Introduction

The importance of antimicrobial compounds has been highlighted recently due to the COVID-19 pandemic outbreak [1,2,3]. Quaternary ammonium salts (QASs) are antiseptics that have shown a wide spectrum of antibacterial activity [4,60]. PC with a ratio of 2:1 was mixed with a resin of triethylene glycol dimethacrylate, bisphenol A glycerolate dimethacrylate, and 2-hydroxyethyl methacrylate (1:3:4 ratio respectively), containing 5% MAE-DB. The positive control group was made from 5% MAE-DB cured resin lacking PC, and the negative control group had neither PC nor MAE-DB. Calcium hydroxide and mineral trioxide aggregates were used as commercial controls [60]. S. mutans growth in culture media and biofilm formation on material surfaces were tested based on CFUs and metabolic activity after one day incubation of over-aged and freshly prepared water samples for six months. Live/dead staining and SEM techniques were used to assess the biofilm formation of S. mutans on the experimental material. Viability staining, metabolic activity, CFU counts, and morphology were similar to the obtained results of biofilms in the positive control group. No influence on the bacterial growth in the solutions was observed [60]. Aged experimental materials retained the contact-inhibition of biofilm formation. There was significant biofilm formation on calcium hydroxide and MTA. With PC as a support material in the formation of mineralized tissue and MAE-DB, Fig. 8, as the antimicrobial agent, it was found that the synthesized material with HEMA-BisGMA-TEDGMA resin inhibited S. mutans biofilm formation despite water aging for six months. However, inhibition of bacteria in the solution was not observed [60].

Fig. 8
figure 8

Structure of QAS monomer MAE-DB

Full size image

Acids from cariogenic biofilms often cause demineralized lesions in tooth enamel around orthodontic brackets. Antibacterial orthodontic cement was developed by incorporating a QAS monomer dimethylaminododecyl methacrylate (DMADDM) commercial orthodontic cement to investigate its effect on enamel bond strength and microcosm biofilm response. DDAM is an antibacterial monomer that was mixed with orthodontic cement at ratios of 0%, 1.5%, 3%, and 5% by mass fractions [61]. Bracket bond strength to the enamel was measured. Metabolic activity, lactic acid production, and CFU were measured using a microcosm biofilm model. Results showed that shear bond strength did not decrease at 3% DMADDM (p < 0.1), however, it was slightly decreased with the use of 5% DMADDM compared with 0% DMADDM. 3% DMADDM orthodontic cement substantially inhibited biofilm viability [61]. Lactic acid production, biofilm metabolic activity, and CFU were observed to be much lower with the use of orthodontic cement, which contained DMADDM versus the control group cement (p < 0.05). It can be concluded that 3% DMADDM orthodontic cement inhibited oral biofilms without decreasing the enamel bond strength [61].

Surface coating applications

Surface immobilized and tethered QASs onto hyperbranched polyurea coatings can build the antimicrobial contact-killing coating. This allows the surface to kill adhering bacteria via partial envelo** [62]. Even after thorough washings, the coatings instigated high rate of contact killing against S. epidermis in both culture-based assays. The bacterial membrane damage was confirmed using confocal laser scanning microscopy. Contact-killing in culture-based tests at 1600 CFU/cm2 was reported [62]. Dissolved QASs working mechanisms are based on bacterial membrane interdigitation [62]. QASs on hyperbranched polyurea partially envelope to clinging bacteria on contact, an indication of this was due to the observation that staphylococcal adhesion forces to hyperbranched QAS coatings were very high, Fig. 9 [62].

Fig. 9
figure 9

Killed bacteria by QASs coatings on different surfaces (glass slide, Si-HB: hyperbranched polyurea coatings, and Si-HB-PEI+: polyethyleneimine anchored Si-HB by converting amino groups of Si-HB-PEI into hydrophobic, polycationic species) number of killed bacteria was proportional to the treatment duration by QASs [62]

Full size image

Bacterial death is the result of these strong adhesion forces, and then removing the membrane lipids [63]. A hybrid coating using Q4N+-Si(OR)3 and tetraethoxysilane can be prepared on glass surfaces using a sol-gel process. Gram-positive and Gram-negative bacteria were used in investigating smooth surfaces covered in the transparent coating. A rapid decrease of both strains was observed within three days. There was a significant correlation between Q4N+-Si(OR)3 concentration and antibacterial activity [63].

In another study, the “grafting onto” technique was used to prepare the antimicrobial surface. Block copolymers contained poly(3-(trimethoxysilyl)propyl-methacrylate) and poly(2-dimethylamino)ethyl methacrylate). The corresponding random copolymers were synthesized using atom transfer radical polymerization method [64]. This was followed by covalent attachment to the glass surface by reacting the surface silanol with the trimethoxysilyl groups. The immobilization time and polymer solution concentration demonstrated a direct correlation with the density of QAS that can bind to small molecules in the solution. The PDMAEMA97-b-PTMSPAx diblock copolymers of fixed lengths of PDMAEMA with a degree of polymerization of 97 and various lengths of PTMSPMA was observed with a maximally available surface charge when the DPPSMAEMA to DPPTMSPMA ratio was 5:1. Ethyl bromide was reacted with tertiary amino groups that are in immobilized PDMAEMA segments to form quaternary ammonium groups. PDMAEMA segments were pre-quaternized by block copolymers and alternatively, these copolymers were attached to the surfaces. The biocidal activity of the surfaces in the presence of grafted polymers against E. coli displayed an increase in the density of QASs on the surface. When the density of QASs was increased from 1.0 × 1014 to 6.0 × 1014 unit/cm2, the surface killing of bacteria showed an uptake from 0.06 × 105 to 0.6 × 10 unit/cm2. With a killing efficiency of QASs on all surfaces being similar at 1.0 × 1010 units needed to kill a single bacterium, the AFM analysis indicated that surface grafting resulted in small patches of the highly concentrated polymer. The patches appeared to have increased the killing efficiency when comparing the grafted surfaces to a more uniformly distributed surface with a similar polymer density [64].

In addition to the antibacterial surfaces/coatings of QASs, they were used to replace the commonly used disinfectants since they do not efficiently kill infections with non-tuberculous Mycobacteria (NTM) [65]. While these infections have become rare in people with AIDS, they are critical opportunistic infections after surgical procedures. Venezuela has seen several NTM soft tissue infections after minor surgeries. Some have been related to using commercial disinfectants based on QASs. Its activity and other QASs on different NTM have been studied using dnaA-gfp fusion to transform the mycobacteria. Then the fluorescence was monitored to gauge the capacities of different QASs of inhibiting bacterial growth [65]. The MIC varied for the differing QASs. Still, it was found that M. terrae was more resistant than M. bovis while M. abscessus and Mycobacterium chelonae (M. chelonae) were consistently more resistant than M. smegmatis.

Additional studies on QASs against S. aureus concluded that didecyldimethylammonium chloride (DDAC) and alkyldimethylbenzylammonium chloride (ADBAC) were both active membrane agents [66]. DDAC and ADBAC displayed a similar MIC behavior range on an inoculum scale of 1 × 105 to 1 × 109 CFU/mL at 35 °C with values ranging from 0.4 to 1.8 ppm. Both salts at a concentration of 2 × 109 CFU/mL had an increased rapidity in S. aureus killing at 35 °C compared with 25 °C. Exponents of concentration for killing were less than 2.5 for both agents, with the temperature being an influencing factor in the concentration exponent value [66]. After studying the leakage and kill data, a single leakage marker did not indicate that cell death was evident. DDAC showed a Langmuir isotherm while ADBAC possessed a high-affinity uptake isotherm. DDAC molecules formed a second monolayer of cell coverage at the end of the initial uptake, whereas ADBAC only created a single monolayer. There was rapid cell leakage occurring at the bactericidal concentrations. ADBAC and DDAC exhibited autolysis at concentrations of 9 μg/mL (0.0278 mM for ADBAC and 0.0276 mM for DDAC), and together, about 30% of the internal potassium pool was depleted. Autolysis contributed to DDAC and ADBAC lethality [66].

QASs are common additives in cleaning products, however, the concern is rising because of the increased antibiotic resistance. QAS activity against dominant drain bacteria (15 genera, 17 species) was determined [67]. A drain microcosm was exposed to 12 days dosing period and 3 month dosing period with QAS containing domestic detergent. There was a two-fold decrease (six species) and an increase (three species) in QAS susceptibility because of the exposure to isolated cultures. After 14 consecutive QAS passages, Ralstonia sp. s. susceptibility was significantly decreased. Denaturing gradient gel electrophoresis analysis and culture testing supported that the dynamic stability of control drain microcosm biofilms could be maintained [67]. Despite half of the QAS-containing detergents causing a 10-fold viability decrease, bacterial population densities were unaffected over the short-term exposure to regular levels of the detergent. Denaturing gradient gel electrophoresis analysis identified the dominant microcosm genera such as Pseudomonas, Psuedoalteromonas, Erwinia, and Enterobacter [40]. The analysis supported that the abundance of acromonads increased from 10 to 50% QAS-containing detergents. The patterns of antimicrobial susceptibility were not altered by long-term exposure to detergents [67].

Environmental applications

A significant obstacle in applying membrane technology to wastewater treatment is biofouling. A study that used anti-biofouling polyvinylidene fluoride (PVDF) membrane motivated investigating QAS dosage activity on anti-biofouling and physicochemical properties of the membrane [68]. Individual organic model foulants were used in anti-fouling tests, which evidenced that modified membranes held higher membrane fouling rates against organic matters. When put in a membrane bioreactor, the modified membranes showed explicit anti-fouling activity. This suggests concurrent efficiency in controlling organic matters and microorganisms-induced fouling which is attributed to the QASs contract-killing effects and binary interactions between organic foulants and bacteria in the membrane bioreactor. Figure 10 shows proposed antimicrobial mechanisms for QASs modified membranes [68].

Fig. 10
figure 10

Proposed antimicrobial mechanisms of QASs modified membranes [68]

Full size image

A study investigated monocationic and polycationic ammonium salts as agents and ionic liquids for the synthesis of antimicrobial surfaces. Ionic liquids are anhydrous salts that are usually liquid at room temperature and have melting points below 100 °C [21]. They are non-flammable, thermally stable, and have low vapor pressure at standard temperature and pressure. The ionic liquids developed and used are based on chiral and racemic forms of 3-chloro-1,2-propanediol. Various methods of application to porous and nonporous surfaces used in the environment to render the chiral ionic liquids antimicrobial were developed concerning the antimicrobial properties of QASs. Carbohydrate-based polycationic salts were synthesized with the ability to gel water and alcohol. The materials used in the study were diazabicylcooctane-based cationic lipophilic salts. The resulting gels showed significant antimicrobial activity against the Gram-positive bacterium S. aureus. DABCO salts successfully modified mannose functionalized PAMAM dendrimers and polyester fabrics. These materials exhibited antibacterial activity against Gram-positive and Gram-negative bacteria [21].

Wastewater treatment also demonstrated the potential of QASs. QAS-modified chitosan effectively inactivated E. coli. Its antibacterial activity increased when acetic acid was added to the treatment system [4]. Chitosan, a biodegradable, naturally occurring, nonallergenic/toxic bio polysaccharide derived from chitin, demonstrated superior antimicrobial activity. Certain QAS derivatives of chitosan have a history of being highly effective antibacterial materials [4]. One study used substituted chitosan derivatives that were quaternized using Quat-188. Antibacterial activities of the quaternized derivatives of substituted chitosan were tested on liquid cultures of S. aureus and E. coli with results expressed with MIC. There was a comparison with unsubstituted chitosan quaternized derivatives where the results indicated that hydrophobic substituents enhanced activity. Γ-Octanic lactone addition resulted in deficient levels of substitution, ranging from less than 1.5% up to 30% feed; yet the derivatives gained using this method resulted in excellent antibacterial activity (MIC = 16 μg/mL), versus 128 μg/mL for unsubstituted quaternized chitosan. Also, heptanoic anhydride produced materials ranging from 1.5 to 2.4% substitution, demonstrating high antibacterial activity (MIC = 32 μg/mL). However, no substitution control was realized using the anhydride route.

Biological environment applications

The activity of biocidal disinfectants including QASs of poly(hexamethylene biguanide), PHMB, on fish is relatively unknown. A study investigated the cytotoxicity of Barquat (commercial antimicrobial), Barquat and BAC, BAC, and Barquat, along with PHMB and glutaraldehyde [69]. In addition, the study included the assessment of ternary/binary mixtures on human liver cells and zebrafish. The molecular effects were analyzed using PCR (polymerase chain reaction) in vitro and targeted gene expression for zebrafish eleuthero-embryos, Fig. 11. In both cell lines, QAS toxicity was reported using EC50 values in the lower μg/mL ranges. PHMB and glutaraldehyde were not as quite cytotoxic. A mixture that combined the five compounds showed potent cytotoxicity. Target genes with transcriptional alterations that had relation to the endoplasmic reticulum and inflammatory action, apoptosis, and general stress were also determined. Endoplasmic reticulum stress genes occurred at non-cytotoxic concentrations of Barquat, glutaraldehyde, and BAC in zebrafish liver cells [69]. Analogous transcriptional alterations were discovered due to its exposure to zebrafish eleuthero-embryos for five days. Glutaraldehyde resulted in endoplasmic reticulum stress genes with BAC inducing apoptosis genes, which was also the case with the highest BAC concentration [69].

Fig. 11
figure 11

Biocidal activity of QASs on zebrafish [69]

Full size image

Alkyl chain effects of QAS surfactants on aerobic biodegradability and aquatic toxicity were investigated [70]. Alkyl benzyl dimethyl ammonium halides and alkyl trimethyl ammonium were used in this study. For Daphnia magna and Photobacterium phosphoreum, acute toxicity tests took place, giving EC50 values within a range of 0.1–1.0 mg/L. Significant toxicity difference between homologs of varying chain lengths was not noted, despite a methyl group replacing a benzyl group, which increased the toxicity. Similar results were obtained in the biodegradability of diverse homologs in both standard conditions and coastal water. It was found that a reduction in the biodegradation rate was due to increasing alkyl chain lengths and replacing a benzyl group with a methyl group. An uptake in bacterioplankton density was correlated with the degradation of the compounds present in coastal waters. This implies that degradation occurred because compounds were used as growth substrates [70].

In addition to the activity of QASs on aquatic species, there are potential threats of QAS exposures since QAS adsorption on sludges was reported [71]. Among QASs, hexadecyl benzyl dimethyl ammonium chloride (C16BDMA), hexadecyl trimethyl ammonium chloride (C16TMA), dodecyl benzyl dimethyl ammonium chloride (C12BDMA), and dodecyl trimethyl ammonium chloride (C12TMA), were applied to sludges from wastewater treatment such as municipal primary sludge, waste activated sludge, and mesophilic/thermophilic digested sludge [71]. The basic adsorption of the four discretely tested QASs onto the municipal primary sludge reached equilibrium in 4 h. The extent of adsorption at a nominal and initial QAS concentration of 300 mg/L with volatile solid concentration at 1 g/L was 13%, 88%, 67%, and 89% for C12TMA, C16TMA, C12BDMA, and C16BDMA, respectively. There was a positive correlation with the QAS hydrophobicity and a negative correlation with the critical micelle concentration. All four sludges had similar adsorption capacities. QASs with low adsorption affinities decreased in binary QAS mixtures when QASs with higher adsorption affinities at high aqueous concentrations were present. At a neutral pH, 40% of the sludge made of C12TMA was desorbed. On the other hand, less than 5% of the C16BDMA sludge was desorbed in 10 days. pH range of 4–10 demonstrated negligible desorption of C12TMA over ten days. Increasing pH of the solution caused 50% desorption of C16BDMA. As 50% of the municipal biosolids used in the study were land applied in the USA, data from this investigation would be of assistance in assessing QASs and their effects on humans and the environment [71].

Conclusions

Quaternary ammonium salts have been extensively used as disinfectants for environmental surfaces in industrial and clinical settings. However, there remain additional studies on the relationships between biocide usage and bacterial resistance amongst isolates from community settings. Many of the studies were focused on the same set of bacteria; more efforts should be taken to focus on different strains of bacteria that also affect humans. QASs have much potential in medicine as active ingredients in treatment and as disinfectants for surfaces and supplies used in hospitals.