Postantibiotic Effect Research Articles

Detection of Antimicrobial-Induced Survival/Dead Bacteria via mEos4b Photoconversion: A Preliminary Study.

The escalating prevalence of hospital-acquired infections poses a critical challenge for healthcare systems worldwide. Effective management requires rapid identification of pathogens and their antibiotic resistance profiles. In this study, we utilized the photoconvertible mEos4b protein, which transitions from green to red fluorescence upon blue light exposure, to distinguish live from dead bacteria. The mEos4b gene was cloned into a prokaryotic vector and expressed in Escherichia coli BL21. The Minimum Inhibitory Concentration (MIC) of the transgenic bacteria was determined for five antibiotics, followed by a post-antibiotic effect assessment over a two-hour exposure period. The optimal photoconversion time for mEos4b was established as 90 seconds, and confocal microscopy was used to visualize live (green) and dead (red) cells post-exposure. The mEos4b-TR system proved highly specific, accurately distinguishing live and dead bacteria without producing false positives, even in control groups, which is a common issue in commercial live-dead kits. By relying on cellular metabolic activity rather than dyes, this system minimizes nonspecific interactions and contamination, making it more reliable than traditional methods prone to false readings. These results highlight the potential of the mEos4b-TR system as a superior alternative for rapid, precise bacterial viability assessments, particularly in determining antibiotic susceptibility. This preliminary study demonstrates the system's differentiation of viable and non-viable cells, suggesting its potential application in future studies involving novel antibacterial agents to refine antibiotic sensitivity testing.

Biological Properties of S. warneri KYS-164 Isolated from Kefir Grains.

Staphylococcus worderi KYS-164, isolated from homemade Tibetan kefir grains, produces bacteriocin-like inhibitory substances (BLIS), which are peptides with antimicrobial properties, but have not been fully characterized. The research on BLIS will lay the foundation for mining new bacteriocins. In this study, the optimal culture conditions for the production of highly active BLIS were found to be incubation at 30 °C and 120 rpm, and the most effective extraction method was ammonium sulfate precipitation (ASP) using ammonium sulfate at 80% saturation. The postantibiotic effect (PAE) of BLIS on Staphylococcus aureus CICC 10384 is significant, with a 4 × MIC BLIS concentration able to prolong the PAE to 2.39 h. BLIS has excellent biosafety, with no deleterious effects observed at 8 × MIC concentration. Gas chromatography-ion mobility spectrometry (GC-IMS) was used to analyze the volatile compounds synthesized by Staphylococcus warneri KYS-164 during its growth. Hydroxycitronellal, ethyl pyruvate, and α-pinene were found to be unique substances produced by this strain, which can provide fresh, refreshing floral and fruity aromas as well as strong pine and resinous aromas in the process of kefir grain fermentation of milk. Analysis of the S. warneri KYS-164 genome provided insights into the major metabolic pathways in which genes expressed in this strain are involved. This study represents the first isolation of S. warneri KYS-164 from kefir grains prepared by Tibetan families, and provides a comprehensive analysis of its physicochemical properties. This research provides a solid foundation for better understanding and utilization of S. warneri KYS-164.

Post-Antibiotic and Post-Antibiotic Sub-Minimum Inhibitory Concentration Effects of Carvacrol against Salmonella Typhimurium.

Carvacrol is a compound present in essential oils with proven antimicrobial activity against numerous pathogens. We firstly determine the post-antibiotic effect (PAE) of carvacrol (1×, 2×, 4× MIC) and post-antibiotic sub-minimum inhibitory concentration (MIC) effect (1× + 0.25× MIC and 2× + 0.25× MIC) for two concentrations of Salmonella Typhimurium ATCC14028 (106 and 108 CFU/mL). Prior to testing, the minimum concentration and exposure time to achieve the bacterial inhibition (MIC 0.6 mg/mL and 10 min) were determined by broth microdilution and time-kill curve methods, respectively. At the MIC, carvacrol did not generate any PAE. At twice the MIC, the PAE was 2 h with the standard inoculum (106 CFU/mL) and 1 h with the high-density inoculum (108 CFU/mL). At 4× MIC concentrations, the PAE was higher in both cases > 43.5 h. Continuous exposure of post-antibiotic phase bacteria (1× and 2× MIC) to carvacrol at 0.25× MIC (0.15 mg/mL) resulted in an increase in PAE (PA-SME) above 43.5 h with both inocula. These results suggest that the PA-SME of carvacrol for S. Typhimurium can be significantly prolonged by increasing the sub-MICs, which would allow dose spacing, reduce adverse effects and improve its efficacy in the treatment of infected animals and as a disinfectant in agri-food facilities.

Improved N-phenylpyrrolamide inhibitors of DNA gyrase as antibacterial agents for high-priority bacterial strains

In this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC50 values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes. All tested compounds show high selectivity towards the human isoform DNA topoisomerase IIα. Compounds 6a, 6d, 6e and 6f show MIC values between 0.031 and 0.0625 μg/mL against vancomycin-intermediate S. aureus (VISA) and Enterococcus faecalis strains. Compound 6g shows an inhibitory effect against the methicillin-resistant S. aureus strain (MRSA) with a MIC of 0.0625 μg/mL and against the E. faecalis strain with a MIC of 0.125 μg/mL. In a time-kill assay, compound 6d showed a dose-dependent bactericidal effect on the MRSA strain and achieved bactericidal activity at 8 × MIC after 8 h. The duration of the post-antibiotic effect (PAE) on the MRSA strain for compound 6d was 2 h, which corresponds to the PAE duration for ciprofloxacin. The compounds were not cytotoxic at effective concentrations, as determined in an MTS assay on the MCF-7 breast cancer cell line.

Screening and Evaluation of Potential Efflux Pump Inhibitors with a Seaweed Compound Diphenylmethane-Scaffold against Drug-Resistant Escherichia coli.

Drug-resistant efflux pumps play a crucial role in bacterial antibiotic resistance. In this study, potential efflux pump inhibitors (EPIs) with a diphenylmethane scaffold were screened and evaluated against drug-resistant Escherichia coli. Twenty-four compounds were docked against the drug-binding site of E. coli multidrug transporter AcrB, and 2,2-diphenylethanol (DPE), di-p-tolyl-methanol (DPT), and 4-(benzylphenyl) acetonitrile (BPA) were screened for their highest binding free energy. The modulation assay was further used for EPI evaluation, revealing that DPE, DPT, and BPA could reduce the drug IC50 value in E. coli strains overexpressing AcrB, indicating their modulation activity. Only DPE and BPA enhanced intracellular dye accumulation and inhibited the efflux of ethidium bromide and erythromycin. In addition, DPE and BPA showed an elevated post-antibiotic effect on drug-resistant E. coli, and they did not damage the permeability of the bacterial outer membrane. The cell toxicity test showed that DPE and BPA had limited human-cell toxicity. Therefore, DPE and BPA demonstrate efflux pump inhibitory activity, and they should be further explored as potential enhancers to improve the effectiveness of existing antibiotics against drug-resistant E. coli.

Screening and characterization of antagonistic Streptomyces sp. JS20 from marine sediment in Vaan Island

In the present study, we have isolated twenty four different Actinomycetia strains from marine sediment in Vaan Island, Tuticorin district (8.83639°N/78.21047°E), Tamil Nadu, India. Among these, the isolate JS20 has been identified as Streptomyces sp. JS20 by 16S rRNA phylogenetic analysis, which exhibited prominent antibacterial activity against panel of WHO priority pathogens mostly, MRSA a clinical pathogen. The bioassay guided fractionation dependent active compound exhibits RT at 3.0 with maximum UV absorption of 250 nm. Furthermore, FT-IR, 1H NMR, 13C NMR and LC-MS spectral analysis confirms the existence of polyketide metabolites with molecular mass of m/z 380.74 [M+Na]+ and m/z 397.00 [M+K]+ adducts. Minimum Inhibitory Concentration (MIC) and Minimum Bacterial Concentration (MBC) value of the purified compound against MRSA was found to be 7.81 µg/mL and 125 µg/mL respectively. Additionally, the anti-MRSA campaign of the purified compound was upheld qualitatively using the high content screening (HCS) and quantitatively by Flow Cytometry (FACS) analysis which indicates concentration of bactericidal activity increases when the concentration of the purified compound was increased. The action of bioactive compound was determined by cell morphology disruption on MRSA bacterial cells as results, the cytoplasm cellular contents and ATP leaky were ahead. Apart from these studies, NaCl tolerant, the multiple passage studies and post antibiotic effect of bioactive compound has been evaluated against the MRSA. Hence, the study revealed that the bioactive compound from the Streptomyces sp. JS20 can be used as a lead compound to inhibit the MRSA in future.

Halicin: A New Horizon in Antibacterial Therapy against Veterinary Pathogens.

It is crucial to discover novel antimicrobial drugs to combat resistance. This study investigated the antibacterial properties of halicin (SU3327), an AI-identified anti-diabetic drug, against 13 kinds of common clinical pathogens of animal origin, including multidrug-resistant strains. Employing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assessments, halicin demonstrated a broad-spectrum antibacterial effect. Time-killing assays revealed its concentration-dependent bactericidal activity against Escherichia coli ATCC 25922 (E. coli ATCC 25922), Staphylococcus aureus ATCC 29213 (S. aureus ATCC 29213), and Actinobacillus pleuropneumoniae S6 (APP S6) after 4 h of treatment at concentrations above the MIC. Halicin exhibited longer post-antibiotic effects (PAEs) and sub-MIC effects (PA-SMEs) for E. coli 25922, S. aureus 29213, and APP S6 compared to ceftiofur and ciprofloxacin, the commonly used veterinary antimicrobial agents, indicating sustained antibacterial action. Additionally, the results of consecutive passaging experiments over 40 d at sub-inhibitory concentrations showed that bacteria exhibited difficulty in developing resistance to halicin. Toxicology studies confirmed that halicin exhibited low acute toxicity, being non-mutagenic, non-reproductive-toxic, and non-genotoxic. Blood biochemical results suggested that halicin has no significant impact on hematological parameters, liver function, and kidney function. Furthermore, halicin effectively treated respiratory A. pleuropneumoniae infections in murine models. These results underscore the potential of halicin as a new antibacterial agent with applications against clinically relevant pathogens in veterinary medicine.

Design, Synthesis, Biological Evaluation, and Molecular Docking Studies of Pleuromutilin Derivatives Containing Thiazole.

In this study, we designed and synthesized a series of pleuromutilin derivatives containing thiazole. The in vitro antimicrobial efficacy of these synthesized compounds was examined by using four strains. Compared with tiamulin (MIC = 0.25 μg/mL), compound 14 exhibited potency in inhibiting MRSA growth (MIC = 0.0625 μg/mL) in these derivatives. Meanwhile, the time-killing kinetics further demonstrated that compound 14 could efficiently inhibit the MRSA growth. After exposure at 4 × MIC, the postantibiotic effect (PAE) of compound 14 was 1.29 h. Additionally, in thigh-infected mice, compound 14 exhibited a more potent antibacterial efficacy (-1.78 ± 0.28 log10 CFU/g) in reducing MRSA load compared to tiamulin (-1.21 ± 0.23 log10 CFU/g). Moreover, the MTT assay on RAW 264.7 cells demonstrated that compound 14 (8 μg/mL) had no significant cytotoxicity. Docking studies indicated the strong affinity of compound 14 toward the 50S ribosomal subunit, with a binding free energy of -9.63 kcal/mol. Taken together, it could be deduced that compound 14 was a promising candidate for treating MRSA infections.

Pharmacokinetic/pharmacodynamic evaluation of gamithromycin against rabbit pasteurellosis

BackgroundGamithromycin is an effective therapy for bovine and swine respiratory diseases but not utilized for rabbits. Given its potent activity against respiratory pathogens, we sought to determine the pharmacokinetic profiles, antimicrobial activity and target pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with therapeutic effect of gamithromycin against Pasteurella multocida in rabbits.ResultsGamithromycin showed favorable PK properties in rabbits, including high subcutaneous bioavailability (86.7 ± 10.7%) and low plasma protein binding (18.5–31.9%). PK analysis identified a mean plasma peak concentration (Cmax) of 1.64 ± 0.86 mg/L and terminal half-life (T1/2) of 31.5 ± 5.74 h after subcutaneous injection. For P. multocida, short post-antibiotic effects (PAE) (1.1–5.3 h) and post-antibiotic sub-inhibitory concentration effects (PA-SME) (6.6–9.1 h) were observed after exposure to gamithromycin at 1 to 4× minimal inhibitory concentration (MIC). Gamithromycin demonstrated concentration-dependent bactericidal activity and the PK/PD index area under the concentration-time curve over 24 h (AUC24h)/MIC correlated well with efficacy (R2 > 0.99). The plasma AUC24h/MIC ratios of gamithromycin associated with the bacteriostatic, bactericidal and bacterial eradication against P. multocida were 15.4, 24.9 and 27.8 h in rabbits, respectively.ConclusionsSubcutaneous administration of 6 mg/kg gamithromycin reached therapeutic concentrations in rabbit plasma against P. multocida. The PK/PD ratios determined herein in combination with ex vivo activity and favorable rabbit PK indicate that gamithromycin may be used for the treatment of rabbit pasteurellosis.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Comparative Evaluation of Residual Antibacterial Substantivity of Chlorhexidine, MTAD and Chitosan Against Enterococcus Faecalis in Human Root Dentin - An In vitro Study.

Complex anatomy of the root canal system results in incomplete debridement with mechanical instrumentation, leaving some areas or root canal walls untouched. There comes the significance of endodontic irrigants with residual antibacterial substantivity which prolongs the post-antibiotic effect, thereby improving the success and predictability of endodontic treatment. To comparatively evaluate the residual antibacterial substantivity of 2% chlorhexidine, Biopure MTAD and 2% chitosan against Enterococcus faecalis at intervals of 1, 14 and 28 days. Seventy-five therapeutically extracted permanent single rooted mandibular premolars teeth were selected. Dentin blocks of 4 mm thickness were prepared and enlarged to 1.6 mm. The autoclaved blocks were inoculated with 24-hour colonies of pure cultures of E. faecalis for 14 days. After the contamination period, canals of each dentin block were irrigated with 5 ml of sterile saline and dried with sterile paper points. A total of 75 dentin blocks were randomly divided into five groups as follows: Group A (21 specimens): 2% chlorhexidine, Group B (21 specimens): Biopure MTAD, Group C (21 specimens): 2% chitosan, Group D (6 specimens): positive control (infected dentin tubes) and Group E (six specimens): negative control (sterile dentin tubes). Then, the lumens of dentin blocks were irrigated with the respective irrigants for 10 mins and were dried using sterile paper points. The specimens were then incubated at 37°C for 28 days to maintain humidity. At experimental intervals of 1, 14 and 28 days, dentin shavings were removed from the canals of respective groups by circumferential filing with sterile no. 35 Hedstrom files. The powdered dentin samples obtained with each dentin block were observed for colony forming units (CFUs) using a Digital Colony counter and were expressed as CFUs/mL. All the irrigants in the study showed a significant decrease in CFUs from day 1 to day 28 indicating that they have residual antibacterial substantivity against E. faecalis. Group B (Biopure MTAD) showed significantly least mean CFUs compared to Group A (2% CHX) and Group C (2% chitosan) against E. faecalis at B1(day 1), B2(day 14) and B3(day 28). Group A (2% CHX) showed significantly higher mean CFUs than Group C (2% chitosan) at A1(day 1). Group C (2% chitosan) showed significantly higher mean CFUs compared to Group A (2% CHX) at C2(day 14) and C3 (day 28). MTAD showed statistically higher residual antibacterial substantivity against E. faecalis at experimental periods of 1, 14 and 28 days. The residual antibacterial effect of 2% chitosan was better compared to 2% CHX at day 1. The residual antimicrobial substantivity of 2% CHX was higher compared to 2% chitosan at intervals of 14 and 28 days.

Pharmacokinetics and pharmacodynamics of antibacterial peptide NZX in Staphylococcus aureus mastitis mouse model

Staphylococcus aureus is associated with dairy mastitis, which causes serious economic losses to dairy farming industry. Antibacterial peptide NZX showed good antibacterial activity against S. aureus. This study aimed to evaluate pharmacokinetics and pharmacodynamics of NZX against S. aureus-induced mouse mastitis. NZX exhibited potent in vitro antibacterial activity against the test S. aureus strains (minimal inhibitory concentration (MIC): 0.23–0.46 μM), low mutant prevention concentration (MPC: 1.18–3.68 μM), and a long post antibiotic effect (PAE: 2.20–8.84 h), which was superior to those of lincomycin and ceftiofur. Antibacterial mechanisms showed that NZX could penetrate the cell membrane, resulting in obvious cell membrane perforation and morphological changes, and bind to intracellular DNA. Furthermore, NZX had a good stability in milk environment (retention rate: 85.36%, 24 h) than that in mammary homogenate (47.90%, 24 h). In mouse mastitis model, NZX (25–400 μg/gland) could significantly reduce the bacterial load of mammary tissue in a dose-dependent manner. In addition, NZX (100 μg/gland) could relieve the inflammatory symptoms of mammary tissue, and significantly decreased its pathological scores. The concentration–time curve of NZX (100 μg/gland) in the mammary tissue was plotted and the corresponding pharmacokinetic parameters were obtained by non-compartment model calculation. Those parameters of Tmax, T1/2, Cmax and AUC were 0.5 h, 35.11 h, 32.49 μg/g and 391 μg·h/g, respectively. Therefore, these results suggest that NZX could act as a promising candidate for treating dairy mastitis disease caused by S. aureus.Key points• NZX could kill S. aureus by dual mechanism involved in membrane and DNA disruption• NZX could relieve S. aureus-induced mouse mastitis• Pharmacokinetic parameters of NZX in mouse mammary gland were obtained

Synergistic Antibacterial Activity of Gallic Acid Based Chalcone Indl 2 by Inhibiting Efflux Pump Transporters.

As a part of novel discovery of drugs from natural resources, present study was undertaken to explore the antibacterial potential of chalcone Indl-2 in combination with different group of antibiotics. MIC of antibiotics was reduced up to eight folds against the different cultures of E. coli by both chalcones. Among the two compounds, the i. e. 1-(3', 4,'5'-trimethoxyphenyl)-3-(3-Indyl)-prop-2-enone (6, Indl-2), a chalcone derivative of gallic acid (Indl-2) was better along with tetracycline (TET) worked synergistically and was found to inhibit efflux transporters as obvious by ethidium bromide efflux confirmed by ATPase assays and docking studies. In combination, Indl-2 kills the MDREC-KG4 cells, post-antibiotic effect (PAE) of TET was prolonged and mutant prevention concentration (MPC) of TET was also decreased. In-vivo studies revealed that Indl-2 reduces the concentration of TNF-α. In acute oral toxicity study, Indl-2 was non-toxic and well tolerated up-to dose of 2000 mg/kg. Perhaps, the study is going to report gallic acid derived chalcone as synergistic agent acting via inhibiting the primary efflux pumps.

Design, synthesis and antibacterial evaluation of low toxicity amphiphilic-cephalosporin derivatives

Global public health is facing a serious problem as a result of the rise in antibiotic resistance and the decline in the discovery of new antibiotics. In this study, two series of amphiphilic-cephalosporins were designed and synthesized, several of which showed good antibacterial activity against both Gram-positive and Gram-negative bacteria. Structure-activity relationships indicated that the length of the hydrophobic alkyl chain significantly affects the antibacterial activity against Gram-negative bacteria. The best compound 2d showed high activity against drug-susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) with MICs of 0.5 and 2–4 μg/mL, respectively. Furthermore, 2d remained active in complex mammalian body fluids and had a longer post-antibiotic effect (PAE) than vancomycin. Mechanism studies indicated that compound 2d lacks membrane-damaging properties and can target penicillin-binding proteins to disrupt bacterial cell wall structure, inhibit the metabolic activity and induce the accumulation of reactive oxygen species (ROS) in bacteria. Compound 2d showed minimal drug resistance and was nontoxic to HUVEC and HBZY-1 cells with CC50 > 128 μg/mL. These findings suggest that 2d is a promising drug candidate for treating bacterial infections.

Expression and characterization of the new antimicrobial peptide AP138L-arg26 anti Staphylococcus aureus

The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in Pichia pastoris X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G+, minimum inhibitory concentration (MIC) 2–16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill Staphylococcus aureus (S. aureus) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in S. aureus. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future.Key points• The AP138L-arg26 was expressed in the P. pastoris expression system with high yield• The AP138 L-arg26 showed high stability and safety in vitro and in vivo• The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism

Dynamic responses of Salmonella Typhimurium to re-exposure to sublethal ciprofloxacin.

This study was designed to evaluate the history-dependent behaviors of Salmonella Typhimurium re-exposed to sublethal levels of ciprofloxacin. The S. Typhimurium cells were pre-exposed to 0 (CON), 1/16 (LOW), 1/8 (MED), and 1/4 (HIGH) minimum inhibitory concentrations (MICs) of ciprofloxacin, followed by re-exposure to the same concentrations. The bacterial growth, postantibiotic effect (PAE), relative fitness, and swimming motility of treatments were evaluated in the absence of ciprofloxacin. The lag phase duration (LPD) was estimate to assess bacterial recovery under ciprofloxacin exposure. A disk diffusion assay was used to determine the cross-resistance and collateral sensitivity of CON, LOW, MED, and HIGH treatments to ciprofloxacin (CIP), ceftriaxone (CEF), erythromycin (ERY), gentamicin (GEN), and polymyxin B (POL). The S. Typhimurium cells pre-exposed to ciprofloxacin were susceptible in antibiotic-free media, showing delayed growth. The highest PAE (>1h) and bacterial fluctuation (CV=5%) were observed at the High treatment compared to the CON. The HIGH treatment had the lowest relative fitness levels (0.87) and swimming motility (55mm). The LPD was significantly decreased at the LOW treatment (1.8h) when re-exposed to 1/16×MIC of ciprofloxacin. The LOW, MED, and HIGH treatments showed the cross-resistance to POL and the collateral sensitivity to CEF, ERY, and GEN. The pre-exposure to ciprofloxacin could induce phenotypic diversity, corresponding to the history-dependent behaviors. These results provide important insights for the dynamic nature of bacterial populations when re-exposed to sublethal concentrations of antibiotics.

The Renaissance of Spiramycin in Clinical Practice

Spiramycin is a natural 16-membered macrolide antibiotic that has been used in clinical practice for about 70 years. Despite long-term use, spiramycin retains its position in clinical practice; the resistance of the main respiratory and urogenital pathogens is lower to spiramycin than to 14- and 15-membered macrolides. The interest in spiramycin is due to the fact that its physico-chemical and pharmacokinetic properties are similar to the last semi-synthetic macrolides, and it lacks the disadvantages of erythromycin. Special biological properties of spiramycin explain its consistently high clinical efficacy that has lasted for 70 years of clinical use in various infections. It is necessary to emphasize the uniqueness of spiramycin as an antibiotic with not only the direct effect on microbes, but also non-antibiotic properties. This allowed the experts at the time to declare the «paradox» of spiramycin — the presence of a higher real clinical efficacy than expected according to in vitro studies. Such properties of spiramycin include pro-antibiotic and post-antibiotic effects, immunomodulatory effect, as well as high intracellular and extracellular concentrations of the antibiotic. The review discusses antimicrobial, pharmacokinetic, and pharmacodynamic characteristics of spiramycin and its place in the treatment of various infections — respiratory, ENT, urogenital, gynecological, and dental.

2170. It takes two to tango: ß-lactam/ß-lactamase inhibitor combinations targeting metallo-ß-lactamase (MBL)- producing Klebsiella pneumoniae.

Abstract Background The emergence of pan drug resistant (PDR) gram-negatives co-producing NDM-1 and CTX-M-15 calls for devising more efficient biocidal regimens. Also, understanding the salient transcriptomic and phenotypic changes that contribute to persistence which aid in pathogen survival is critical to therapeutic preparation in the clinical setting. Methods A 10-day hollow fiber infection model (HFIM) was utilized to simulate humanized β-lactam regimens involving imipenem (IMI) 1g q8h, aztreonam (ATM) 2g q8h, and ceftazidime/avibactam (CAZ/AVI) 2g/500mg alone and in 3-drug combination regimens against a PDR Klebsiella pneumonia isolate co-harboring blaNDM-1 and blaCMY-6, blaCTX-M-15 and blaSHV-2. Static time kills were performed to investigate the interplay between IMI and ATM. At 2h post drug exposure, total RNA was analyzed by qRT-PCR for the expression of pertinent genes. Glutaraldehyde-fixed samples were imaged using fluorescence and scanning electron microscopy. Results In HFIM, IMI contributed synergistically to the antimicrobial action as the average bacterial concentration between 30-120h was 2.1 log10 CFU/mL lower in ATM + CAZ/AVI + IMI than in ATM + CAZ/AVI (2.44 log10 CFU/mL vs. 4.54 log10 CFU/mL, respectively). Adding IMI to ATM+ CAZ/AVI also resulted in an enhanced post-antibiotic effect. Although ATM + IMI reduced bacterial counts by ≥4 log10 CFU/mL in 6h time kill assays, regrowth to the system carrying capacity of ∼8.5 log10 CFU/mL by 24h in all arms rendered these drugs ineffective. By 2h of treatment, ATM alone resulted in long filamentous cells whereas IMI resulted in significant populations of spheroplasts. ATM+IMI caused blebbing in the center of elongating filaments followed by a burst in spheroplast formation. qRT-PCR showed upregulation of spheroplast protein Y (spy) and penicillin binding proteins (pbp1, pbp2, pbp3) for all combinations involving IMI. However, pbp1, pbp3 and spy remained unchanged and pbp2 levels were downregulated by >25% in ATM monotherapy. Conclusion Overall, this PDR clinical isolate persisted in the presence of ATM + CAZ/AVI. The addition of IMI to target PBP2 in β-lactam combinations maximally suppressed the emergence of filamentous persisters and may be a promising strategy to prepare for increasing rates of gram-negative resistance. Disclosures All Authors: No reported disclosures

Inoculum-Based Dosing: A Novel Concept for Combining Time with Concentration-Dependent Antibiotics to Optimize Clinical and Microbiological Outcomes in Severe Gram Negative Sepsis.

Certain classes of antibiotics show "concentration dependent" antimicrobial activity; higher concentrations result in increased bacterial killing rates, in contrast to "time dependent antibiotics", which show antimicrobial activity that depends on the time that antibiotic concentrations remain above the MIC. Aminoglycosides and fluoroquinolones are still widely used concentration-dependent antibiotics. These antibiotics are not hydrolyzed by beta-lactamases and are less sensitive to the inoculum effect, which can be defined as an increased MIC for the antibiotic in the presence of a relatively higher bacterial load (inoculum). In addition, they possess a relatively long Post-Antibiotic Effect (PAE), which can be defined as the absence of bacterial growth when antibiotic concentrations fall below the MIC. These characteristics make them interesting complementary antibiotics in the management of Multi-Drug Resistant (MDR) bacteria and/or (neutropenic) patients with severe sepsis. Global surveillance studies have shown that up to 90% of MDR Gram-negative bacteria still remain susceptible to aminoglycosides, depending on the susceptibility breakpoint (e.g., CLSI or EUCAST) being applied. This percentage is notably lower for fluoroquinolones but depends on the region, type of organism, and mechanism of resistance involved. Daily (high-dose) dosing of aminoglycosides for less than one week has been associated with significantly less nephro/oto toxicity and improved target attainment. Furthermore, higher-than-conventional dosing of fluoroquinolones has been linked to improved clinical outcomes. Beta-lactam antibiotics are the recommended backbone of therapy for severe sepsis. Since these antibiotics are time-dependent, the addition of a second concentration-dependent antibiotic could serve to quickly lower the bacterial inoculum, create PAE, and reduce Penicillin-Binding Protein (PBP) expression. Inadequate antibiotic levels at the site of infection, especially in the presence of high inoculum infections, have been shown to be important risk factors for inadequate resistance suppression and therapeutic failure. Therefore, in the early phase of severe sepsis, effort should be made to optimize the dose and quickly lower the inoculum. In this article, the authors propose a novel concept of "Inoculum Based Dosing" in which the decision for antibiotic dosing regimens and/or combination therapy is not only based on the PK parameters of the patient, but also on the presumed inoculum size. Once the inoculum has been lowered, indirectly reflected by clinical improvement, treatment simplification should be considered to further treat the infection.

Evaluation of antibacterial effect of a cationic porphyrin derivative on Pseudomonas aeruginosa in photodynamic therapy

BackgroundPseudomonas aeruginosa is a gram-negative bacterium without spores, and it is one of the pathogens that easily cause secondary infectious diseases when human immune function is low. The purpose of this study is to explore the inhibitory effect of photodynamic antibacterial chemotherapy-induced by cationic porphyrin derivative on clinical P. aeruginosa and its mechanism. MethodsThe uptake of photosensitizer by P. aeruginosa and L929 cells was measured by an ultraviolet spectrophotometer. Effect of laser energy density on the bacterial activity of P. aeruginosa and post antibiotic effect were measured by bacterial suspension and tenfold dilution method. Flow cytometry and scanning electron microscopy were used to observe the activity and morphological changes of P. aeruginosa after PACT treatment. ResultsThe uptake of Tetra-ATPP-Lys-by P. aeruginosa and L929 was shown as concentration-dependent and time-dependent. However the uptake of L929 cell had a clear difference with P. aeruginosa at the same time and concentration intervals(P < 0.05).The increasing laser energy density had a high inactivation effect of on P. aeruginosa at the same Tetra-ATPP-Lys-concentration(P < 0.05). Post-antibiotic effect of Tetra-ATPP-Lys -PACT was dose-dependent(P < 0.05). Bacterial viability which evaluated by the flow cytometry method demonstrated that the proportion of viable bacteria is decreased with the photosensitizer dose-dependent. The morphology and microstructure of P. aeruginosa after Tetra-ATPP-Lys -PACT was demonstrated by a scanning electron microscope(SEM). After PACT, the morphology of P. aeruginosa was rod-shaped, the outer membrane surface was rough, and the bacteria were dry flat, sunken, shrunk and deformed. ConclusionsCationic porphyrin photosensitizer had a great damage effect on P. aeruginosa under the PACT, which can effectively destroy the microstructure of bacteria and lead to bacterial inactivation and death.