Studied microbial hydrophobicity, adhesion to hydrophobic surfaces, oral microbiology, halitosis (bad breath), body odors. Now at Holon Institute of Technology.
Abstract: We have reported previously that growth on alcohol vapors confers hemolytic properties on certain yeast species and strains ['microbial alcohol-conferred hemolysis' (MACH)]. In a recent study, we analyzed the genetic basis of MACH in Saccharomyces cerevisiae using the EUROSCARF mutant collection. The data suggested that intact mitochondrial and respiratory chain functions are critical for the observed alcohol-mediated hemolysis. We proposed that the uncontrolled cellular uptake of alcohol results in yeast 'hyper-respiration', leading to elaboration of hemolytic molecules such as hydrogen peroxide and lytic lipids. In the current study, we have further analyzed the molecular mechanisms involved in the MACH phenomenon in S. cerevisiae, using DNA microarrays. The patterns of regulation were confirmed by quantitative reverse transcriptase PCR. The results presented here lend further support to this hypothesis, based on upregulation of the genes responsible for coping with vast amounts of hydrogen peroxide produced as a byproduct of excessive oxidation of alcohol. These results, taken together, show that alcohol-mediated hemolysis in yeast appears to be related to the overproduction of hemolytic byproducts, particularly hydrogen peroxide, which accumulates during long-term exposure of S. cerevisiae to both ethanol and n-butanol.
Abstract: In the present investigation we examined the effect of three brands of coffee on microbial volatile sulfur compound (VSC) production using a decarboxylase incubation assay. Stimulated whole saliva was added to decarboxylase medium supplemented with 0.005% hemin. Incubation was carried out anaerobically for 72 h in the presence of powdered coffee at concentrations ranging from 0.5 to 2.0% (w/v), as compared with appropriate controls. VSC levels were determined using OralChromaâ„¢ and Halimeterâ„¢ and malodor was scored by an experienced odor judge. Experimental biofilm was grown with or without coffee and examined for VSC-producing bacteria using confocal laser scanning microscopy. Results showed that VSC and malodor levels were decreased by 85% in the presence of 2% coffee. The data suggest that coffee components reduce malodor production, VSC levels and experimental biofilm VSC-producing bacteria in vitro.
Abstract: It was recently shown that, as in yeast, alcohols selectively increase the hemolytic properties of certain staphylococci strains. This phenomenon has been called 'microbial alcohol-conferred hemolysis'(MACH). Here we present the changes in gene expression by Staphylococcus aureus 8325-4, in response to ethanol. Ethanol upregulated the expression of multiple toxins and increase the pathogen potential of S. aureus strain 8325-4. Ethanol also increased the level of genes considered necessary for production and viability of biofilm, such as: icaAD, sdrDE, pyr, and ure. Increased urease activity appeared to be an important factor in the ethanol response along with macromolecule repair mechanisms. Oxidative-stress responses, such as increased expression of sodA1, sodA2 and upregulation of zinc-containing alcohol dehydrogenase, alcohol-acetaldehyde dehydrogenase (adhE) and two aldehyde dehydrogenases (aldA1, aldA2), which can generate more reducing power, were also induced. Upregulation of fatty acid metabolism appears to be important in enabling the bacteria to handle excess amounts of ethanol which ultimately may lead to synthesis of lytic lypids. The patterns of regulation were confirmed by quantitive reverse transcriptase PCR (QRT-PCR). These results, taken together, suggest that exposure to ethanol increases pathogenic traits and induce oxidative-stress responses.
Abstract: We recently suggested that oral malodor production involves two steps: (i) deglycosylation of glycoproteins and (ii) proteolysis and amino acid utilization of the protein core to yield volatiles such as volatile sulfide compounds (VSCs). Our aim was to test the hypothesis that β-galactosidase activity and VSC production occur in distinct areas of the biofilm by two different bacterial populations. Biofilms were grown anaerobically for seven days with or without antibiotics (i.e. vancomycin and metronidazole). Biofilms were stained for β-galactosidase activity and VSC production and studied using confocal laser scanning microscopy. Results showed that β-galactosidase activity occurs in the outer layers and disappears following vancomycin addition, whereas VSC production occurs deeper within the biofilm and disappears following metronidazole application. These findings suggest that β-galactosidase activity is produced mainly by Gram-positive oral bacteria at the outer portion of the biofilm, and VSC production occurs in the deeper layers by Gram-negative oral bacteria.
Abstract: Previous research has shown that the production of volatile sulfide compounds (VSC) by oral bacteria is associated with oral malodor. In the present study, we report a novel technique (microscopic sulfide assay (MSA)) for the quantification of VSC-producing oral microorganisms. The MSA was performed by overnight incubation of saliva samples in the presence of ferrous sulfate and sodium thiosulfate, followed by digital analysis of cells stained black due to cell-associated precipitation of ferric sulfide. This method was found to correlate significantly with oral malodor parameters, including mean odor judge scores (two judges, r = 0.48 and p = 0.001) and Halimeter® readings (r = 0.53 and p < 0.001), in a group of 42 subjects. As compared with odor judge scores as the gold standard, the new MSA technique yielded a diagnostic accuracy of 0.7 (ROC, p = 0.023). Results indicate that the MSA may serve as a diagnostic technique for assessing oral malodor levels and aid in identifying the particular bacteria involved in this condition.
Abstract: Hemolysis of blood agar is broadly used as a diagnostic tool for identifying and studying pathogenic microorganisms. We have recently shown that alcohol vapors can confer hemolytic properties on otherwise nonhemolytic fungi (microbial alcohol-conferred hemolysis; MACH). Until now, this phenomenon has been found in various yeast strains and other fungi, but only in a few bacterial species (e.g., staphylococci). In the current study we (1) determined the extent of the above phenomenon in various gram-positive and gram-negative laboratory bacterial strains and in clinical bacterial isolates, (2) validated the observed hemolysis using a quantitative technique, and (3) provided evidence that the observed alcohol-mediated hemolysis may, at least in part, be related to synthesis of hemolytic lipids.
Abstract: Bad breath is a common condition, difficult to assess in the general population. In the present study, we tested the hypothesis that a self-administered questionnaire can help identify factors associated with greater risk of oral malodor. Persons (n = 88) undergoing routine medical check-ups completed a questionnaire including 38 questions on general and oral health, dietary habits, and their own oral malodor levels. Oral malodor assessments included odor judge scores, volatile sulfide levels (via a Halimeter, Interscan Corp.), and salivary beta-galactosidase. Among the questionnaire results, 9 responses were significantly associated with odor judge scores (p < 0.05, unpaired t test), including questions on alcohol intake and body mass index (BMI). Predictions of odor judge scores based on these 9 questions (linear multiple regression analysis) yielded R = 0.601; when introduced together with Halimeter and beta-galactosidase scores, the correlation rose to R = 0.843. The results suggest that alcohol intake and BMI may be factors that help predict oral malodor.
Abstract: We have previously reported that growth on alcohol vapors confers hemolytic properties on certain yeast species and strains ('microbial alcohol conferred hemolysis'; MACH). Here, a Saccharomyces cerevisiae deletion library consisting of c. 4800 clones was screened for MACH mutants in the presence of n-butanol vapors; 136 mutants were MACH-negative, and 325 exhibited reduced hemolysis and/or growth. Of the MACH-negative mutants, 35.3% were affected in mitochondrial-related genes. The data suggest that intact mitochondrial and respiratory chain functions are critical for the observed MACH phenomenon. We propose that the uncontrolled cellular uptake of alcohol results in yeast 'hyper-respiration', leading to elaboration of hemolytic molecules such as hydrogen peroxide and hemolysis-causing lipids. To support this premise, we showed that: (1) exogenous catalase and glutathione reduce alcohol-conferred hemolysis in S. cerevisiae BY4741 and Candida tropicalis 59445; (2) C. tropicalis produces hydrogen peroxide following growth on ethanol and n-butanol, as shown using xylenol orange; and (3) a lysophospholipid-containing lipid extract from alcohol-grown C. tropicalis specifically causes hemolysis.
Abstract: It was recently found that alcohols can confer hemolytic properties on certain species of yeast. Here, it is reported that alcohol can promote hemolysis by various species of staphylococci, including strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis. In order to study this novel phenomenon in S. aureus and S. epidermidis, strains that exhibit this phenomenon (e.g. S. aureus 8325-4, COL, SH1000, S. epidermidis), as compared with strains that exhibited little alcohol-enhanced hemolysis (e.g. S. aureus 8325-4 DeltaTRAP, RN6911) were examined. Both ethanol and n-butanol caused upregulation of the virulence regulator-RNAIII, with a concomitant increase in the production of alpha, beta and gamma-hemolysins in strain 8325-4. In S. aureus COL and SH1000, there was an increase in RNAIII but no change in transcription levels of alpha, beta and gamma hemolysins. Staphylococcus epidermidis stain sofi exhibited increased RNAIII and beta hemolysin production. Staphylococcus aureus mutant strains (8325-4 DeltaTRAP and RN6911) showed no change in the transcription level of the RNAIII regulator and the above hemolysins. Increased hemolysis in S. aureus COL, SH1000 and mutant strains may be caused by other hemolysins (not regulated by RNAIII) or through other mechanisms such as hyperoxidation or cytotoxic lipids.
Abstract: Although the contribution of the oral microbiota to oral malodor is well-documented, the potential role of Gram-positive micro-organisms is unclear. In the current study, we tested the hypothesis that Gram-positive micro-organisms contribute to malodor production by deglycosylating oral glycoproteins, rendering them susceptible to subsequent proteolysis. To this end, we examined the effect of Streptococcus salivarius on Porphyromonas gingivalis-mediated putrefaction of a model glycoprotein (pig gastric mucin). Malodor was scored by two odor judges, and volatile sulfides were determined with the use of a sulfide monitor. Mucin degradation was followed by electrophoresis on SDS-PAGE. Results showed that the addition of S. salivarius or beta-galactosidase promoted mucin degradation and concomitant malodor production. Addition of glycosidic inhibitors (p-APTG and glucose) inhibited this process. These results suggest that Gram-positive micro-organisms such as S. salivarius contribute to oral malodor production by deglycosylating salivary glycoproteins, thus exposing their protein core to further degradation by Gram-negative micro-organisms.
Abstract: Twenty-five years ago this past autumn, we published a short article entitled 'Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity' in Volume 9 of FEMS Microbiology Letters. Together with my Ph.D. supervisors, Eugene Rosenberg and David Gutnick, we proposed a method of measuring bacterial cell surface hydrophobicity based on bacterial adherence to hydrocarbon ('BATH', later known as 'MATH', for microbial adhesion to hydrocarbon). The method became popular soon after it was published, and the paper was, for at least the following decade, the Journal's most cited article. It became an ISI 'citation classic' in 1991. This minireview is a rather personal look at the development of the method and its various modifications and other scientific offspring, with the perspective of a quarter-century.
Abstract: The purpose of the present study was to evaluate the extent of self-reported bad breath in an Israeli population of young army recruits and to assess its relationship with other self-reported parameters, as well as general dental status. Self-reported parameters included smoking status, bad taste, gingival bleeding and the presence of tonsilloliths. The study comprised 426 young adults recruits (ages 18-19), almost all males (95%), all of whom agreed to answer a questionnaire. All participants underwent a dental screening and were divided into three groups regarding to their dental status (do not need treatment, need moderate treatment, need extensive treatment). Statistical analysis included Pearson's chi square test of association using BMDP statistical software. Among the recruits, 142 (33%) were active smokers. Thirty-five participants (8.2%) reported bad breath as well as bad taste. Twenty-seven (6.3%) reported being told that they had bad breath. Tonsilloliths were reported by 31 participants (7.3%) and gingival bleeding by 80 (18.8%). Self-reported bad breath was positively associated with bad taste, gingival bleeding, the presence of tonsilloliths and general dental status (p<0.05). The study indicates that self reports of bad breath are associated by objective factors (e.g. dental status, tonsilloliths) as well as subjective parameters (bad taste).
Abstract: The aim of the present study was to determine whether oral malodour and periodontal disease parameters are associated with one another in 71 Israeli subjects (mean age 36.2 +/- 18.4; ages ranging from 15 to 65). Parameters measured included whole mouth odour judge scoring, Halimeter, OK to Kiss test, gingival index, plaque index and probing depth. Odour judge scores were significantly associated with Halimeter (r = 0.55; P < 0.001), as well as the OK to Kiss test (r = 0.52; P < 0.001). However, neither gingival index, plaque index nor probing depth was significantly associated with odour judge scores or Halimeter scores. Logistic regression analysis showed that both Halimeter and OK to Kiss scores factored significantly (P = 0.005 and 0.018, respectively, odds ratios 14.9 and 2.7, respectively) in predicting the severity of oral malodour. Results suggest that in the population studied, periodontal health and oral malodour are not associated with one another.
Abstract: The purpose of this review was to assess the relationship between mean organoleptic scores (using a 0-to-5 scale) and concentrations of putative odorants representative of those thought to be important in oral malodor, as well as to propose a simple model that explains the dose-response curves obtained from a group of odor judges.
Abstract: The 0-5 organoleptic scale is used widely in breath research and in trials to measure the efficacy of anti-odor agents. However, the precise relationship between odor scores and gas concentrations of target odorants is unknown. The purpose of this study was to relate mean organoleptic scores from odor judges (n = 7) for pure odorants (n = 8) representative of those found in oral malodor. Judges used a common 0-5 scale to report the odor intensity of sample sets in random order of concentration. Regression analysis of data showed that odor score was proportional to the log concentration of odorant, and comparison of slopes showed H(2)S to be the most significant in terms of odor power. Detection thresholds (mol.dm(-3)) were: Skatole (7.2 x 10(-13)) < methylmercaptan (1.0 x 10(-11)) < trimethylamine (1.8 x 10(-11)) < isovalerate (1.8 x 10(-11)) < butyrate (2.3 x 10(-10)) < hydrogen sulphide (6.4 x 10(-10)) < putrescine (9.1 x 10(-10)) < dimethyl disulphide (5.9 x 10(-8)). The study demonstrates the exponential nature of the olfactory response and shows that any single compound's contribution to malodor depends on odor power and threshold in addition to concentration.
Abstract: Although yeast are generally non-haemolytic, we have found that addition of alcohol vapour confers haemolytic properties on many strains of yeast and other fungi. We have called this phenomenon 'microbial alcohol-conferred haemolysis' (MACH). MACH is species- and strain-specific: whereas all six Candida tropicalis strains tested were haemolytic in the presence of ethanol, none among 10 C. glabrata strains tested exhibited this phenomenon. Among 27 C. albicans strains and 11 Saccharomyces cerevisiae strains tested, ethanol-mediated haemolysis was observed in 11 and 4 strains, respectively. Haemolysis is also dependent on the alcohol moiety: n-butanol and n-pentanol could also confer haemolysis, whereas methanol and 2-propanol did not. Haemolysis was found to be dependent on initial oxidation of the alcohol. Reduced haemolysis was observed in specific alcohol dehydrogenase mutants of both Aspergillus nidulans and S. cerevisiae. MACH was not observed during anaerobic growth, and was reduced in the presence of pararosaniline, an aldehyde scavenger. Results suggest that initial oxidation of the alcohol to the corresponding aldehyde is an essential step in the observed phenomenon.
Abstract: Halitosis (oral malodour or breath odour) is a fairly common complaint. Halitosis is most often a consequence of oral bacterial activity, typically from anaerobes. Occasional causes include systemic disease, and some patients have a psychogenic background to the complaint. The management is outlined in this paper.
Abstract: Aspiration of infected oropharyngeal content is the main cause of aspiration pneumonia. This complication, mainly related to gram-negative bacteria, threatens percutaneous enterogastric tube as well as nasogastric tube (NGT) fed patients. The objective of this study was to examine the oral microbiota of tuboenterally fed patients and compare it with that of orally fed counterparts.
Abstract: Present volumetric or gravimetric techniques for measuring saliva output are often cumbersome and, therefore, not generally used. In the present study, a simple approach to study the weight loss of a standard hard sugar candy after 3 minutes of passive incubation between tongue dorsum and palate was tested.
Abstract: Deglycosylation of oral mucins may be a critical initial step leading to their subsequent proteolysis and putrefaction. The present study was undertaken to determine whether activity in saliva of a major glycosidic enzyme (beta-galactosidase) is associated with oral malodor in a group of 64 subjects. Enzyme activity was detected by the use of a chromogenic substrate (X-Gal) impregnated on paper discs. Malodor-related measurements included two odor judges' assessments of whole-mouth and tongue malodor, and volatile sulfide levels measured by a portable sulfide monitor (Interscan Corp.). Beta-galactosidase assay scores were significantly associated with both odor judges' scores for whole-mouth (p < or = 0.002; Spearman) and tongue malodor (p < or = 0.001; Spearman). Beta-galactosidase activity and sulfide monitor measurements both factored significantly into multiple regression equations for odor judge scores, yielding multiple r-values ranging from 0.47 (p = 0.0007) to 0.60 (p < 0.0001). Analysis of the data presented indicates that beta-galactosidase activity in saliva is correlated with oral malodor.
Abstract: Bad breath, also known as halitosis, is a common concern for millions of people. Yet there is almost no reliable way for people to properly assess their breath odor. While many develop faulty perceptions about having bad breath that affect their entire lives, others who have halitosis are unaware of their condition.
Abstract: Oral malodor is a common complaint in Western society and is an important reason why adults seek dental counsel. In the present study, an attempt was made to evaluate the contribution of psychopathologic traits and of body-image char acteristics on participants' self-perception of breath odor. 60 participants without any specific complaint concerning breath odor (55% men: M age 35.5 yr., SD= 10) were evaluated. Variables included self-evaluation of participants' own breath odor (gener ally and current), an organoleptic evaluation of an impartial judge of odor, measurement of the volatile sulfide level in the oral cavity, and questionnaires referring to psychopathologic symptoms and body-image characteristics. Stepwise regression analysis showed that in addition to impartial measurements, self-perception of breath odor among noncomplaining subjects can be predicted by their feelings and attitudes toward the body and by their hostility.
Abstract: In an initial study, subjects complaining of bad breath were generally unable to score the level of their own oral malodor in an objective fashion. Subjects were taught several techniques for self-measurement of bad breath. One year following the initial consultation, subjects were recalled to determine whether their ability to assess their own oral malodor had improved.
Abstract: Bad breath is a common phenomenon, usually the result of bacterial metabolism in the oral cavity. It is generally accepted that Gram-negative bacteria are responsible for this problem, largely through degradation of proteinaceous substances. In initial experiments, screening of malodorous isolates following outgrowth of samples obtained from saliva, periodontal pockets, and the tongue dorsum yielded enterobacterial isolates. Clinical studies were conducted to examine the prevalence of such bacteria in four different populations: orthodontic patients, malodor clinic patients, complete-denture wearers, and a healthy young population. The prevalence of Enterobacteriaceae in the oral cavities of the denture-wearing population was very high (48.0%) as compared with the other groups: 27.1% in the malodor clinic patients, 16.4% in the normal population, and 13% among orthodontic patients. Isolates of Klebsiella and Enterobacter emitted foul odors in vitro which resembled bad breath, with concomitant production of volatile sulfides and cadaverine, both compounds related to bad breath. When incubated on a sterile denture, enterobacterial isolates produced typical denture foul odor. Isolates exhibited cell-surface hydrophobic properties when tested for adhesion to acryl and aggregation with ammonium sulphate. The results, taken together, suggest that Klebsiella and related Enterobacteriaceae may play a role in denture malodor.
Abstract: The main purpose of the study was to examine the anti-malodor properties of oxidizing lozenges, as compared to breath mints and chewing gum. Healthy, young adult volunteers (N = 123; mean age 24.5 years) were measured for oral malodor-related parameters (whole mouth odor measured by 2 judges; tongue dorsum posterior odor using the spoon test; volatile sulphide levels; salivary levels of cadaverine and putrescine; and 2 versions of an oral rinse test) on the first afternoon of the study. They were then assigned randomly to one of 6 groups (2 brands of breath mints, chewing gum with no active ingredients, regular and full-strength oxidizing lozenges, and a no-treatment control), and instructed to employ the treatment before bedtime, the next morning, and in the early afternoon 3 hours prior to measurements, which were carried out 24 hours following baseline measurements. Volunteers also estimated the level of their own whole mouth and tongue odors at baseline and post-treatment. The data showed that, among treatments, only the full-strength oxidizing lozenge significantly reduced tongue dorsum malodor, as determined by the spoon test. The full-strength lozenge also yielded a significant increase in the modified oral rinse test, presumably due, at least in part, to residual oxidizing activity retained in the oral cavity. Self-estimations of whole mouth and tongue malodor by volunteers were significantly correlated with corresponding-judge assessments, suggesting some degree of objectivity in assessing one's own oral malodor.
Abstract: Bad breath typically originates in the mouth, often from the back of the tongue. Nasal problems also can cause bad breath; odor generated in this manner can be easily distinguished from mouth odor by comparing the odor exiting the mouth and nose. In most cases, good professional oral care combined with a daily regimen of oral hygiene--including interdental cleaning, deep tongue cleaning and optional use of an efficacious mouthrinse---will lead to improvement. This article discusses common causes of oral malodor as well as methods to assess the extent of the problem.
Abstract: Oral malodor (halitosis) is a common concern in Western society. As with other human perceptions, emotional as well as cognitive variables play a major role in one's sensation and complaint. To study factors potentially associated with the complaint of oral malodor, periodontal and psychological evaluations were carried out on 38 subjects (66% female, mean age 43 years) with a complaint of oral malodor. Subjects underwent evaluation of their periodontal status, odor evaluation by an odor judge, and psychopathological symptom survey by means of the SCL-90 questionnaire. The patient's self-rating of oral odor was significantly higher than the evaluation of an objective odor judge and was not associated with their periodontal status. The SCL-90 profile of subjects was relatively higher than that of an age- and gender-matched reference group of dental patients. The results suggest that the complaint of oral malodor may be related to psychopathological symptoms as recorded by the SCL-90 questionnaire.
Abstract: The purpose of the study was to examine the anti-malodor, anti-gingivitis, and plaque reducing properties of a 2 phase oil:water mouthrinse compared with a control mouthrinse. Fifty subjects rinsed with one of the two rinses for 30 seconds twice a day over 6 weeks, while continuing their normal oral hygiene habits. Measurements were made at time zero (prior to beginning the rinsing regimen), and > or = 9 hours following rinsing, at intervals of 1, 3, and 6 weeks. Malodor of whole mouth, as well as tongue dorsum anterior and posterior, was assessed on a 0 to 5 semi-integer scale by two odor judges. Volatile sulphide compounds (VSC) were determined using a sulphide monitor. Gingival, plaque, and bleeding indices were recorded for Ramfjord teeth. Oral microbial levels were assessed using the oratest. Salivary levels of diamines (putrescine and cadaverine) were analyzed by HPLC. Results were analyzed by 2-tailed covariant ANOVA, with the time zero value as covariant. Dramatic improvements were observed in parameters associated with malodor, periodontal health, plaque accumulation, and microbial levels in both groups. As compared to time zero scores, whole mouth odor, tongue dorsum anterior and posterior odors decreased continuously over time, attaining 80%, 79% and 70%, reductions, respectively following 6 weeks, in the 2-phase mouthrinse group, versus 70%, 77% and 59% for the control group. For whole mouth and tongue dorsum posterior, the reductions observed in the 2-phase mouthrinse group were significantly greater than those obtained with the control mouthrinse (P = 0.026 and P = 0.025, respectively), suggesting that the 2-phase mouthrinse is superior to the control mouthrinse in long-term reduction of oral malodor. For bleeding index, gingival index, oral microbial levels, and VSC, differences between the groups were not significant. Diamine levels were not significantly reduced in either group. The control mouthrinse reduced plaque index more significantly than the 2-phase mouthrinse (P < 0.005). The results of this randomized clinical trial suggest that the 2-phase oil:water mouthrinse formulation is superior to the control mouthrinse in long-term reduction of oral malodor.
Abstract: In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli, E. coli DH5 alpha cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized component(s) on the transformant outer surface.
Abstract: Bad breath (halitosis, oral malodor) is a common condition, usually the result of microbial putrefaction within the oral cavity. Often, people suffering from bad breath remain unaware of it, whereas others remain convinced that they suffer from foul oral malodor, although there is no evidence for such. The purpose of the present investigation was to determine whether objective self-measurement of oral malodors is possible. Each of 52 volunteers was asked to sample the odor from his/her mouth, tongue, and saliva. Results were compared with (i) self-assessments prior to (preconception) and following (post-measurement) self-measurements; (ii) odor judge scores; (iii) dental-measurements (plaque index, gingival index, and probing depth); (iv) volatile sulphide levels; (v) salivary cadaverine levels; and (vi) intra-oral trypsin-like activity. Among the self-measurements, only saliva self-scores yielded significant correlations with objective parameters. Despite the partial objectivity of saliva self-estimates, subsequent post-measurement self-assessments failed to correlate with objective parameters. The results suggest that (i) preconceived notions confound the ability to score one's own oral malodors in an objective fashion; and (ii) partial objectivity can be obtained in the case of saliva self-measurement, presumably because the stimulus is removed from the body proper.
Abstract: Most investigations of mechanisms accounting for intergeneric coaggregation have emphasized stereospecific rather than nonspecific interactions. The purpose of this investigation was to determine the relative importance of lectin-carbohydrate and nonspecific hydrophobic and ionic interactions, using a model based on strains with one of the most well understood specific coaggregation mechanisms, the lactose-reversible coaggregation of Actinomyces naeslundii and Streptococcus oralis. The kinetics of coadhesion and desorption of coadherent bacteria were studied using S. oralis 34 bound to hexadecane droplets as an affinity support for the adhesion of A. naeslundii WVU 398A. Light, confocal microscopy and transmission electron microscopy confirmed that A. naeslundii cells adhered only to the S. oralis cells, not to exposed hexadecane between the streptococci. Coadhesion was inhibited by lactose concentrations as low as 2.0 mM. The rate of coadhesion was halved at 60 mM lactose. The hydrophobicity inhibitors bovine serum albumin and defatted bovine serum albumin and the salts LiCl and KCl failed to inhibit coadhesion in the hexadecane assay, and bovine serum albumin also failed to inhibit coaggregation in a bacterial aggregation assay on glass slides. High concentrations of the salts achieved a 50% rate decrease in A. naeslundii adhesion to the S. oralis-coated droplets only when they were combined with > 20 mM lactose. Sodium dodecyl sulfate (SDS) and Tween 20 inhibition was tested by the slide coaggregation assay because they tended to emulsify the droplets; SDS was inhibitory. Lactose selectively desorbed A. naeslundii from S. oralis-coated droplets at low concentrations equivalent to those that inhibited coadhesion. Neither LiCl nor KCl desorbed A. naeslundii from the droplets, even at 500 mM. At low concentrations, SDS but not Tween 20 eluted both A. naeslundii and S. oralis from the droplets. Although the SDS results might suggest a degree of cooperative charge interactions, the results support the hypothesis that stereospecific, beta-galactoside-sensitive interactions have a much greater impact than nonspecific interactions on the coadhesion of A. naeslundii and S. oralis.
Abstract: Associations between oral malodor, measures of periodontal disease, and trypsin-like activity of periodontal pathogens on tongue and teeth were examined in 127 subjects. Volatile sulphur compound (VSC) measurements were made with a portable sulphide monitor; oral malodor was also estimated by organoleptic methods. Measurements repeated one week apart indicated that steady-state VSC levels (r = 0.72; P = 0.0001) and peak VSC levels (r = 0.63; P = 0.0001) were reproducible but these r values were not significantly different (P > 0.1). There was a significant correlation between tongue odor and peak VSC levels (r = 0.40; P = 0.0001) and between tongue odor and whole mouth organoleptic measures (r = 0.55; P = 0.0001). To study the effect of reducing microbial colonization on oral malodor, chlorhexidine gluconate (0.2%) rinsing was prescribed for 7 days. Reductions of VSC levels were significant for both peak (37%) and steady-state (41%) data (P = 0.0001). Anaerobic periodontal pathogens on the tongue estimated by the proportions of positive BANA tests were reduced 19% (P = 0.001) and this was concomitant with a 40% (P = 0.0001) decrease in organoleptic measurement of the tongue dorsum. Mean pH measurements of the tongue dorsum showed large reductions from 6.9 initially to 6.3 post-treatment (P = 0.0001). Subjects were divided into periodontitis/no periodontitis based on periodontal inflammation and probing depth (> or = 5 mm). Of the 37 subjects with periodontitis, 23 had oral malodor whereas 52 out of 90 periodontally healthy subjects exhibited malodor. Chi square analysis comparing halitosis in subjects with and without periodontitis showed no statistically significant association (chi 2 = 0.208; P 0.65) between these two factors although the intensity of malodor as based on VSC concentration in periodontally healthy subjects was 19% less (mean = 111 ppb) than in subjects with periodontitis (mean = 136 ppb). The odds ratio was 1.2, indicating that oral malodor was not associated with periodontitis. These data indicate that a large proportion of individuals with oral malodor are periodontally healthy and that the mucosal surface of the tongue is a major site of oral malodor production.
Abstract: In a previous study (M. Barki, Y. Koltin, M. Yanko, A. Tamarkin, and M. Rosenberg, J. Bacteriol. 175:5683-5689, 1993), a 3.3-kb DNA fragment from Candida albicans which confers adhesion and autoaggregation in Saccharomyces cerevisiae was isolated and partially characterized. In this report, evidence is presented that the adhesion-autoaggregation phenotype observed in S. cerevisiae cells transformed with the candidal DNA fragment is due to expression of a C. albicans surface antigen. Rabbit antiserum, prepared against transformant S. cerevisiae cells, was adsorbed with S. cerevisiae bearing the vector alone. Immunofluorescence micrography showed that the adsorbed antiserum bound to the surface of transformant S. cerevisiae cells as well as to C.albicans cells, but only marginally to the S. cerevisiae control. The absorbed antiserum specifically inhibited autoaggregation of transformant cells. Further adsorption of the antiserum with transformant cells eliminated both inhibition and immunofluorescence. Autoaggregative activity and immunofluorescence of transformant cells were abolished following proteolytic treatment. Western blot (immunoblot) analysis of candidal extracts revealed that the absorbed antiserum recognized a major candidal antigen of ca. 30 kDa which was present on both yeast-phase and germ tube cells. The data suggest that the observed adhesion-autoaggregation phenotype is due to the presence of a specific candidal antigen on the outer surface of the transformant cells.
Abstract: Whereas previous studies have shown correlations between volatile sulphur compounds (VSC) and bad breath levels, it is probable that other compounds found in the oral cavity may contribute to oral malodor. In the present investigation, the possibility that diamines (cadaverine and putrescine) are associated with oral malodor parameters was assessed. Saliva samples from 52 subjects were analyzed for cadaverine and putrescine by HPLC. Oral malodor of whole mouth, tongue, and saliva of the subjects was recorded by an experienced judge on a continuous 10-cm scale; peak and steady-state VSC intraoral levels were measured by the Interscan 1170 sulphide monitor. Log-transformed VSC and diamine levels were compared with odor judge measurements by Pearson analysis and stepwise forward multiple regression. Putrescine scores were not significantly associated with odor judge parameters or with VSC levels (p > 0.1). However, highly significant correlations (p < or = 0.003) were found between cadaverine levels and all three odor judge assessments. In contrast, associations between cadaverine and VSC measurements were non-significant. In an attempt to correlate odor judge results in terms of both VSC and diamines, we carried out stepwise forward multiple regression. Results showed that VSC and cadaverine both factor significantly in explaining each of the odor judge measurements, with multiple r values ranging from 0.545 (p = 0.0002) to 0.604 (p < 0.0001). The results suggest that cadaverine levels are associated with oral malodor, and that this association may be independent of VSC.
Abstract: The purpose of the present investigation was to test the association between the BANA test (Perioscan, Oral-B), and oral malodor parameters. The subject population consisted of 52 Israeli adults, 43 of whom complained of oral malodor. Oral malodor measurements consisted of peak and steady-state volatile sulphide measurement by a portable sulphide monitor (Interscan Corp., model 1170), as well as organoleptic measurements of malodor from whole mouth, tongue, and saliva. Samples for the BANA test were obtained from four loci (shallow pocket, deep pocket, tongue dorsum, saliva); results were scored as negative (0), weak (1), or strong (2). BANA scores were significantly associated with odor-judge ratings, with the highest association obtained when BANA saliva scores and odor-judge saliva assessment were compared (r = 0.500; p < 0.001). BANA tests from the different loci were not significantly associated with sulphide monitor levels. Stepwise multiple-regression analysis of odor-judge measurements in terms of sulphide levels and average BANA scores showed that both log peak sulphide levels as well as BANA scores were significantly factored into the equations, yielding, in all cases, highly significant correlations (multiple r = 0.57, 0.50, and 0.59, respectively, with significance levels of 0.0001, 0.001, and < 0.0001, for whole mouth, tongue, and saliva malodor, respectively). The results suggest that the BANA scores are associated with a component of oral malodor which is independent of volatile sulphide measurements and suggest its use as an adjunct test to volatile sulphide measurement.
Abstract: Candida albicans is an opportunistic pathogen which may give rise to superficial and systemic infections. In the present study, C. albicans adhesion was studied by expression of C. albicans DNA sequences encoding adhesion functions in a nonadherent strain of Saccharomyces cerevisiae. Adherent transformant cells of S. cerevisiae harbouring a C. albicans genomic library cloned in a yeast-Escherichia coli shuttle vector were selected by using tissue culture-treated polystyrene as the attachment substratum. One transformant exhibited enhanced adhesion to treated and untreated polystyrene as well as autoaggregation, unlike control cells bearing the vector alone. Analysis of this clone revealed an insert of ca. 4.5 kb from C. albicans. Curing of the plasmid resulted in loss of adhesion and autoaggregation properties. A subclone bearing a reduced insert of 3.3 kb retained the ability to autoaggregate, to bind to treated and untreated polystyrene, and to adhere to buccal epithelial cells, unlike appropriate controls. Further subcloning of the insert to 2.7- and 1.9-kb fragments resulted in incremental decreases in adhesion and autoaggregation, whereas smaller fragments did not confer these properties. Hybridization of the 2.7-kb segment with C. albicans and S. cerevisiae DNA confirmed its origin as a single-copy sequence in the C. albicans genome as well as the absence of a homologous sequence in the genome of S. cerevisiae. The data suggest that the adhesion and aggregation phenomena of the transformant cells are related to expression of a C. albicans surface antigen encoded by the cloned DNA fragment.
Abstract: Measurement of oral malodor is complicated by a variety of parameters including complexity of gaseous molecular species, sampling difficulties, temporal variations, choice of suitable subject populations, and lack of agreement on reference standards. Since oral malodor is a perceived olfactory stimulus, direct sampling and assessment by human judges may be the most logical measurement approach. However, as with other psychophysical assessments, human malodor measurement by the human nose may vary widely among and between judges, and consequently cannot be confidently reproduced in other laboratories. Such shortcomings have led several investigators to propose quantitative approaches based on measurement of volatile sulfide compounds which are associated with oral malodor. Highly sensitive and discriminatory measurements of volatile sulfides can be made using gas chromatography although for rapid sampling of larger subject populations, portable sulfide monitors may be more appropriate. Future research in this field should consider: 1) improved and simplified instrumentation for more rapid through-put and reliability; 2) development and definition of reference standards for oral malodor assessment; 3) formulation of clinical studies with appropriately sized, well-defined patient populations; and 4) further development of within mouth, site-specific measurements.
Abstract: Few scientific investigations have addressed the ability of mouthrinses to reduce oral malodor for periods longer than 3 hours. In the present report, we have employed simple, recently described techniques to assess the day-long reduction in oral malodor of a novel 2-phase oil:water mouthrinse (TPM), as compared to a corresponding placebo rinse, and to a commercial 0.2% chlorhexidine mouthrinse. Sixty dental students were divided randomly into 3 groups, and instructed to use one of the rinses prior to bedtime and the following morning. Measurements carried out in the late afternoon, about 8 to 10 hours following rinsing, were compared with baseline measurements carried out in the late afternoon of the previous day. Volatile sulphide levels were measured using a portable industrial sulphide monitor. Microbial levels were estimated using a simple rinsing technique employing sterilized milk. These quantitative techniques were corroborated by organoleptic (hedonic) ratings of a single odor judge. Both TPM and chlorhexidine brought about significant decreases in volatile sulphides (P less than 0.05) as compared to the placebo group. These results were corroborated by the organoleptic data. Similarly, both chlorhexidine and TPM were highly effective in reducing microbial levels as measured by the rinsing technique, in comparison to the placebo group. Chlorhexidine appeared to be more effective than TPM in all measurement categories, although only in the case of microbial activity was there a significant (P less than 0.05) difference between the two groups.
Abstract: The Diaslide urine culture device consists of a hinged case containing two opposing agar media separated by a sampler with a handle at one end and two bent sampler tips at the opposite end. The tips of the sampler are first dipped into the urine. The sampler is then pulled out through the casing, simultaneously inoculating both agar surfaces with a streaking dilution. As a result, individual colonies can be observed even when bacterial concentrations exceed 10(6) CFU/ml. The number of colonies on the Diaslide correlated linearly with CFU per milliliter as determined by dilution plating. The clinical performance of the Diaslide was compared with those of ordinary dipslides and conventional cultures with a sample of 473 prescreened hospital urine specimens. The sensitivity, specificity, and positive predictive value of Diaslide versus those of culture at the 10(4)-CFU/ml cutoff level were 97.5, 98.3, and 98.3%, respectively, compared with 98.8, 95.7, and 97.2%, respectively, for dipslide versus culture. Similar results were found at the 10(5)-CFU/ml cutoff level. Only 5.5% of the Diaslides required subculturing, compared with 14.7 and 9.4% of the dipslides and conventional cultures, respectively. The Diaslide proved more convenient than an ordinary dipslide for sampling low volumes of urine. These data suggest that the Diaslide is a simple, effective device for culturing of urine specimens.
Abstract: Previous studies have established that hydrogen sulphide and mercaptans are the primary components of halitosis (bad breath). In the present investigation, we report a simple, rapid technique for measurement of halitosis-related sulphides. The technique is based on a portable instrument generally used for environmental safety applications. Seventy-five volunteers were measured using this technique, and the results (in peak ppb hydrogen sulphide equivalents) compared with organoleptic assessment by 7 judges. A highly significant overall correlation (r = 0.603; P less than 0.001) was obtained between these 2 methods. Moreover, in most cases, the organoleptic ratings of the individual judges correlated more highly with sulphide monitor values than with one another. The simplicity of the technique suggests its use in clinical studies as well as in diagnosis and treatment of patients with this complaint.
Abstract: Forty-one subjects with bad breath were assessed for oral malodor and periodontal status on three occasions, at intervals of approximately one week. Oral malodor was assessed by measurement of peak and steady-state volatile sulphide levels with a portable sulphide monitor and by organoleptic measurement of whole-mouth, tongue dorsum, and interproximal dental odors by two independent judges. Reproducibility of measurements, assessed by paired t tests and Kappa testing, demonstrated no significant differences between any of the test results from the first and second appointments. Steady-state sulphide levels were the most reproducible of all tests. The ability of the tests to detect an expected reduction of malodor following a 0.2% chlorhexidine mouthrinse regimen was investigated by comparison of test values between the second and third appointments. Following the mouthrinsing treatment, 43% reductions of peak, 47% reductions of steady-state volatile sulphide levels, and 15-58% reductions in all other measurement categories were observed. The majority of the participants (22/41) had no pockets greater than 5 mm and exhibited both moderate gingival inflammation (Mean Gingival Index = 1.17) and moderate plaque accumulation (Mean Plaque Index = 1.84). Plaque Index and presence of pockets greater than 7 mm were weakly related to sulphide measurements. Whereas assessment of steady-state sulphide levels by the sulphide monitor does not constitute a direct measure of oral malodor, its relation to organoleptic measurement, superior reproducibility, objectivity, and sensitivity support the use of the sulphide monitor in clinical studies.
Abstract: Microbial hydrophobicity has been studied since 1924. During the last decade, various techniques have become available for measuring hydrophobic surface properties of microbial cells. This has led to a surge in investigations suggesting a role for hydrophobicity in adhesion of bacteria to an array of surfaces (oral surfaces, mineral particles, fatty meat, epithelial cells, phagocytes, biomaterials), partitioning at interfaces, as well as gliding mobility. The present manuscript comprises a critical, chronological look at the origins of microbial hydrophobicity research, its development, origins, and applications. Emphasis is placed on microbial adhesion to hydrocarbons, a technique with which the author has the most experience and research interest.
Abstract: Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.
Abstract: Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.
Abstract: A simple and rapid test for measuring oral hygiene was recently developed. It is based on the rate of oxygen consumption of oral expectorates of milk. This investigation modified the test to study denture hygiene. The dentures of 20 patients were immersed in 10 mL of sterile milk. After a 2-minute agitation, 3 mL of milk was added to test tubes containing methylene blue. The time required for color change at the bottom of the test tube, which is indicative of the rate of oxygen consumption, was recorded. For comparison with visual plaque accumulation, the dentures were coated with disclosing solution and the extent of plaque was scored by three examiners. A correlation was found between the plaque index scores and results of the milk test (r = -0.64; p less than 0.005). The data suggests the use of this test to monitor denture hygiene.
Abstract: A simple, non-invasive test (the Oratest) has recently been proposed, which provides an estimate of oral microbial levels based on the rate of oxygen depletion in expectorated milk samples. Following 30 seconds of vigorous rinsing with sterilized milk, 3 ml of the expectorate is added to a test tube containing the redox indicator, methylene blue, and the time required for a color change from blue (i.e., aerobic conditions) to white (anaerobic conditions) at the bottom of the test tube is recorded. In the present study, Oratest scores were compared to clinical parameters (Plaque Index [PI] and Gingival Index [GI]) in a group of 49 volunteers. Significant correlations were found between the logarithm of Oratest results and PI (r = -0.58; P = 0.001) as well as GI (r = -0.66; P = 0.001). The data indicate that the Oratest provides a reliable estimate of gingival inflammation, thus extending the previously reported strong correlations between Oratest scores and microbial counts. The data suggest that the Oratest may have potential as a clinical and research tool.
Abstract: Microbial adhesion at the oil-water interface is a subject of both basic interest (e.g., as a technique for the measurement of hydrophobicity) and applied interest (e.g., for use in two-phase oil-water mouthwashes for the desorption of oral microorganisms). In general, surfactants inhibit microbial adhesion to oils and other hydrophobic surfaces. In the present study, we demonstrated that the cationic surfactant cetylpyridinium chloride (CPC) significantly enhanced microbial adhesion to hexadecane and various oils, as well as to the solid hydrophobic surface polystyrene. CPC increased adhesion to hexadecane of Escherichia coli, Candida albicans and Acinetobacter calcoaceticus MR-481 and of expectorated oral bacteria from near 0% to over 90%. The CPC concentration required for optimal enhancement of adhesion was a function of the initial cell density. This phenomenon was inhibited by high salt concentrations and, in the case of E. coli, by a low pH. CPC-pretreated cells were able to bind to hexadecane, but CPC-pretreated hexadecane was unable to bind untreated cells. Another cationic, surface-active antimicrobial agent, chlorhexidine gluconate, was similarly able to promote microbial adhesion to hexadecane. The results suggest that (i) CPC enhances microbial adhesion to hexadecane by binding via electrostatic interactions at the cell surface, thus diminishing surface charge and increasing cell surface hydrophobicity, and (ii) this phenomenon may have applications in oral formulations and in the use of hydrocarbon droplets as a support for cell immobilization.
Abstract: The adhesion of Streptococcus sanguis to hydroxylapatite is a process involving several adhesins and receptors. Binding isotherms and Scatchard plots of the adhesion suggest that cooperative interactions occur at low cell densities. It was found that sulfolane, a hydrophobic-bond diluent, was capable of inhibiting the cooperative adhesion of S. sanguis to saliva-coated hydroxylapatite beads. Sodium thiocyanate, a chaotropic agent, inhibited not only cooperative adhesion, but also the adhesion thought to result from noncooperative interactions. It is suggested that strong chaotropic agents may not only inhibit adhesin-receptor complexes, but also may influence the secondary/tertiary structures of interacting species.
Abstract: Serratia marcescens RZ has been previously shown to possess pronounced cell-surface hydrophobicity, as evidenced by its affinity for hydrocarbons and polystyrene. The present report suggests the involvement of a 70 kDa protein, serraphobin, in this phenomenon. The 70 kDa protein was recovered from both the cell surface and culture supernatant of hydrophobic wild-type cells, but was either totally absent or present in minor quantities in hydrophobicity-deficient mutants. Similarly, loss of hydrophobicity of RZ cells following growth at 39 degrees C was accompanied by loss of the protein. Serraphobin was capable of binding to hexadecane droplets following a brief mixing procedure, and could be desorbed by solidifying and melting the hexadecane phase.
Abstract: The cell surface hydrophobicity of Serratia marcescens appears to be an important factor in its adhesion to and colonization of various interfaces. The cell surface components responsible for mediating the hydrophobicity of S. marcescens have not been completely elucidated, but may include prodigiosin and other factors. In the present report we have investigated the potential role of serratamolide, an amphipathic aminolipid present on the surfaces of certain S. marcescens strains, in modulating cell surface hydrophobicity. The hydrophobic properties of a serratamolide-producing strain (NS 38) were compared with those of a serratamolide-deficient mutant (NS 38-9) by monitoring the kinetics of adhesion to hexadecane. Serratamolide production was monitored by thin-layer chromatography and the wetting activity of washed-cell suspensions on polystyrene. Wild-type NS 38 cells were far less hydrophobic than the serratamolide-deficient mutant cells were; the removal coefficients were 48 min-1 for the mutant, as compared with only 18 min-1 for the wild type. The data suggest that the presence of serratamolide on S. marcescens cells results in a reduction in hydrophobicity, presumably by blocking hydrophobic sites on the cell surface.
Abstract: In the present study, we examined the potential roles of cell surface hydrophobicity and mannose-sensitive (MS) interactions in blood clearance of Serratia marcescens in mice. Hydrophobic strain RZ, partially hydrophobic mutant 3162, and nonhydrophobic mutant 3164 were coinoculated into BALB/c male mice, and blood samples were plated out at different time intervals; colonies of the three strains were distinguished by their different morphologies. All three strains were cleared from the blood stream at similar rates, despite their large relative differences in cell surface hydrophobicity. Clearance from blood was subsequently studied by coinoculating two clinical isolates which differ in their abilities to adhere via MS interactions. MS+ strain 1785 was cleared much more rapidly than MS- strain 3255; moreover, in the presence of D-mannose, clearance of strain 1785 was inhibited to a rate similar to that of MS- strain 3255. When D-glucose was substituted for D-mannose, inhibition was not observed. The results suggest that MS, rather than hydrophobic, interactions are primarily responsible for the rapid clearance of S. marcescens from blood observed.
Abstract: The cell surface hydrophobicity of 10 pigmented and 4 nonpigmented clinical Serratia marcescens strains was studied, based on the ability of the strains to adhere to hydrocarbons and to polystyrene. The cell surface hydrophobicity depended greatly on growth temperature; all of the strains tested were adherent following growth at 30 degrees C, whereas none was adherent following growth at 38 degrees C. In previous studies, the pigment prodigiosin has been cited as responsible for cell surface hydrophobicity in various Serratia strains. However, the observed ability of the nonpigmented strains to adhere to the test hydrocarbons and to polystyrene indicates that Serratia strains can possess hydrophobic surface properties in the absence of this pigment. Moreover, strain 1785 cells were adherent whether they were grown at 30 or 36.5 degrees C, even though pigment was not synthesized at the higher temperature. In Escherichia coli correlations have been noted between increased cell surface hydrophobicity and the presence of mannose-specific adhesins; no such relationship was found in the S. marcescens strains tested. The expression of cell surface hydrophobicity in clinical S. marcescens strains at 30 degrees C and the loss of hydrophobicity at host temperatures raise the possibility that infective cells from the environment are initially hydrophobic, but lose this property upon subsequent proliferation within a host.
Abstract: A method is proposed for the relatively simple and rapid separation of amphipathic biopolymers, based on adsorption onto and desorption from the surface of hexadecane droplets. Adsorption to the hexadecane:water interface was carried out by mixing hexadecane with aqueous protein solutions at room temperature. Desorption was performed by consecutive solidification and melting of the liquid hydrocarbon (m.p. 18 degrees C), resulting in coalescence of the droplets and reappearance of the desorbed moiety in the bulk aqueous phase. Of interest was the observation that lysozyme remains enzymatically active following this procedure.
Abstract: Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface factors.
Abstract: One of the advantages in using the carbon dioxide laser in medicine is the sterilization of the wound at the site of surgical intervention. In microbial studies, using the Sharplan Model 743 Medical Laser, we found substantial contamination of the area directly below the probe by viable bacteria and fungi. The levels of contamination varied from experiment to experiment, but were always substantial. The contamination is likely due to the stream of nitrogen gas emitted during and following laser irradiation in order to cool the lens. Following the implementation of several simple prophylactic procedures, including insertion of a filter on the end of the tube emitting the nitrogen gas, contamination by the gas stream was reduced to insignificant levels.
Abstract: The cell surface hydrophobicity of bacteria obtained directly from human tooth surfaces was assayed by measuring their adherence to liquid hydrocarbons. Fresh samples of supragingival dental plaque were washed and dispersed in buffer. Adherence of the plaque microorganisms to hexadecane, octane, and xylene was tested turbidimetrically and by direct microscopic observation. The results clearly show that the vast majority of bacteria comprising dental plaque exhibit pronounced cell surface hydrophobicity. These data support the hypothesis that hydrophobic interactions play a major role in mediating bacterial adherence on tooth surfaces.
Abstract: Acinetobacter calcoaceticus RAG-1 and BD413, as well as Streptococcus pyogenes M-5, adhered to octane. Adherence was inhibited by emulsan (100 micrograms/ml), the polymeric emulsifying agent produced by A. calcoaceticus RAG-1. Emulsan also inhibited adherence of S. pyogenes and RAG-1 to buccal epithelial cells. The mean values of bound S. pyogenes per epithelial cell were 57.2 and 20.7 for the control and emulsan-containing suspensions, respectively; mean values of bound RAG-1 per epithelial cell were 221 for the control and 40 for the suspension containing 100 micrograms of emulsan per ml. Desorption of previously bound RAG-1 from epithelial cells by emulsan was concentration dependent: a maximum of 80% desorption was obtained with 200 micrograms of emulsan per ml. The data showing that emulsan desorbed 70% of the indigenous bacterial flora from buccal epithelial cells suggest that hydrophobic interactions mediate not only the in vitro adherence of laboratory strains to epithelial cells, but actually govern the adherence of the majority of the bacteria that colonize this surface. The advantages of using emulsan as an antiadherence agent include its chemical purity, stability, and polymeric nature.
Abstract: Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.
Abstract: Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.
Abstract: A simple replica method is described for the rapid identification of colonies of bacteria which adhere to polystyrene. A correlation was found between the adherence of bacterial strains to polystyrene and cell surface hydrophobicity, suggesting the use of this technique in screening for cell surface mutants and in the isolation of hydrophobic bacteria from nature.
Abstract: The ability of Acinetobacter calcoaceticus RAG-1 to adhere to human epithelial cells was investigated and compared with its ability to adhere to a test hydrocarbon (hexadecane). RAG-1, a microorganism originally isolated for growth on hydrocarbon, adhered to epithelial cells when grown under conditions which promote its adherence to hexadecane; similarly, RAG-1 cells adhered poorly to epithelial cells when grown under conditions which cause the cells to possess low affinity towards hexadecane. A mutant derived from RAG-1, MR-481, deficient in its ability to adhere to hydrocarbon, was similarly unable to adhere to epithelial cells. RAG-1 adherence to epithelial cells was not blocked by a number of sugars tested. Streptococcus pyogenes, whose adherence to epithelial cells has been previously attributed to hydrophobic interactions, was also able to adhere to hexadecane. Results suggest that hydrophobic interactions mediate adherence of the strains studied to both epithelial cells and hydrocarbon.
Abstract: The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.
