Listeria monocytogenes strains selected on ciprofloxacin or the disinfectant benzalkonium chloride exhibit reduced susceptibility to ciprofloxacin, gentamicin, benzalkonium chloride, and other toxic compounds

Listeria monocytogenes is a leading agent for severe food-borne illness and death in the United States and other nations. Even though drug resistance has not yet threatened therapeutic interventions for listeriosis, selective pressure associated with exposure to antibiotics and disinfectants may result in reduced susceptibility to these agents. In this study, selection of several L. monocytogenes strains on either ciprofloxacin (2 μg/ml) or the quaternary ammonium disinfectant benzalkonium chloride (BC; 10 μg/ml) led to derivatives with increased MICs not only to these agents but also to several other toxic compounds, including gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenylphosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC. Inclusion of strains harboring the large plasmid pLM80 revealed that MICs to ciprofloxacin and gentamicin did not differ between the parental and plasmid-cured strains. However, ciprofloxacin-selected derivatives of pLM80-harboring strains had higher MICs than those derived from the plasmid-cured strains. Susceptibility to the antimicrobials was partially restored in the presence of the potent efflux inhibitor reserpine. Taken together, these data suggest that mutations in efflux systems are responsible for the multidrug resistance phenotype of strains selected on ciprofloxacin or BC.     

Impact of benzalkonium chloride,benzethonium chloride and chloroxylenol on bacterial antimicrobial resistance

This review examined 3655 articles on benzalkonium chloride (BKC), benzethonium chloride (BZT) and chloroxylenol (CHO) aiming to understand their impact on antimicrobial resistance. Following the application of inclusion/exclusion criteria, only 230 articles were retained for analysis; 212 concerned BKC, with only 18 for CHO and BZT. Seventy-eight percent of studies used MIC to measure BKC efficacy. Very few studies defined the term ‘resistance’ and 85% of studies defined ‘resistance’ as <10-fold increase (40% as low as 2-fold) in MIC. Only a few in vitro studies reported on formulated products and when they did, products performed better. In vitro studies looking at the impact of BKC exposure on bacterial resistance used either a stepwise training protocol or exposure to constant BKC concentrations. In these, BKC exposure resulted in elevated MIC or/and MBC, often associated with efflux, and at time, a change in antibiotic susceptibility profile. The clinical relevance of these findings was, however, neither reported nor addressed. Of note, several studies reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BKC concentration. BKC exposure was shown to reduce bacterial diversity in complex microbial microcosms, although the clinical significance of such a change has not been established. The impact of BKC exposure on the dissemination of resistant genes (notably efflux) remains speculative, although it manifests that clinical, veterinary and food isolates with elevated BKC MIC carried multiple efflux pump genes. The correlation between BKC usage and gene carriage, maintenance and dissemination has also not been established. The lack of clinical interpretation and significance in these studies does not allow to establish with certainty the role of BKC on AMR in practice. The limited literature and BZT and CHO do not allow to conclude that these will impact negatively on emerging bacterial resistance in practice.     

Effect of benzalkonium chloride stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

Addition of benzalkonium chloride (BKC) at 0, 0.3, 0.6 and 1.0 mg l(-1) to tryptic soy broth (TSB) had no effect on growth of Lactococcus garvieae, a bacterial pathogen of the giant freshwater prawn Macrobrachium rosenbergii. However, injection of the cultured cells into prawns at a dose of 4 x 10(6) colony-forming units (cfu) prawn(-1) resulted in significantly higher mortality at 120 h (p < 0.05) in prawns injected with cells grown in the absence of BKC than in prawns injected with cells grown in the presence of BKC. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 3 x 10(5) cfu prawn(-1)) and then held in water containing BKC at 0, 0.3, 0.6 and 1.0 mg l(-1). After 120 h, mortality was significantly higher in all the BKC treatments than in the control without BKC. Prawns showed no significant differences in total hemocyte count (THC) or differential hemocyte count (DHC) amongst treatment and control groups. However, 96 h exposure to 0.3 mg l(-1) BKC or more resulted in a decrease in phenoloxidase activity and an increase in respiratory burst to levels considered to be cytoxic. In summary, exposure of L. garvieae to BKC at 0.3 mg l(-1) or more decreased its virulence to M. rosenbergii, while exposure of M. rosenbergii to BKC at 0.3 mg l(-1) or more increased its susceptibility to L. garvieae infection.     

Mechanism of resistance to benzalkonium chloride by Pseudomonas aeruginosa

The mechanisms of resistance of Pseudomonas aeruginosa to benzalkonium chloride (BC) were studied. The effluence of cell components was observed in susceptible P. aeruginosa by electron microscopy, but resistant P. aeruginosa seemed to be undamaged. No marked changes in cell surface potential between Escherichia coli NIHJC-2 and a spheroplast strain were found. The contents of phospholipids (PL) and fatty and neutral lipids (FNL) in the cell walls of resistant P. aeruginosa were higher than those in the cell walls of susceptible P. aeruginosa. The amounts of BC adsorbed to PL and FNL of cell walls of BC-resistant P. aeruginosa were lower than those for BC-susceptible P. aeruginosa. Fifteen species of cellular fatty acids were identified by capillary gas chromatography and gas chromatography-mass spectrometry. The ability of BC to permeate the cell wall was reduced because of the increase in cellular fatty acids. These results suggested that the resistance of P. aeruginosa to BC is mainly a result of increased in the contents of PL and FNL. In resistant P. aeruginosa, the decrease in the amount of BC adsorbed is likely to be the result of increases in the contents of PL and FNL.     

Mechanism of resistance to benzalkonium chloride by Pseudomonas aeruginosa.

The mechanisms of resistance of Pseudomonas aeruginosa to benzalkonium chloride (BC) were studied. The effluence of cell components was observed in susceptible P. aeruginosa by electron microscopy, but resistant P. aeruginosa seemed to be undamaged. No marked changes in cell surface potential between Escherichia coli NIHJC-2 and a spheroplast strain were found. The contents of phospholipids (PL) and fatty and neutral lipids (FNL) in the cell walls of resistant P. aeruginosa were higher than those in the cell walls of susceptible P. aeruginosa. The amounts of BC adsorbed to PL and FNL of cell walls of BC-resistant P. aeruginosa were lower than those for BC-susceptible P. aeruginosa. Fifteen species of cellular fatty acids were identified by capillary gas chromatography and gas chromatography-mass spectrometry. The ability of BC to permeate the cell wall was reduced because of the increase in cellular fatty acids. These results suggested that the resistance of P. aeruginosa to BC is mainly a result of increased in the contents of PL and FNL. In resistant P. aeruginosa, the decrease in the amount of BC adsorbed is likely to be the result of increases in the contents of PL and FNL.     

Postadaptational resistance to benzalkonium chloride and subsequent physicochemical modifications of Listeria monocytogenes

Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.     

Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli

Aims: To investigate the effect of oxygen limitation, glucose-starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results: The effect of BAC on planktonic and sessile cells were investigated using the gfp-tagged E. coli K-12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l⁻¹ BAC). Decreasing cell densities increased the decay rate for BAC-exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane-compromised cells than biofilms grown in aerobic medium. Image analyses of BAC-exposed biofilms showed that membrane-compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions: Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study: The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.     

Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride

The present study investigated and compared the effect of growth temperature on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells, to benzalkonium chloride (BAC). This study also highlights the impact of BAC on the bacterial physiology and the role of membrane fluidity regulation as a bacterial resistance mechanism. The minimum inhibitory concentration of BAC was characterized with micro-dilution growth inhibition assay. The BAC treatment was performed on S. aureus cultured at 20 °C and 37 °C, for 24 h. The morphology of S. aureus cells was examined using scanning electron microscopy. The loss of bacterial membrane integrity after BAC treatment was studied by monitoring the intracellular potassium ion leakage using the atomic absorption spectroscopy. The bacterial membrane total fatty acid composition, controlling the membrane fluidity, was analyzed by GC/MS. The results showed that the resistance of S. aureus cells to BAC increased with the increase of growth temperature. The planktonic cells were more susceptible to BAC than biofilm-detached ones. The rise of growth temperature resulted in an increase of S. aureus membrane rigidity. Furthermore, a higher membrane fluidity was observed in planktonic cells when compared to that in the biofilm-detached ones. The resistance of S. aureus seems to depend on the growth temperature. Compared to planktonic cells, biofilm-detached cells showed a greater resistance to BAC. The BAC targets and disturbs the bacterial membrane. Membrane fluidity modulation is likely a one of resistance mechanisms for S. aureus to BAC at the cellular scale. Therefore, disinfection procedures, in food sector, should be adapted for bacteria detached from biofilm.     

Effect of temperature and benzalkonium chloride on nitrate reduction

The effect of temperature and benzalkonium chloride (BAC) on nitrate reduction was investigated in batch assays using a mixed nitrate reducing culture. Nitrate was transformed completely, mainly through denitrification, to dinitrogen at 5, 10, 15 and 22 °C. In the absence of BAC, reduction of individual nitrogen oxides had different susceptibility to temperature and transient nitrite accumulation was observed at low temperatures. When the effect of BAC was tested up to 100 mg/L from 5 to 22 °C, denitrification was inhibited at and above 50 mg BAC/L with transient nitrite accumulation at all temperatures. The effect of BAC was described by a competitive inhibition model. Nitrite reduction was the denitrification step most susceptible to BAC, especially at low temperatures. BAC was not degraded during the batch incubation and was mostly biomass-adsorbed. Overall, this study shows that low temperatures exacerbate the BAC inhibitory effect, which in turn is controlled by adsorption to biomass.     

Factors contributing to resistance and susceptibility of cotton and other hosts to the boll weevil, Anthonomus grandis

A large portion of the world's germ plasm of cotton has been screened at the USDA Boll Weevil Research Laboratory, State College, Mississippi during the period 1962–68 utilizing developed techniques.An oviposition suppression factor causing 25–40% reduction in number of eggs laid by the weevil has been found in Gossypium barbadense and successfully moved into upland cotton, G. hirsutum.Two years field data with frego cottons indicate that the frego character contributes a significant degree of non-preference for egg laying by the boll weevil under field conditions. The frego character is currently being combined with selected lines of G. hirsutum carrying the oviposition suppression factor.A seedling screening technique utilizing field cages and greenhouse sand flats has been shown to have significant value in screening for individual resistant plants within a commercial variety. A number of selections with 25% or more resistance to oviposition are under extensive investigation.A number of biologically active materials have been detected in the cotton plant and other hosts of the boll weevil which significantly influences feeding stimulation, feeding deterrency, repellency and attractancy of the weevil to its hosts.A highly active square abscission factor has been detected and extracted successfully from 2nd and 3rd instar larvae. The material injected into cotton squares causes 95% abscission of squares within 48–60 hours.

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Zusammenfassung Ein großer Teil des Weltsortiments der Baumwolle wurde in den Jahren 1962–68 im USA-Baumwollkäfer-Institut der Staatshochschule von Mississippi mit modernen Methoden getestet.Ein die Eiablage unterdrückender Faktor, der die Anzahl der abgelegten Eier um 25–40% herabsetzt, wurde bei Gossypium barbadense gefunden und erfolgreich in Hochland-Baumwolle, G. hirsutum, eingekreuzt.Zweijährige Erfahrungen mit frego-Baumwolle weisen darauf hin, daß das frego-Merkmal unter Feldbedingungen in einem signifikanten Ausmaße zur Nonpräferenz für die eierablegenden Käfer beiträgt. Das frego-Merkmal wird laufend mit selektierten Linien von G. hirsutum kombiniert, die den Faktor für Eiablage-Unterdrückung tragen.Eine Sämlings-Prüfungstechnik, die Feldkäfige und Gewächshaus-Sandbetee benutzt, hatte nachweislich signifikanten Wert für die Auslese resistenter Einzelpflanzen innerhalb einer Handelssorte. Eine Anzahl Selektionslinien mit 25 oder mehr Prozent Eiablage-Resistenz wird intensiv geprüft.Eine Anzahl biologisch aktiven Materials wurde bei Baumwolle und anderen Wirtspflanzen endeckt, das Fraßstimulation, Fraßabschreckung, Repellenz und Attraktanz der Käfer in bezug auf ihre Wirte signifikant beeinflußt.Ein hochaktiver Kapsel-Abwurf-Faktor wurde entdeckt und erfolgreich aus Larven des 2. und 3. Stadiums extrahiert. In Baumwollkapseln injiziert, verursachte dieses Material innerhalb 48–60 Stunden Abwurf von 95% der Kapseln.

     

Pathology of benzalkonium chloride toxicity and its effect on body weight gain in broiler birds

Four groups comprising 16 broiler birds each were given benzalkonium chloride (BC) at 100, 300, 500 and 700 ppm in drinking water for 40 days and one group of 16 birds (control) was kept on plain water. Clinical signs in higher dose groups were respiratory distress, drooling of saliva, difficulty in deglutition, inappetence, apathy, lethargy and loss of body weight. Better body weight gain was recorded in 100 ppm dose rate. At 300 ppm, no significant body weight variation was recorded, whereas, at 500 and 700 ppm dose rates, significantly poor body weight gain was recorded. Major pathological changes were seen in 500 and 700 ppm groups, which exhibited formation of yellow diphtheritic plaques in the buccal cavity, swollen and pale commissures of beak and shortening of tongue. Minute necrotic and ulcerative foci were seen in oesophagus and crop. Hyperplastic and hypertrophic alterations were seen in mucosa of the upper digestive tract. Crop of 300 ppm group revealed formation of well developed epithelial nest with pseudoepitheliomatous hyperplasia at the margin of the lesion. Serum alanine transaminase, urea and uric acid in 500 and 700 ppm groups were elevated whereas no significant variations were observed in the 100 and 300 ppm groups. BC could enhance performance of broiler birds at 100 ppm dose rate. It should not be used beyond 300 ppm.     

Effect of chlorhexidine and benzalkonium chloride on bacterial biofilm formation

AIM: To study the effect of antiseptics on bacterial biofilm formation. METHODS AND RESULTS: Biofilm formation and planktonic growth were tested in microtiter plates in the presence of antiseptics. For Escherichia coli G1473 in the presence of chlorhexidine or benzalkonium chloride, for Klebsiella pneumoniae CF504 in the presence of chlorhexidine and for Pseudomonas aeruginosa PAO1 in the presence of benzalkonium chloride, biofilm development and planktonic growth were affected at the same concentrations of antiseptics. For PAO1 in the presence of chlorhexidine and CF504 in the presence of benzalkonium chloride, planktonic growth was significantly inhibited by a fourfold lower antiseptic concentration than biofilm development. For Staphylococcus epidermidis CIP53124 in the presence of antiseptics at the minimal inhibitory concentration (MIC), a total inhibition of biofilm formation was observed. For Staph. epidermidis exposed to chlorhexidine at 1/2, 1/4 and 1/8 MIC, or to benzalkonium chloride at 1/8, 1/16 or 1/32 MIC, biofilm formation was increased from 11.4% to 22.5% without any significant effect onto planktonic growth. CONCLUSIONS: Chlorhexidine and benzalkonium chloride inhibited biofilm formation of different bacterial species but were able to induce biofilm development for the Staph. epidermidis CIP53124 strain at sub-MICs. SIGNIFICANCE AND IMPACT OF THE STUDY: Sublethal exposure to cationic antiseptics may contribute to the persistence of staphylococci through biofilm induction.     

Haemophilus influenzae: the current status of its in vitro susceptibility to antimicrobials

Continuous surveillance of the antimicrobial susceptibility patterns of strains of Haemophilus influenzae (H. i.) is part of a routine surveillance of bacterial sensitivity to antibiotics. In this study a major attention was given to the clinically important antimicrobials used in the treatments of H. i. infections, namely ampicillin, chloramphenicol and some cephalosporin preparations. Antimicrobial susceptibility determinations carried out in 1983 and before on H.i. strains isolated in different Czech regions show a clear-cut tendency towards the higher incidence of strains resistant to ampicillin and its derivative amoxycillin. The rise in resistance to erythromycin was also observed. Susceptibility to chloramphenicol continues to remain unchanged. Of the cephalosporin antimicrobials tested (cephalothin, ceftriaxone, cefaperazone, cefsulodine and cefadroxil) the most active were ceftriaxone and cefaperazone. The need of a further continuation of surveillance of H.i. drug susceptibility patterns is strongly emphasized; to ensure effective treatment results antibiograms should be done in each case of H.i. infection.     

ClinicalPseudomonas aeruginosa: Potential factors of pathogenicity and resistance to antimicrobials

Resistance to 17 antimicrobials, surface hydrophobicity, motility, biofilm, production ofN-acylhomoserine lactone signal molecules (N-butyrylhomoserine lactone andN-3-oxolauroylhomoserine lactone) and response to oxidative stress were analyzed in 47 clinicalPseudomonas aeruginosa strains. In addition to natural resistance, the strains demonstrated the greatest level of resistance to cefotaxime (91.5 %). Isolates in the range of 44.7–57.4 % were resistant to aminoglycosides and ciprofloxacin, of 25.5–36.2 % to cephalosporins. On the other hand, 97.9 % remained susceptible to meropenem, 93.6 % to piperacillin + tazobactam and 87.2% to piperacillin. The majority of the strains (72.3 %) manifested their hydrophilic character. Higher zones of motility showed 12 isolates (in average 54.8 mm) as compared to the others (30.2 mm). Approximately 1/3 of the strains (29.8 %) produced a higher amount of biofilm quantified by measuring the absorbance of solubilized crystal violet (0.20–0.46) than the rest of isolates (0–0.19). All but two strains producedN-3-oxolauroylhomoserine lactone and in 48.9 % of samplesN-butyrylhomoserine lactone were detected. Only four isolates with higher biofilm production showed both types of homoserine lactone. Majority of the strains (70.2 %) manifested higher resistance to H₂O₂ than the rest of the strains. The group of strains resistant to aminoglycosides and ciprofloxacin revealed a significantly higher number of hydrophobic strains (compared with the sensitive ones). In contrast, higher number of strains sensitive to aminoglycosides and ciprofloxacin or only to ciprofloxacin producedN-butyrylhomoserine lactone and biofilm (compared to the resistant ones). Such association was not found among the rest of the tested parameters. The results indicate that the resistance to antimicrobials inP. aeruginosa isolates was not generally associated with changes in the production of the pathogenicity factors.     

Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.