant S. Typhimurium CCARM 8009 (STCCARM) in association with hydrophobicity, auto-aggregation, motility, protein content, extracellular DNA, and depolymerase activity. The affinity to hexadecane was significantly increased in STWT, STKCCM, and STCCARM cells after P22 infection. All strains tested showed relatively higher auto-aggregation abilities in the presence of P22 than the absence of P22. STKCCM showed the greatest auto-aggregative ability (23%) in the presence of P22, while STWT showed the least auto-aggregative ability (9%) in the absence of P22. The bacterial swimming motility affected the bacterial attachment at the early stage of biofilm formation. The red, dry and rough morphotype was observed for all strains tested. The numbers of STWT, STCIP, and STKCCM planktonic cells were considerably reduced by 7.2, 5.0, and 5.0 log CFU/ml, respectively, and STWT, STCIP, and STKCCM biofilm-forming cells were reduced by 5.8, 4.5, and 4.9 log, respectively, after 24 h of phage infection. The depolymerase produced by phages were confirmed by the presence of outer rim of plaques. Phages could be considered as promising alternatives for the control of biofilms due to their advantages including enzymatic degradation of extracellular biofilm matrix. The study would provide useful information for understanding the dynamic interactions between phages and biofilms and also designing the effective phage-based control system as an alterative strategy against biofilms.

Assessment of cooperative antibiotic resistance of Salmonella Typhimurium within heterogeneous population.

This study was designed to investigate the cooperative resistance in the mixed culture of antibiotic-sensitive and antibiotic-resistant Salmonella Typhimurium. Strains of S. Typhimurium ATCC 19585 (STS) and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STR) grown in single and mixture with 1 × MIC ceftriaxone (CEF) were used to determine the viability, ß-lactamase activity, and gene expression. The MIC50 values of STR to CEF was increased by more than 5-fold with increasing inoculum densities from 102 to 107 CFU/mL. STS was resistant to 1 × MIC CEF in the mixed culture of STS and STR, showing the more than 108 CFU/mL after 20 h of incubation at 37 °C. The highest ß-lactamase activity was 18 µmol/min/mL in the mixed culture, corresponding to the highest relative expression of ß-lactamase-related genes (blaTEM). These results shed new light on the cooperative resistance of antibiotic-sensitive bacteria within a heterogeneous population including ß-lactamase-producing bacteria.

Assessment of cross-resistance potential to serial antibiotic treatments in antibiotic-resistant Salmonella Typhimurium.

This study was designed to evaluate the cross-resistance of Salmonella Typhimurium ATCC 19585 (STWT), ciprofloxacin-resistant S. Typhimurium (STCIP), and clinically isolated S. Typhimurium CCARM 8009 (STCLI) serially exposed to a half MIC of ceftriaxone (CEF), ciprofloxacin (CIP), polymyxin B (POL), or tetracycline (TET), followed by 1 MIC of CEF, CIP, POL, and TET. The viability changes of STWT, STCIP, and STCLI exposed to different classes of antibiotics were measured to evaluate the development of cross-resistance. The relative fitness was also estimated to evaluate the reproductive success of STWT, STCIP, and STCLI consecutively exposed to a half and 1 MIC of CEF, CIP, POL, or TET. The expression levels of efflux pump-related genes (acrA, acrB, mdsA, mdsB, mdtB, and tolC) were evaluated by using a quantitative RT-PCR assay. The MIC of CIP against STCIP was increased by 128-fold compared to STWT. STCLI was highly resistant to POL (MIC = 8 µg/ml) and TET (MIC = 256 µg/ml). STWT exposed to CIP [1/2] was susceptible to POL [1] than CEF [1], CIP [1], and TET [1], STCIP exposed to CIP [1/2] was susceptible to CEF [1] and POL [1], and STCLI exposed to CIP [1/2] was susceptible to POL [1]. STCIP was cross-resistant to CEF [1/2]-CIP [1], CIP [1/2]-CIP [1], POL [1/2]-CIP [1], and TET [1/2]-CIP (Ochoa et al., 2020) [1]. The efflux pump-related genes (acrA, acrB, mdsA, mdsB, mdtB, and tolC) were overexpressed in STWT, STCIP, and STCLI exposed to serial antibiotic treatments, which was related to the development of cross-resistance. The antibiotic susceptibility profiles of STWT, STCIP, and STCLI exposed to serial antibiotic treatments are worth understanding the evolution of antibiotic resistance and developing effective therapeutic regimens against antibiotic-resistant bacteria.

Assessment of antibiotic resistance in bacteriophage-insensitive Klebsiella pneumoniae.

This study was design to evaluate the physiological properties of bacteriophage-insensitive Klebsiella pneumoniae (BIKP) mutants in association with the antibiotic cross-resistance, ß-lactamase activity, and gene expression. Klebsiella pneumoniae ATCC 23357(KPWT), ciprofloxacin-induced antibiotic-resistant K. pneumoniae ATCC 23357 (KPCIP), and clinically isolated antibiotic-resistant K. pneumoniae 10263 (KPCLI) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive K. pneumoniae ATCC 23357 (BIKPWT), ciprofloxacin-induced K. pneumoniae ATCC 23357 (BIKPCIP), and clinically isolated antibiotic-resistant K. pneumoniae CCARM 10263 (BIKPCLI). BIKPWT, BIKPCIP, and BIKPCLI were resistant to Klebsiella bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKPWT. The relative expression levels of vagC was increased by more than 8-folds in BIKPWT, corresponding to the increased ß-lactamase activity. The aac(6')-Ib-cr was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with ß-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.

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.

Phenotypic and Genotypic Responses of Foodborne Pathogens to Sublethal Concentrations of Lactic Acid and Sodium Chloride.

The aim of this study was to evaluate the phenotypic and genotypic responses of Salmonella Typhimurium ATCC 19585 (ST) and Staphylococcus aureus KACC 13236 (SA) preadapted to sublethal concentrations of lactic acid (LA) and sodium chloride (NaCl) for 48 hr at 37°C, followed by re-exposure to lethal concentrations of LA and NaCl for 24 hr at 37°C. ST and SA treated in a sequential and ordered manner with LA and NaCl were assigned as LA-LA, LA-NaCl, NaCl-LA, and NaCl-NaCl. The treatments, LA-LA, LA-NaCl, NaCl-LA, and NaCl-NaCl, were evaluated by antimicrobial susceptibility, bacterial fluctuation, relative fitness, zeta potential, and gene expression. The MICt/MICc ratios of LA, NaCl, CIP, GEN, and TET against ST treated with LA-LA were 1.0 to 0.8, 0.8, 0.3, 0.4, and 0.5, respectively. The MICt/MICc ratios of NaCl, CIP, GEN, and TET were between 0.5-0.8 for SA treated with LA-LA. ST treated with LA-LA and SA treated with LA-NaCl exhibited the highest coefficient of variance. The lowest relative fitness was observed at ST treated with LA-LA (0.5). ST and SA treated with LA-LA showed the lowest zeta potential. The transporter-, toxin-antitoxin system-, chaperone protein-, and SOS response-related genes were suppressed at ST and SA treated with LA-LA. The transporter-, toxin-antitoxin system-, and chaperone protein-related genes were overexpressed in SA treated with LA-NaCl, NaCl-LA, and NaCl-NaCl. The results suggest that ST and SA treated with LA-LA, LA-NaCl, NaCl-LA, and NaCl-NaCl could induce collateral sensitivity and cross-resistance.

Assessment of phenotypic heterogeneity in Salmonella Typhimurium preadapted to ciprofloxacin and tetracycline.

Bacterial population exposed to stressful antibiotic conditions consists of various subpopulations such as tolerant, persister, and resistant cells. The aim of this study was to evaluate the phenotypic heterogeneity of Salmonella Typhimurium preadapted to sublethal concentrations of antibiotics. Salmonella Typhimurium cells were treated with 1/2 × MIC of antibiotics for the first 48 h and successively 1 × MIC for the second 24 h at 37°C, including untreated control (CON), no antibiotic and 1 × MIC ciprofloxacin (NON-CIP), 1/2 × MIC ciprofloxacin and 1 × MIC ciprofloxacin (CIP-CIP), 1/2 × MIC tetracycline and 1 × MIC ciprofloxacin (TET-CIP), no antibiotic and 1 × MIC tetracycline (NON-TET), 1/2 × MIC ciprofloxacin and 1 × MIC tetracycline (CIP-TET), and 1/2 × MIC tetracycline and 1 × MIC tetracycline (TET-TET). All treatments were evaluated by antibiotic susceptibility, ATP level, relative fitness, cross-resistance, and persistence. S. Typhimurium cells were more susceptible to non-adapted NON-CIP and NON-TET (>3-log reduction) than pre-adapted CIP-CIP, TET-CIP, CIP-TET, and TET-TET. CON exhibited the highest ATP level, corresponding to the viable cell number. The relative fitness levels were more than 0.95 for all treatments, except for NON-CIP (0.78). The resistance to ciprofloxacin and tetracycline was increased at all treatments with the exception of NON-TET. The persister cells were noticeably induced at CIP-TET treatment, showing more than 5 log CFU mL-1. The results suggest that the antibiotic preadaptation led to heterogeneous populations including persisters that can develop to resistance. This study provides new insight in the bacterial persistence associated with their potential risk and paves the way to design antibiotic therapy targeting dormant bacteria.

Bacterial Stress Responses as Potential Targets in Overcoming Antibiotic Resistance.

Bacteria can be adapted to adverse and detrimental conditions that induce general and specific responses to DNA damage as well as acid, heat, cold, starvation, oxidative, envelope, and osmotic stresses. The stress-triggered regulatory systems are involved in bacterial survival processes, such as adaptation, physiological changes, virulence potential, and antibiotic resistance. Antibiotic susceptibility to several antibiotics is reduced due to the activation of stress responses in cellular physiology by the stimulation of resistance mechanisms, the promotion of a resistant lifestyle (biofilm or persistence), and/or the induction of resistance mutations. Hence, the activation of bacterial stress responses poses a serious threat to the efficacy and clinical success of antibiotic therapy. Bacterial stress responses can be potential targets for therapeutic alternatives to antibiotics. An understanding of the regulation of stress response in association with antibiotic resistance provides useful information for the discovery of novel antimicrobial adjuvants and the development of effective therapeutic strategies to control antibiotic resistance in bacteria. Therefore, this review discusses bacterial stress responses linked to antibiotic resistance in Gram-negative bacteria and also provides information on novel therapies targeting bacterial stress responses that have been identified as potential candidates for the effective control of Gram-negative antibiotic-resistant bacteria.

Application of DNA barcoding for ensuring food safety and quality.

With increasing international food trade, food quality and safety are high priority worldwide. The consumption of contaminated and adulterated food can cause serious health problems such as infectious diseases and allergies. Therefore, the authentication and traceability systems are needed to improve food safety. The mitochondrial DNA can be used for species authentication of food and food products. Effective DNA barcode markers have been developed to correctly identify species. The US FDA approved to the use of DNA barcoding for various food products. The DNA barcoding technology can be used as a regulatory tool for identification and authenticity. The application of DNA barcoding can reduce the microbiological and toxicological risks associated with the consumption of food and food products. DNA barcoding can be a gold-standard method in food authenticity and fraud detection. This review describes the DNA barcoding method for preventing food fraud and adulteration in meat, fish, and medicinal plants.

Variability in Adaptive Resistance of Salmonella Typhimurium to Sublethal Levels of Antibiotics.

This study was designed to evaluate the adaptive resistance of Salmonella Typhimurium under continuous sublethal selective pressure. Salmonella Typhimurium ATCC 19585 (STATCC) and S. Typhimurium CCARM 8009 (STCCARM) were sequentially cultured for 3 days at 37 °C in trypticase soy broth containing 1/2 × MICs of cefotaxime (CEF1/2), chloramphenicol (CHL1/2), gentamicin (GEN1/2), and polymyxin B (POL1/2). The STATCC and STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were evaluated using antibiotic susceptibility, cross-resistance, and relative fitness. The susceptibilities of STATCC exposed to GEN1/2 and POL1/2 were increased by a 2-fold (gentamicin) and 8-fold (polymyxin B) increase in minimum inhibitory concentration (MIC) values, respectively. The MIC values of STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were increased by 4-fold (cefotaxime), 2-fold (chloramphenicol), 2-fold (gentamicin), and 8-fold (polymyxin B). The highest heterogeneous fractions were observed for the STATCC exposed to CEF1/2 (38%) and POL1/2 (82%). The STCCARM exposed to GEN1/2 was cross-resistant to cefotaxime (p < 0.05), chloramphenicol (p < 0.01), and polymyxin B (p < 0.05). The highest relative fitness levels were 0.92 and 0.96, respectively, in STATCC exposed to CEF1/2 and STCCARM exposed to POL1/2. This study provides new insight into the fate of persistent cells and also guidance for antibiotic use.

Role of Efflux Pump-Mediated Antibiotic Resistance in Quorum Sensing-Regulated Biofilm Formation by Salmonella Typhimurium.

This study was designed to assess the influence of efflux pump activity on the biofilm formation in Salmonella Typhimurium. Salmonella enterica subsp. enterica serovar Typhimurium ATCC 19585 (STWT) and clinically isolated S. Typhimurium CCARM 8009 (STCI) were treated with ceftriaxone (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), norfloxacin (NOR), and tetracycline (TET) in autoinducer-containing media in the absence and presence of phenylalanine-arginine ß-naphthylamide (PAßN) to compare efflux pump activity with biofilm-forming ability. The susceptibilities of STWT and STCI were increased in the presence of PAßN. ERY+PAßN showed the highest decrease in the minimum inhibitory concentration (MIC) of ERY from 256 to 2 µg/mL against STWT and STCI. The antimicrobial activity of NOR against planktonic cells was significantly increased in the presence of PAßN, showing the lowest numbers of STWT (3.2 log CFU/cm2), and the TET+PAßN effectively inhibited the growth of STCI (5.2 log CFU/cm2). The lowest biofilm-forming abilities were observed at NOR+PAßN against STWT (biofilm-forming index, BFI < 0.41) and CEF+PAßN against STCI (BFI = 0.32). The bacteria swimming motility and relative fitness varied depending on the antibiotic and PAßN treatments. The motility diameters of STWT were significantly decreased by NOR+PAßN (6 mm) and TET+PAßN (15 mm), while the lowest motility of STCI was observed at CIP+PAßN (8 mm). The significant decrease in the relative fitness levels of STWT and STCI was observed at CIP+PAßN and NOR+PAßN. The PAßN as an efflux pump inhibitor (EPI) can improve the antimicrobial and anti-biofilm efficacy of antibiotics against S. Typhimurium. This study provides useful information for understanding the role of efflux pump activity in quorum sensing-regulated biofilm formation and also emphasizes the necessity of the discovery of novel EPIs for controlling biofilm formation by antibiotic-resistant pathogens.

Effectiveness of Antibiotic Combination Treatments to Control Heteroresistant Salmonella Typhimurium.

This study was designed to assess the interactions between antibiotics in combination treatments of Salmonella enterica serovar Typhimurium in association with the development of antibiotic heteroresistance. Salmonella Typhimurium ATCC 19585 (STWT), ciprofloxacin (CIP)-induced S. Typhimurium ATCC 19585 (STCIP), and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 (STCLI) treated with antibiotic alone (cephalothin [CEP], CIP, ceftriaxone [CEF], and tobramycin [TOB]) and combination antibiotics (CEP-CIP and CEF-TOB) were used to compare the antibiotic susceptibility, resistance fitness, and cross-resistance. The susceptibilities of STWT, STCIP, and STCLI to CEP were not significant differences between CEP and CEP-CIP treatments, whereas those of STWT, STCIP, and STCLI to TOB were significant differences between TOB and CEF-TOB treatments. The interactions between CEP and CIP in the combination treatment showed mutually synergistic activities against STWT and STCLI. For the CEF-TOB combination treatments, TOB helped enhance the antibiotic activity of CEF against STWT, showing directional synergistic effect. The CEF-TOB combination treatment increased bactericidal activity against STWT, STCIP, and STCLI without causing injured cells. The combination antibiotic treatments (CEP-CIP and CEF-TOB) increased the fitness cost (relative fitness = 0.7) and decreased the cross-resistance of STWT, STCIP, and STCLI when exposed to different classes of antibiotics. This study provides new insight for designing combination antibiotic regimens that can synergistically enhance the antimicrobial activity against antibiotic-resistant Salmonella and inhibit the emergence of cross-resistance to different classes of antibiotics.

Effects of Incubation Time and Inoculation Level on the Stabilities of Bacteriostatic and Bactericidal Antibiotics against Salmonella Typhimurium.

This study was designed to evaluate the stability of chloramphenicol, erythromycin, tetracycline, cephalothin, ciprofloxacin, and tobramycin against antibiotic-sensitive Salmonella Typhimurium (ASST) and antibiotic-resistant S. Typhimurium (ARST) during the broth microdilution assay. The antimicrobial activity in association with antibiotic stability was measured by using antibiotic susceptibility, time-delayed inoculation, time-extended incubation, and inoculum effect assays. The loss of the antimicrobial activity of cephalothin against ASST exposed to 1 MIC was observed for the 10 h delayed inoculation. The antimicrobial activities of tetracycline and ciprofloxacin against ASST and ARST exposed to ½ MIC were significantly decreased after the 10 h delayed inoculation. All antibiotics used in this study, except for ciprofloxacin, showed the considerable losses of antimicrobial activities against ASST and ARST after 40 h of incubation at 37 °C when compared to the 20 h of incubation during AST. Compared to the standard inoculum level (6 log CFU/mL), the MIC0.1 values of bactericidal antibiotics, ciprofloxacin and tobramycin against ASST were increased by more than 4-fold at the high inoculum level of 9 log CFU/mL. This would provide practical information for better understanding the clinical efficacy of the currently used antibiotics by considering the antibiotic stability during incubation time at different inoculum levels.

The Role of Bacterial Membrane Vesicles in the Dissemination of Antibiotic Resistance and as Promising Carriers for Therapeutic Agent Delivery.

The rapid emergence and spread of antibiotic-resistant bacteria continues to be an issue difficult to deal with, especially in the clinical, animal husbandry, and food fields. The occurrence of multidrug-resistant bacteria renders treatment with antibiotics ineffective. Therefore, the development of new therapeutic methods is a worthwhile research endeavor in treating infections caused by antibiotic-resistant bacteria. Recently, bacterial membrane vesicles (BMVs) have been investigated as a possible approach to drug delivery and vaccine development. The BMVs are released by both pathogenic and non-pathogenic Gram-positive and Gram-negative bacteria, containing various components originating from the cytoplasm and the cell envelope. The BMVs are able to transform bacteria with genes that encode enzymes such as proteases, glycosidases, and peptidases, resulting in the enhanced antibiotic resistance in bacteria. The BMVs can increase the resistance of bacteria to antibiotics. However, the biogenesis and functions of BMVs are not fully understood in association with the bacterial pathogenesis. Therefore, this review aims to discuss BMV-associated antibiotic resistance and BMV-based therapeutic interventions.

Development of de novo resistance in Salmonella Typhimurium treated with antibiotic combinations.

This study was designed to evaluate the evolution of antibiotic resistance in Salmonella enterica serovar Typhimurium treated with the combination of antibiotics. The experimental evolution of antibiotic resistance of S. Typhimurium was evaluated either under single antibiotic (kanamycin, KAN; penicillin, PEN; erythromycin, ERY) or in combination of two antibiotics (KAN + PEN or KAN + ERY) as measured by fractional inhibitory concentrations (FICs), stepwise resistance selection, cross-resistance evaluation, resistance fitness and relative gene expression. KAN + PEN and KAN + ERY showed the synergistic effect against S. Typhimurium (FIC index < 0.5). KAN + ERY delayed the induction of de novo mutations in S. Typhimurium. The cross-resistance of S. Typhimurium to all antibiotics except ERY and tetracycline was observed in KAN and PEN alone. The fitness cost was lower in single antibiotic treatments than combinations. The highest relative fitness was 0.91 in PEN, followed by KAN (0.84) and ERY (0.78), indicating the low fitness costs in single antibiotic treatments. The overexpression of efflux pump-related genes (acrA and acrB), outer membrane-related gene (ompC) and adherence-related gene (csgD) were observed in the single antibiotic treatments. Our results suggest that KAN + PEN and KAN + ERY could be used as a potential therapeutic treatment by decreasing the evolution of antibiotic resistance in S. Typhimurium and reusing conventional antibiotics.