Cheap, Fast, Safe, Sustainable, and Positively Irresistible: The Future of Cationic Amphiphilic Disinfectant Development.

The increasing demand for orthopedic surgeries, including joint replacements, is driven by an aging population and improved diagnosis of joint conditions. Orthopedic surgeries carry a risk of infection, especially in patients with comorbidities. The rise of antibiotic resistance exacerbates this issue, necessitating alternatives like in vitro bioengineered antimicrobial peptides (AMPs), offering broad-spectrum activity and multiple action mechanisms. This review aimed to assess the prevalence of antimicrobial potential and the yield after purification among recombinant AMP families. The antimicrobial potential was evaluated using the Minimum Inhibitory Concentration (MIC) values against the most common bacteria involved in clinical infections. This systematic review adhered to PRISMA guidelines, focusing on in vitro studies of recombinant AMPs. The search strategy was run on PubMed, Scopus and Embase up to 30th March 2023. The Population, Exposure and Outcome model was used to extract the data from studies and ToxRTool for the risk of bias analysis. This review included studies providing peptide production yield data and MIC values against pathogenic bacteria. Non-English texts, reviews, conference abstracts, books, studies focusing solely on chemical synthesis, those reporting incomplete data sets, using non-standard MIC assessment methods, or presenting MIC values as ranges rather than precise concentrations, were excluded. From 370 publications, 34 studies on AMPs were analyzed. These covered 46 AMPs across 18 families, with Defensins and Hepcidins being most common. Yields varied from 0.5 to 2,700 mg/L. AMPs were tested against 23 bacterial genera, with MIC values ranging from 0.125 to >1,152 μg/mL. Arenicins showed the highest antimicrobial activity, particularly against common orthopedic infection pathogens. However, AMP production yields varied and some AMPs demonstrated limited effectiveness against certain bacterial strains. This systematic review emphasizes the critical role of bioengineered AMPs to cope infections and antibiotic resistance. It meticulously evaluates recombinant AMPs, focusing on their antimicrobial efficacy and production yields. The review highlights that, despite the variability in AMP yields and effectiveness, Arenicins and Defensins are promising candidates for future research and clinical applications in treating antibiotic-resistant orthopedic infections. This study contributes significantly to the understanding of AMPs in healthcare, underscoring their potential in addressing the growing challenge of antibiotic resistance. Systematic review registration:https://osf.io/2uq4c/.

Quaternary ammonium compounds (QACs) are a group of compounds of great economic significance. They are widely used as emulsifiers, detergents, solubilizers and corrosion inhibitors in household and industrial products. Due to their excellent antimicrobial activity QACs have also gained a special meaning as antimicrobials in hospitals, agriculture and the food industry. The main representatives of the microbiocidal QACs are the benzalkonium chlorides (BACs), which exhibit biocidal activity against most bacteria, fungi, algae and some viruses. However, the misuses of QACs, mainly at sublethal concentrations, can lead to an increasing resistance of microorganisms. One of the ways to avoid this serious problem is the introduction and use of new biocides with modified structures instead of the biocides applied so far. Therefore new BAC analogues P13–P18 with pyridine rings were synthesized. The new compounds were characterized by NMR, FT-IR and ESI-MS methods. PM3 semiempirical calculations of molecular structures and the heats of formation of compounds P13–P18 were also performed. Critical micellization concentrations (CMCs) were determined to characterize the aggregation behavior of the new BAC analogues. The antimicrobial properties of novel QACs were examined by determining their minimal inhibitory concentration (MIC) values against the fungi Aspergillus niger, Candida albicans, Penicillium chrysogenum and bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The MIC values of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides for fungi range from 0.1 to 12 mM and for bacteria, they range from 0.02 to 6 mM.

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.

Development of resistance to disinfectant substances in nosocomial microorganisms is an important problem encountered during disinfectant practices. Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant cause of hospital-acquired infections. Besides being resistant to several antimicrobial agents, MRSA strains can also become resistant to some disinfectant substances. Resistance to disinfectant substances may develop due to the misuse of disinfectants. This may either be due to the frequent use of disinfectant substances or use in lower concentrations than recommended. MRSA strains may harbour the qacA/B disinfectant resistance genes that may cause resistance to quarternary ammonium compounds and some cationic disinfectants. These resistance genes are found in plasmids and are responsible for decreased susceptibility or resistance. In this study, a total of 69 nosocomial MRSA strains isolated from clinical specimens in our hospital were tested for disinfectant activity and the presence of qacA/B disinfectant resistance genes in these isolates was investigated by polymerase chain reaction. We determined whether the presence of these genes caused phenotypic resistance to chlorhexidine and benzalkonium chloride by the use of bactericidal and bacteriostatic tests. For this purpose, the minimum inhibitory concentration (MIC) values of these disinfectants against MRSA isolates were detected by microdilution method with the proposals of CLSI, and bactericidal effects of these disinfectants were also detected by using quantitative suspension test according to EN13727:2003 European Standard. It has been found that 11.6% (8/69) of the isolates harbored qacA/B resistance genes. MIC values for chlorhexidine and benzalkonium chloride were found in the range of 2-8 µg/ml. Although it was observed that MIC values were higher in five of the qacA/B gene positive isolates, statistically significant difference was not found between gene positive and gene negative groups. Both 1% chlorhexidine and 1% benzalkonium chloride were found bactericidal against the isolates including the ones carrying the qacA/B resistance genes. It was concluded that the presence of the qacA/B disinfectant resistance genes did not lead to resistance to the disinfectant substances at the concentrations used in clinical practices. Furthermore, tested disinfectants still exhibited bactericidal activity even with high MIC values.

1281. An Evaluation of Tebipenem In Vitro Activity Against a Panel of Pseudomonas aeruginosa Isolates with Efflux, AmpC, and OprD Mutations

Bacteria have acquired resistance against almost all antibiotics because of the misuse of antibacterial agents and long periods of treatment. Antimicrobial peptides (AMPs) are one of the most encouraging candidates to solve this problem, as they possess high prokaryotic selectivity, and affect the bacteria by a unique mode of action. Novel cyclic undecapeptides (QNRNFYFNRNQ and QNRNFHFNRNQ) and their linear counterparts were investigated for their antibacterial activity against virulent strains. The minimal inhibitory concentration (MIC) values showed that tyrosine and histidine AMPs have promising antibacterial activity against virulent bacteria. The MIC values against the P. aeruginosa PA14, E. coli O157:H7 CR3, S. aureus 209P, and B. subtilis ATCC 6633 bacterial strains were evaluated for the cyclic peptide containing tyrosine, and their values were 6.25, 12.5, 12.5, and 12.5 µM, respectively. Meanwhile, for the linear form, they were 9.3, 12.5, 12.5, and 12.5 µM, respectively. The cyclic-peptide–containing histidines’ MIC values were 6.25, 3.1, 6.25, and 3.1 µM, respectively. Meanwhile, for the linear form, they were 3.1, 3.1, 3.1, and 6.25 µM, respectively. The antibacterial activities of the new AMPs were compared with that of gentamicin sulfate, and showed relatively higher potencies. Time-inhibition studies demonstrated the rapid antibacterial effects of the novel AMPs, which were more likely to be concentration-dependent, rather than time-dependent. At double the MIC concentration, all of the tested peptides exhibited relatively stable antibacterial effects up to 24 h, especially the peptides containing tyrosine, which showed an improved antibacterial effect.

Objective To investigate the mechanism of efflux pump AcrAB-TolC involved in the cross-resistance between quaternary ammonium compounds (QAC) and fluoroquinolones (FQ) in Escherichia coli. Methods Seventy-eight Escherichia coli strains were isolated from clinical samples and then tested for the sensitivity to benzalkonium bromide by ager dilution method. Six strains were randomly selected and induced with benzalkonium bromide. Changes in the sensitivity of these strains to benzalkonium bromide and ciprofloxacin were analyzed after induction. Expression of the efflux pump genes acrA, acrB and tolC at mRNA level in the induced strains and their parent strains were detected by quantitative real-time PCR. Results Among the 78 strains, the minimum inhibitory concentrations (MIC) ranged from 8 μg/ml to 64 μg/ml and 47.4% of strains have higher MIC than the standard strain. Compared with the parent strains, the induced strains showed higher expression of acrA and tolC genes, and the differences between the two groups were statistically significant. The MIC values of ciprofloxacin to the six induced strains were 4-8 times higher than those to their parent strains. Conclusions It was speculated that the increased expression of acrA and tolC genes at transcription level in the induced strains were related to the cross-resistance between quaternary ammonium compounds and fluoroquinolone antibiotics. Key words: Escherichia coli; Quaternary ammonium compound (QAC); Disinfectant; Efflux pump

Our aim was to assess the effects of step-wise exposure to didecyl dimethyl ammonium chloride (DDAC) on the antimicrobial (antibiotics and biocides) susceptibilities of food-associated bacterial strains. Adaptive responses of bacterial strains were investigated by exposing the strains daily to increasing subinhibitory concentrations of DDAC for 7days. Following adaptation to DDAC, a threefold increase in the minimum inhibitory concentration (MIC) values for this biocide was observed in 48% of the Escherichia coli and Listeria monocytogenes strains, and 3% of the Salmonella strains. Reduced susceptibility to other biocides was found with the most important increase in MIC for benzalkonium chloride (BC) and a commercial biocide formulation (Galox Horizon) containing DDAC and glutaraldehyde, for all species except Salmonella. Increase in antibiotic MIC values was more pronounced in E.coli in terms of antibiotic numbers and of magnitude (from 4- to 32-fold increase) and, to a lesser extent, in Salmonella strains. Most of these strains had acquired resistance to ampicillin, cefotaxime, ceftazidime, chloramphenicol and ciprofloxacin. The effects of exposure to DDAC on biocides and antibiotics susceptibilities depend upon the bacteria species. Extensive use of DDAC at subinhibitory concentrations may lead to the development of antibiotic-resistant bacteria and may represent a public health issue.

Disinfectants may have fungicidal or fungistatic effects against fungal cells. The mechanism of action of disinfectants on fungal cells believed to be similar to the antibacterial activity. The aim of this study was to demonstrate the efficacy of some disinfectants against Candida albicans and to investigate the relationship between virulence and disinfectant resistance. In this study, the susceptibility of 417 clinical C.albicans and reference isolates against disinfectants were determined. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of disinfectants were obtained by using broth microdilution (BMD) assay. Epidemiological cut-off values (ECVs) were determined by using the MIC and MFC values. Crystal violet assay was carried out to investigate membrane permeability in disinfectant resistant and susceptible isolates. Rhodamine 6G (R6G) flourescence stain was used to show the increase in the number of efflux pumps among selected isolates. The relationship between virulence and disinfectant resistance was determined by in vitro and in vivo investigations. Virulence factors secretory acid proteinase (SAP), phospholipase, esterase, hemolytic activity and slime factor production were examined in vitro. In vivo virulence assay was performed by infecting Galleria mellonella larvae. The relationship between virulence factors and disinfectant resistance was evaluated according to the mortality rates of G.mellonella larvae. The range of MIC values for benzalkonium chloride (BZC) and chlorhexidine digluconate (CHX), triclosan (TRC) and sodium hypochlorite (SHC) were 0.25-8 mg/L, 0.06-4 mg/L and 256-16.384 mg/L, respectively. ECV values for BZC, CHX, TRC and SHC were determined as 4, 2, 1 and 4096 mg/L, respectively. The rate of crystal violet uptake was found between 26.5-57.6% for disinfectant susceptible isolates, and between 33-79.2% for resistant isolates. It is concluded that the disinfectant resistance was related with efflux pumps. Due to the lack of number of isolates that were used in this assay, the relationship between disinfectant resistance and virulence factors could not be assessed. There was no difference in the mortality of larvae infections caused by disinfectant resistant and susceptible isolates. As a result, in this study, resistant isolates against BZC, CHX, SHC and TRC were found among 417 isolates. Input and output of disinfectants were found to be associated with the cell membrane efflux pumps of C.albicans.

In the past, several methods have been developed for predicting antibacterial and antimicrobial peptides, but only limited attempts have been made to predict their minimum inhibitory concentration (MIC) values. In this study, we developed predictive models for MIC values of antibacterial peptides against Escherichia coli (E. coli), comprised of 3143 peptides for training and 786 peptides for validation, with experimentally determined MIC values. We found that the Composition Enhanced Transition and Distribution (CeTD) attributes significantly correlate with MIC values. Initially, we attempted to estimate MIC using BLAST similarity searches but found them inadequate. Subsequently, we employed machine learning regression models that integrated various features, including peptide composition, binary profiles and embeddings from large language models. Feature selection techniques, particularly mRMR, were utilized to refine our model inputs. Our Random Forest regressor built using default parameters achieved a correlation coefficient (R) of 0.78, R2 of 0.59, and RMSE of 0.53 on the validation set. Our best model outperformed existing methods when benchmarked on an independent dataset of 498 anti-E. coli peptides. Additionally, we screened anti-E. coli proteins in the proteomes of three probiotic bacterial strains and created a web-based platform, “EIPpred”, enabling users to design peptides with desired MIC values (https://webs.iiitd.edu.in/raghava/eippred).

Quaternary ammonium surfactants derived from leucine and methionine: Novel challenging surface active molecules with antimicrobial activity

Recent data show an emergence of resistance in the Bacteroides fragilis group against several antimicrobial agents and inducible resistance against metronidazole in nim-positive strains. The aim of the present study was to investigate inducible metronidazole resistance in nim-positive as well as in nim-negative B. fragilis group strains. Of 18 B. fragilis strains (including four nim-positive reference strains and one ATCC strain), two Bacteroides ovatus strains, and one Bacteroides thetaiotaomicron DSM strain minimum inhibitory concentration (MIC) values for metronidazole were determined by Etest and analyzed for nim genes (nimA to -G) by PCR. For this purpose bacterial suspensions were incubated on supplemented Columbia agar plates containing metronidazole at twice the MIC value of the specific strain and incubated under anaerobic conditions for 48 hours. After incubation, growing bacteria were harvested and thereafter incubated at four times the original MIC. This procedure was repeated with increasing antibiotic concentrations. The resulting MIC values were confirmed by Etest. The MIC values for metronidazole of the four nim-positive reference strains ranged from 3 to 8 mg/l. The B. fragilis ATCC 25285 strain and the B. thetaiotaomicron DSM 2255 strain were nim negative with MIC values of 0.19 mg/l and 0.75 mg/l, respectively. Three clinical isolates of B. fragilis strains showed MIC values of > 256 mg/l. In all three strains, nim genes were detected by PCR. The other clinical isolates were nim negative. In these strains, MIC values ranged from 0.19 to 0.75 mg/l. After several passages on metronidazole containing agar, all B. fragilis group strains exhibited MIC values of > 256 mg/l determined by Etest. Metronidazole resistance can be selected not only in nim-positive strains but also in nim-negative strains, suggesting that mechanisms other than nim genes are involved. These findings and the emerging resistance of the B. fragilis group against several antimicrobial agents underscore the importance of susceptibility testing of anaerobes even in routine laboratories.

We compared the values of the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) values ​​of three antimicrobial agents for 72 bovine isolates ofPasteurella multocida, 80 swine isolates ofP. multocida, 80 bovine isolates ofEscherichia coli, 80 swine isolates ofE. coli, and 80 isolates ofStaphylococcus aureusfrom bovine mastitis. The ratio of MIC90​​/MPC90which limited mutant selection window (MSW) was ≤ 0.12/4 mg/l for enrofloxacin, 0.5/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin in bovineP. multocidaisolates, ≤ 0.12/2 mg/l for enrofloxacin, 0.5/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin in swineP. multocidaisolates, 1/16 mg/l for enrofloxacin, 8/≥ 64 mg/l for florfenicol and 8/≥ 128 mg/l for tulathromycin in bovineE. coliisolates, 0.5/16 mg/l for enrofloxacin, ≥ 64/≥ 64 mg/l for florfenicol and 8/≥ 128 mg/l for tulathromycin in swineE. coliisolates, and 0.25/16 mg/l for enrofloxacin, 4/≥ 64 mg/l for florfenicol and 4/≥ 128 mg/l for tulathromycin inS. aureusisolates. These findings indicate that the dosage of antimicrobial agents to achieve serum concentration equal to or higher than MPC could reduce selection of resistant bacterial subpopulation.

Effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical E. coli isolates on antibiotic susceptibilities and clinical outcomes

BackgroundFosfomycin combination therapy is a potential approach for treatment of multidrug-resistant (MDR) PA infections despite a lack of approved susceptibility breakpoints for this organism. While DD testing is commonly used for fosfomycin, growth of discrete inner colonies (IC) within the zone of inhibition has been observed for multiple organisms following DD. Criteria recommended by CLSI and EUCAST are contradictory for interpreting these inner colonies. We therefore sought to determine the frequency of inner colonies and MIC differences between PA parent-inner colony pairs from an international isolate collection.MethodsA convenience collection of 198 clinical PA isolates from three U.S institutions (n = 82), two Australian institutions (n = 72), and the CDC & FDA Antibiotic Resistance Isolate Bank (n = 44) were included. Fosfomycin MIC values were determined in duplicate on separate days by DD and broth microdilution (BMD) testing. For parent isolates with discrete IC observed during DD, IC isolates were subcultured and MIC values were determined and then compared to their corresponding parent isolates. MIC values were interpreted using CLSI Escherichia coli (EC) breakpoints (susceptible: MIC ≤ 64 μg/mL, intermediate: MIC = 128, resistant: MIC ≥ 256 μg/mL).ResultsParent isolate BMD MIC values ranged from < 4 to > 256 μg/mL while IC isolate BMD MIC values ranged from 128 to > 1024 μg/mL. MIC50/90 values were 128/256 μg/mL and > 1024/ > 1024 μg/mL for the parent and IC isolates, respectively. A high frequency of 45% (89/198) of parent isolates displayed discrete IC which also demonstrated a higher frequency of resistance (97.8%) compared to the parent isolates (23.7%).ConclusionIC MIC values were higher overall compared to parent MIC values, with an average fold difference of ~18 between the parent-inner colony pairs. The frequency of IC found in this study (45%) is considerably higher than previously observed in EC clinical isolates. These data highlight the need to further investigate the importance of these IC and warrant caution for extrapolation of EC breakpoints for fosfomycin susceptibility testing against PA.DisclosuresAll Authors: No reported disclosures