Gram Negative Microorganisms and Oral Malodor*

ORIGINAL PAPERS Dent. Med. Probl. 2004, 41, 2, 235 239 ISSN 1644 387X ROMAN GANCARZ 1, IRENA MALISZEWSKA 1, ANNA FRĄCKOWIAK 1, BARBARA BRUZIEWICZ MIKŁASZEWSKA 2 Gram Negative Microorganisms and Oral Malodor* Bakterie Gram ujemne i halitoza 1 Institute of Organic Chemistry, Biochemistry and Biotechnology, Department of Chemistry, Wroclaw University of Technology, Poland 2 Department and Chair of Dental Prosthodontics Wroclaw Medical University, Wroclaw, Poland Abstract Background. Halitosis, also known as oral malodor, affects a large population of patients. It is generally considered as a result of volatile sulphur compounds (VSC), amines and carboxylic acids, produced by Gram negative anaero bic bacteria. Material and Methods. The studied material was taken from the tongue and periodontal pockets of the 12 patients and various strains of microorganisms (Gram negative and Gram positive) were isolated. In the experiments the authors used five strains of bacteria, namely: Enterobacter cloacae, Enterobacter amnigenes, Pseudomonas puti da, Comomonas testosteroni and Cedecea sp. All of these microorganisms were grown in aerobic conditions on mineral medium containing cysteine, metionine or caseine as the only source of carbon. In this manner the authors were able to monitor the level of smell as a function of a substrate in the medium. Results. The best source of carbon for all studied bacteria was caseine, whereas the highest level of smell was observed when cysteine or methionine was present in the medium. The presence of hydrogen sulphide and methyl sul phide in the air above the medium was confirmed by chemical analysis. No detectable amount of amines and volatile carboxylic acids were observed as monitored by GC MS analysis. Conclusions. These results suggest that Gram negative facultatively anaerobic rods may play a role in oral mal odor production (Dent. Med. Probl. 2004, 41, 2, 235 239). Key words: halitosis, GC MS, Gram negative microorganisms, volatile sulphur compounds. Streszczenie Wprowadzenie. Choroba jamy ustnej halitoza dotyka znaczną część populacji ludzi. Powszechnie uważa się, że nieprzyjemny zapach z ust jest następstwem tworzenia się lotnych związków siarki, amin i lotnych kwasów kar boksylowych. Związki te są wytwarzane przez bakterie kolonizujące jamę ustną, a zwłaszcza język. W literaturze zgodnie twierdzi się, że odpowiedzialne za ten efekt są przede wszystkim bakterie Gram ujemne. Materiał i metody. Użyty w eksperymentach materiał badawczy został wyizolowany z próbek pochodzących z ję zyka i kieszonek zębowych 12 pacjentów dotkniętych halitozą. Wyizolowano zarówno bakterie Gram dodatnie, jak i Gram ujemne. Zbadano 5 szczepów bakterii Gram ujemnych: Enterobacter cloacae, Enterobacter amnigenes, Pseudomonas putida, Comomonas testosteroni i Cedecea sp. Bakterie hodowano na podłożu mineralnym, zawie rającym odpowiednio cysteinę, metioninę i kazeinę jako jedyne źródło węgla. Wyniki. Najlepszy wzrost drobnoustrojów obserwowano, gdy w podłożu była obecna kazeina, najwyższy poziom nieprzyjemnego zapachu stwierdzono natomiast, gdy w podłożu była obecna metionina i cysteina. Obecność siar kowodoru i siarczku metylu potwierdzono metodą analizy chemicznej. Natomiast metodami GC MS nie wykazano obecności lotnych amin i kwasów karboksylowych. Wnioski. Uzyskane wyniki badań sugerują, że względnie beztlenowe Gram ujemne pałeczki mogą uczestniczyć w wytwarzaniu nieprzyjemnego zapachu z ust (Dent. Med. Probl. 2004, 41, 2, 235 239). Słowa kluczowe: halitoza, GC MS, Gram ujemne bakterie, lotne produkty siarkowe. * This study was supported by KBN research grant no. 3 PO5D 103 23.

236 R. GANCARZ, I. MALISZEWSKA, A. FRĄCKOWIAK, B. BRUZIEWICZ MIKŁASZEWSKA Halitosis affects a large proportion of the pop ulation and may be the cause of a significant social and psychological problems [1, 2]. Bad breath (halitosis, fetor ex ore) is generally considered to result from the production of malodor compounds by oral bacteria [3]. Mainly volatile sulphur com pounds, but also amines (cadaverine, putrescine) and volatile carboxylic acids [4 6] were found in the saliva of the patients. Studies of oral malodor have contributed to our understanding of microbial colonization of oral soft tissues, particularly the dorsum of the tongue. These studies indicated that a wide range of bacteria species, for example Treponema denti cola, P. gingivalis, Bacteroides forsythus, Prevo tella melaninogenica, P. intermedia, Fusobacte rium, Streptococcus, Lactobacillus, Rothia, Cap nocytophaga and Actinomyces spp. could be found in samples taken from the tongue [7]. These oral isolates produce volatile sulphur compounds (VSC) including hydrogen sulphide and methyl mercaptan during growth in vitro [8]. Also cadaverine, which is produced in vitro by the decarboxylation of lysine, was shown to be signif icantly associated with the organoleptic scores derived from the saliva and tongue [4]. Many other malodorous compounds produced by the oral bac teria are difficult to identify. Among the oral microorganisms, a range of Gram negative bacte ria is considered to be particularly odorigenic [9]. Goldberg et al. [10] postulated that members of Enterobacteriaceae family, Klebsiella in partic ular, could participate in the production of oral malodor. However, Enterobacteriaceae are not generally considered as important members of the oral microbiota. In this contribution the authors present results of examination malodor production by oral Gram negative facultative anaerobic bacteria. The malodor producing properties of isolated rods were compared under various conditions. Material and Methods Identification of the Bacteria Strains The 12 patients (6 males, 6 females) were selected from those who have apparent halitosis according to examiners at the general dental health screening. All of the selected subjects agreed to participate in the study. They were aged from 4 to 70. The material taken from the tongue and peri odontal pockets of the subjects were analysed and various strains of microorganisms (Gram negative and Gram positive) were isolated. Isolation and identification of microorganisms were carried out according to the standard procedure [11, 12] at the Bacteriological Laboratory Korczak s Children Hospital in Wroclaw. In vitro Production of Oral Malodour Malodor production was analysed in vitro. In the studies authors tested five Gram negative bac terial strains isolated from malodor clinic patients. The studied strains were: Enterobacter cloacae, Enterobacter amnigenes, Pseudomonas putida, Comomonas testosteroni and Cedecea sp. The bacteria were inoculated into broth medium and/or modified minimal Davis [13] medium con taining cysteine, methionine or caseine as the car bon source and incubated at 37 C for 4 5 days. The growth of bacteria was determined as OD 550 (optical density). The volatile compounds were determined as described below. Identification of the Malodor Compounds The Volatile Metabolites, Other than Sulphur Compounds All potential volatile metabolites (amines, car boxylic acids) were collected by passing the nitro gen gas through the flask containing the growing bacteria, for 45 minutes at the nitrogen pressure 0.1 bar, which corresponds to the flow 1.2 10 4 m 3 /min followed by absorption in 5 ml of ethyl alcohol cooled down to 78 C (a dry ice bath). The solution was then analysed by GC MS process. In this method the identities of the compounds present in the solution were identified by the composition pattern and comparison with literature data. The Hydrogen Sulphide Analysis (Method A) A stream of nitrogen gas passed through the flask containing the growing bacteria and then throuh the solution of lead (IV) acetate (20 ml of 1M solution) for 45 minutes at the nitrogen pres sure 0.1 bar, which corresponds to the flow 1.2 10 4 m 3 /min. Then the precipitate (lead sulphide) was centrifuged for 15 minutes at 800 rad/s. The precipitate was dried to the constant weight. Each measurement was repeated three times.

Gram Negative Microorganisms and Oral Malodor 237 The Hydrogen Sulphide Analysis (Method B) The hydrogen sulphide was collected in man ner similar to the method A but it was collected in the 20 ml of water (in the ice bath). To the obtained solution the known amount of iodine (0.1M) was added and the excess of iodine was titrated by 0.1M solution of sodium thiosulphate. Estimation of the Smell Level The smell intensity was estimated by the organoleptic method using scale from 1 to 3, where: 1 very low, at the detection limit, 2 mo derate, 3 strong. Results Bacteria Growth Analysis of the material taken from the exam ined patients led to isolation of various microor ganisms (Tab. 1). There is a general consensus that Gram negative oral microorganisms are responsi ble for most cases of oral malodor. In the experiments, five bacterial strains, name ly Gram negative rods: Enterobacter cloacae, Enterobacter amnigenes, Pseudomonas putida, Comomonas testosteroni and Cedecea sp. were studied. All these strains were able to grow in the mineral medium containing: cysteine, methionine or caseine as the carbon source (Tab. 2). In this manner the authors were able to analyse the differ ences in production of volatile compounds as a function of the substrate in the medium. The growth of the bacteria was analysed by measuring the opti cal density at 550 nm and these data are presented in Table 2. The best source of carbon for all studied bacteria was caseine. Comomonas testosteroni is the slowest growing strain for all studied media. Cysteine and methionine is a good substrate for Cedecea sp. but worse for Enterobacter amnigenes Table 2. Optical density (OD 550 ) of tested bacteria Tabela 2. Gęstość optyczna (OD 550 ) hodowli testowanych bakterii Carbon source (Źródło węgla) Strains cysteine methionine caseine 1% (Szczepy) (cysteina) (metionina) (kazeina) 12 mm 12 mm OD 550 Enterobacter 0.17 0.16 0.20 cloacae Enterobacter 0.10 0.13 0.22 amnigenes Pseudomonas 0.22 0.23 0.22 putida Comomonas 0.05 0.12 0.13 testosteroni Cedecea sp. 0.24 0.19 0.23 Table 1. Oral microorganisms isolated from subjects Tabela 1. Mikroorganizmy wyizolowane z jamy ustnej pacjentów Microorganisms Number of subjects in (Mikroorganizm) which bacteria were found (Liczba pacjentów, u któ rych znaleziono bakterie) Enterobacter cloacae 2 Enterobacter amnigenes 1 Cedecea sp. 1 Pseudomonas putida 1 Comomonas testosteroni 1 Staphylococcus aureus 1 Streptococcus oralae 12 Peptostreptococcus prevotii 1 Prevotella oralis 2 Veillonella sp. 8 Neisseria sp. 12 Candida sp. 3 Actinomyces 1 Table 3. The odor estimation in the air above the mineral medium containing bacteria and cysteine, methionine or caseine as a carbon source Tabela 3. Określenie poziomu nieprzyjemnego zapachu w powietrzu nad pożywką mineralną zawierającą bakterie rosnące na cysteinie, metioninie lub kazeinie jako źródle węgla Carbon source (Źródło węgla) Strains cysteine methionine caseine 1% (Szczepy) (cysteina) (metionina) (kazeina) 12 mm 12 mm Poziom zapachu (Smell level) Enterobacter 3 2 0 1 cloacae Enterobacter 2 2 0 1 amnigenes Pseudomonas 2 3 0 1 putida Comomonas 2 2 0 1 testosteroni Cedecea sp. 3 2 3 0 1 1 very low, at the detection limit (bardzo niski, w grani cach detekcji), 2 moderate (średni), 3 strong (silny).

238 R. GANCARZ, I. MALISZEWSKA, A. FRĄCKOWIAK, B. BRUZIEWICZ MIKŁASZEWSKA Table 4. Identification of VSC produced by isolated bacteria method A Tabela 4. Identyfikacja lotnych związków siarki wytwarzanych przez wyizolowane bakterie metoda A Carbon source Enterobacter amnigenes Cedecea sp. (Źródło węgla) precipitate precipitate sulphur precipitate precipitate sulphur colour weight amount colour weight amount (kolor osadu) (masa osadu) (zawartość (kolor osadu) (masa osadu) (zawartość mg osadu) mg osadu) g/m 3* g/m 3* Cysteine black (czarny) 35.4 0.88 black (czarny) 32.4 (Cysteina) Methionine white (biały) 33.4 + white (biały) 67.8 (Metionina) Caseine traces no (brak) + gray (szary) 66.3 (Kazeina) (ilości śladowe) * Sulphur amount in the air above medium g/m 3 (ilość siarki w powietrzu nad pożywką) g/m 3. Table 5. Identification of VSC produced by isolated bacteria method B Tabela 5. Identyfikacja lotnych związków siarki wytwarzanych przez wyizolowane bakterie metoda B Carbon source Enterobacter amnigenes Cedecea sp. (Źródło węgla) absorbance VSC concentration absorbance VSC concentration (absorbancja) (stężenie lotnych (absorbancja) (stężenie lotnych związków siarki) związków siarki) mol/dm 3 mol/dm 3 Cysteine 0.975 1.029 10 4 1.044 1.10 10 4 (Cysteina) Methionine 0.047 4.960 10 6 0.894 9.43 10 5 (Metionina) Caseine 0.020 2.11 10 6 0.018 1.89 10 6 (Kazeina) in comparison with the caseine. These two strains were then selected for metabolites analysis. Smell Level Parallely with the analysis of the growth of the bacteria, the level for smell above the medium was analysed by organoleptic method. Data are pre sented in Table 3. As shown in Table 3, the highest level of smell was observed in all cases when the caseine was used as a source of carbon. When methionine and cysteine was the only carbon source for growing bacteria the strong smell was detected in case of Cedecea sp. whereas the lowest one in the case of Enterobacter amnigenes. These facts were addi tional reason that those two bacteria strains were taken for identification of the metabolites. Sulphur Metabolites Production The total amount of hydrogen sulphide in air above mineral medium containing cysteine/ methionine or caseine in which the selected bacte ria were grown is given in Tables 4 and 5. Other than Sulphur Volatile Metabolites Analysis of the samples, as described in the Material and Methods section, by the GC MS process did not indicate production of volatile amines or carboxylic acids are produced by the tested bacte ria when grown on cysteine, methionine or caseine. Discussion The studied oral Gram negative bacteria can metabolize methionine and cysteine leading to only sulphur volatile products. None of carboxylic acids or amines were detected in the air above the culture medium. The main bacteria which were found to be odorogenic were anaerobic strains: Peptostrepto coccus, Prevotella, Veillonella, Fusobacterium. It was postulated [10] that oral enerobacterial iso

Gram Negative Microorganisms and Oral Malodor 239 lates (Klebsiella, Enterobacter) may also play a role in the oral cavity. It is well established that Gram negative, growing anaerobically, microorganisms are res ponsible for the most cases of bad breath [14, 15]. It was shown that the tongue is the main source of oral bacteria. The oral surfaces are colonized by over 500 bacterial species [16], which can degrade proteins, peptides and amino acids (cysteine, methionine) to volatile sulphur compounds (VSC). Persson et al. [17] found that the most active producers of SC in vitro were: Peptostreptococcus, Eubacterium, Bacteroides, Selenomonas, Fuso bacterium, Prevotella, Porphyromonas, Trepo nema. These species do not utilize the same nutrient. For example Peptostreptococcus might produce H 2 S only from cysteine, but not from the me thionine or peptides. A few years ago, Goldberg et al. [10] postulat ed that not only anaerobic rods might participate in the production of oral malodor. They found that facultatively anaerobic bacteria Klebsiella and related Enterobactericeae may play a role in bad breath. Several observation in the present study tend to support a role for enterobacterial species in oral malodor production. In the experiments the stud ied bacteria can metabolize all substrates exam ined in aerobic conditions, leading to sulphur volatile products. None of carboxylic acids or amines were detected in the air above the culture medium. References [1] PRINZ H.: Offensive breath. Its causes and prevention. Dent. Cosmos 1930, 72, 700 707. [2] TONZETICH J.: Production and origin of oral malodor. A review of mechanism and methods of analysis. J. Periodontol. 1977, 48, 13 20. [3] LOESCHE W. J., KAZOR C.: Microbiology and treatment of halitosis. Periodontology 2000, 28, 256 279. [4] STEENBERGHE D., ROSENBERG M. (eds.): Bad breath a multidisciplinary approach. Leuven University Press, Belgium 1966. [5] IWAKURA M., YASUNO K., SHIMURA M., SAKAMOTO S.: Clinical characteristic of halitosis. J. Dent. Res. 1994, 73, 9, 1568 1574. [6] CLARK G.T., NACHNANI S., MESSADI D.V.: Detecting and treating oral and nonoral malodors. J. Calif. Dent. Assoc. 1997, 25, 133 144. [7] MAGER D. L., XIMENEZ FYVIE L. A., HAFFAJEE A. D., SOCRANSKY S. S.: Distribution of selected bacterial species on intraoral surfaces. J. Clin. Periodontol. 2003, 30, 644 654. [8] GOODSON J. M.: Antimicrobial strategies for treatment of periodontal disease. Periodontology 2000, 5, 142 168. [9] GOLDBERG S., KOZLOVSKY A., GORDON D., GELERNTER I., SINTOV A., ROSENBERG M.: Cadaverine as a putative component of oral malodor. J. Dent. Res. 1994, 73, 1168 1172. [10] GOLDBERG S., CARDASH H., BROWNING H. III, SAHLY H., ROSENBERG M.: Isolation of Enterobacteriaceae from the mouth and potential association with malodor. J. Dent. Res. 1997, 76, 11, 1770 1775. [11] Bergey s Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore 1989. [12] ZAREMBA M. L., BOROWSKI J.: Mikrobiologia lekarska. PZWL, Warszawa 1997. [13] DAVIS B. D., MIGNOLI E. S.: Isolation of E. coli K12 B 12 deficient mutants. J. Bacteriol. 1950, 17, 60 69. [14] YAEKI K., SANADA K.: Biochemical and clinical factors influencing oral malodor in periodontal patients. J. Periodontol. 1992, 63, 9, 783 789. [15] DE BOEVER E. H., LOESCHE W. J.: Assessing the contribution of anaerobic microflora of the tongue to oral mal odor. J. Am. Dent. Assoc. 1995, 126, 1384 1393. [16] MIYAZAKI H., SAKAO S., KATOH Y., TAKEHARA T.: Correlation between volatile sulphur compouds and certain oral health measurements in the general population. J. Periodontol. 1995, 66, 679 684 [17] PERSSON S., EDLUND M. B., CLAESSON R., CARLSSON J.: The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol. Immunol. 1990, 5, 195 201. Address for correspondence: Roman Gancarz Institute of Organic Chemistry, Biochemistry and Biotechnology Department of Chemistry, Wrocław University of Technology Wybrzeże Wyspiańskiego 27 50 370 Wrocław Poland tel.: (+48 71) 320 38 90 e mail: roman.gancarz@pwr.wroc.pl Received: 19.01.2004 Revised: 29.01.2004 Accepted: 11.03.2004 Praca wpłynęła do Redakcji: 19.01.2004 r. Po recenzji: 29.01.2004 r. Zaakceptowano do druku: 11.03.2004