INT J TUBERC LUNG DIS 18(8):988–991 Q 2014 The Union http://dx.doi.org/10.5588/ijtld.14.0026
Bacterial contamination of different components of the waterpipe H. Safizadeh,* M. Moradi,† M. Rostami Rad,‡ N. Nakhaee§ *Research Center for Social Determinants of Health, Institute for Futures Studies in Health, †Research Center for Tropical and Infectious Diseases, ‡Medical Student Research Committee, and §Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran SUMMARY S E T T I N G : Waterpipe smoking is an emerging topic in tobacco research that may have unrecognised health hazards. O B J E C T I V E : To determine whether waterpipes are a source of bacterial contamination. M E T H O D S : A total of 15 restaurants and waterpipe caf´es were randomly selected from the list of locations serving waterpipe tobacco in Kerman city, Iran. Different parts of the waterpipe devices were sampled, including the disposable mouthpiece, the mouthpiece of the hose and the water in the bowl of the waterpipe. The samples were smeared onto bacterial culture media, including eosin methylene blue, blood agar and MacConkey agar growth media, and incubated at 378C. After 24–48 h, they were examined for colony growth.
Of 285 samples from different parts of the waterpipes, 236 (82.8%) showed positive cultures; the rate of contamination ranged from 69% in the fixed mouthpiece to 96% in bowl water. Coagulase-negative staphylococci (32.9%), Streptococcus spp (26.9%), Neisseria spp (13.7%) and Escherichia coli (9.4%) were the most frequent contaminants. C O N C L U S I O N : Waterpipes are frequently contaminated with microorganisms. This study revealed potential microbial hazards in waterpipes that may contribute to respiratory tract colonisation. K E Y W O R D S : bacterial contamination; hookah; lung infection
THE ADVERSE HEALTH EFFECTS of waterpipe smoking have been more evident in the past few decades than ever before.1 The history of waterpipe smoking goes back more than 400 years.2 Although until recently waterpipe smoking was limited to elderly and retired men in traditional coffee shops, mainly in the Mediterranean region, we are now facing an increasing rate of consumption, especially among adolescents and youths in different regions around the world, including Western countries such as the United Kingdom, France, Russia, Canada, Australia and America.2,3 Waterpipes come in various shapes, but generally have the following components: a head, where tobacco and coal are placed, isolated by a perforated aluminum foil; a body that fixes the head to a glass bottle (bowl); a bowl that contains water through which the smoke passes; and a hose, a flexible connecting tube with a fixed mouthpiece.2 A disposable plastic mouthpiece may be attached to the fixed mouthpiece at the end of the hose (Figure 1). The average length of each session of waterpipe
smoking is 45–60 min,4,5 but can last up to 6 h.6 The results of recent studies have shown significant harmful effects from waterpipe smoking, as well as potential for addiction.1,3–5 Due to waterpipe smoking in social situations and misconceptions about their safety,6,7 waterpipe sharing has become such a common custom that in some countries use of a shared mouthpiece has been reported among 90– 100% of consumers.4 In some cultures, refusal to share a waterpipe is even regarded as a disrespectful act.4 Present evidence indicates that waterpipe smoking increases the risk of lung cancer, respiratory illness, low birth weight and periodontal disease,5 and there is particular emphasis on the role of waterpipe smoking in pulmonary disease.8–10 In a systematic review, waterpipe smokers showed poorer spirometry indices and lung function than non-smokers, while they showed no significant difference with cigarette smokers in this regard.8 It has been observed in a case-control study that waterpipe dependence significantly (odds ratio [OR] 3.7) increases the risk of
R E S U LT S :
Correspondence to: Nouzar Nakhaee, Professor of Community Medicine, Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Somayeh Cross, PO Box 76175-113, Kerman, Iran. Tel: (þ98) 341 226 4196. Fax: (þ98) 341 226 4198. e-mail: [email protected]; [email protected] Article submitted 10 January 2014. Final version accepted 16 April 2014.
Bacterial contamination of the waterpipe
Coal Tobacco
Fixed mouthpiece
Body
Hose
Disposable mouthpiece
Bowl
Figure 1 Different parts of a waterpipe device. This image can be viewed online in colour at http://www.ingentaconnect.com/ content/iuatld/ijtld/2014/00000018/00000008/art000020
chronic bronchitis.10 In addition to non-infectious diseases attributed to waterpipe smoking, there is a potential risk of transfer of infectious diseases, because a waterpipe is commonly used in small groups of people using the same mouthpiece and hose.4 Some review articles have mentioned an increased risk of infection transfer due both to using common waterpipes and to the limited space in restaurants and coffee shops where they are used.2,4 Three studies have reported a risk of Helicobacter pylori infection,11 pulmonary aspergillosis in immunocompromised patients12 and tuberculosis transmission13 due to waterpipe smoking. Despite the probability of increased risk of infection transmission due to using a common waterpipe, to our knowledge no studies have been published on bacterial contamination of waterpipes. The present study therefore aimed to investigate bacterial contamination of waterpipes used in restaurants and coffee shops.
989
selected places and explaining the aim of the study, informed consent was obtained from the owners and the waterpipes were sampled. The owners were assured of the confidentiality of the results. The research protocol was approved by the Ethical Committee of the Kerman Neuroscience Research Center, Kerman University of Medical Sciences. Sampling was performed from the disposable mouthpiece, the mouthpiece of the hose and the water in the bowl of the waterpipe (Figure 1). In the 15 restaurants and coffee shops studied, the water in the bowl was reported to be changed from ‘at least once a day’ to ‘after every session’ (i.e., 3–4 times/ day). The fixed mouthpiece and the hose were rinsed once daily to once monthly. Disposable mouthpieces were replaced after each session. Each waterpipe device was used one to four times a day. Samples were collected from the disposable and fixed mouthpieces of the hose using a wet sterile swab and cultured in bacteriological growth media, including eosin methylene blue (EMB), blood agar and MacConkey agar. For collecting samples of the bowl content, 5 ml was taken with a sterile syringe and transferred to the laboratory. Aseptic practices were followed in all sampling processes. In the laboratory, the collected samples were grown on EMB, blood agar and MacConkey agar growth media and incubated at 378C. After 24–48 h, the colonies were studied and identified. A colony count was performed and the samples were investigated further for the presence and number of pathogenic bacteria, including Staphylococcus aureus, Pseudomonas and Enterobacteriaceae. Samples taken from the inside of the hose were centrifuged and 0.1 ml of the concentrated sample was cultured in each of blood agar, EMB and MacConkey media. After 24–48 h incubation, colonies were counted and identified. To identify the isolated bacteria, first one smear was prepared from each sample and Gram staining was performed. For the identification of staphylococci, catalase and coagulase tests, and for susceptible colonies, growth in Manitol salt agar, were performed; all three tests are positive for S. aureus. To identify intestinal gram-negative bacillus, for each gramnegative bacillus with negative oxidase test, biochemical tests such as growth in triple sugar iron, Simmon citrate, mobility, indole, urea, lysine decarboxylase and methyl red and Voges-Proskauer (MR&VP) were used. We categorised sites as clean (,10 colony-forming units [cfu] per ml), mildly contaminated (10–100 cfu) and heavily contaminated (.100 cfu).
MATERIALS AND METHODS This cross-sectional study was performed in Kerman, the centre of the largest province in Iran. A total of 15 restaurants and coffee shops in different parts of the city were randomly selected. After contacting the
RESULTS A total of 285 samples from different waterpipe parts were microbiologically investigated. The fixed mouthpiece had the highest number of samples
990
The International Journal of Tuberculosis and Lung Disease
Table 2 Number of colony-forming units per cultured specimen Colony-forming units n ¼ 285 Specimen Fixed mouthpiece Disposable mouthpiece Water in bowl Overall
Figure 2 Microbial colonisation of different segments of a waterpipe.
(131, 46.0%), 96 (33.7%) were from disposable mouthpieces and 58 (20.3%) were from bowl water (Figure 2). In total, 236 samples (82.8%) were positive on culture; the rate of contamination ranged from 69% in fixed mouthpieces to 96% in bowl water (Figure 2). A total of 438 strains were isolated from 236 samples with positive cultures (Table 1). The highest numbers of isolates were related to coagulase-negative staphylococci (32.9%), followed by Streptococcus (26.9%) and Neisseria spp (13.7%). In fixed and disposable mouthpieces, the highest numbers of isolates were coagulase-negative Staphylococcus and Streptococcus, but in bowl water Escherichia coli and Klebsiella had the highest prevalence rates (Table 1). More than two thirds (64.8%) of culture-positive samples showed multiple contamination. The highest rate was related to the disposable mouthpieces (76.4%), followed by bowl water (60.0%) and fixed mouthpieces (56.0%). To determine the degree of contamination, the total number of microorganisms in each milliliter of sample was determined, and accordingly the highest frequency of contamination was related to samples with a count of .102 cfu/ml (Table 2).
DISCUSSION The present study showed bacterial contamination of Table 1
,10 n (%) 51 17 7 75
(17.9) (6.0) (2.5) (26.3)
10–100 n (%) 2 (0.7) 0 0 2 (0.7)
.100 n (%) 78 79 51 208
(27.4) (27.7) (17.9) (73.0)
the mouthpiece (either fixed or disposable) and/or bowl in 83% of cases. Many of the isolates, such as P. aeruginosa, Klebsiella spp and Streptococcus spp, are included amongst the potential causes of bacterial pneumonia. Coagulase-negative staphylococci (32.9%), Streptococcus spp (26.9%) and Neisseria spp (13.7%) were the most prevalent bacteria. The most prevalent contaminant in the bowl water was E. coli (42.3%). The contamination pattern of the mouthpieces, regardless of fixed or disposable type, was similar in that while no case of E. coli contamination was found in the culture medium of samples from these two sources, coagulase-negative staphylococci and Streptococcus spp were found in both samples (Table 1). Bacteria such as P. aeruginosa and Klebsiella spp, which are relatively uncommon human pathogens,14 were found in almost none of the samples from the fixed and disposable mouthpieces (Table 1). Although coagulase-negative staphylococci commonly inhabit the skin of humans and mucous membranes, they are considered important pathogens, as this group of organisms is known as one of the main causative agents of nosocomial infections.15 In out-patient settings, coagulase-negative staphylococci have been identified as one of the major causes of prosthetic cardiac valve infection and of urinary tract infections in women.15 Some Streptococcus species are capable of causing streptococcal pharyngitis, bacterial pneumonia, meningitis and skin infections in human.16 Neisseria species usually colonise the mucosal surface of the nasopharynx; N.
Distribution of bacterial isolates from waterpipe
Organism Coagulase-negative staphylococci Streptococcus spp Staphylococcus aureus Klebsiella spp Neisseria spp Pseudomonas aeruginosa Bacillus spp. Escherichia coli
Fixed mouthpiece n ¼ 160 n (%) 70 50 4 0 25 0 11 0
(43.8) (31.2) (2.5) (0) (15.6) (0) (6.9) (0)
Disposable mouthpiece n ¼ 181 n (%) 68 67 3 2 34 0 7 0
(37.6) (37.0) (1.6) (1.1) (18.8) (0) (3.9) (0)
Water in bowl n ¼ 97 n (%) 6 1 0 32 1 14 2 41
(6.2) (1.0) (0) (33.0) (1.0) (14.4) (2.1) (42.3)
Overall n ¼ 438 n (%) 144 118 7 34 60 14 20 41
(32.9) (26.9) (1.6) (7.7) (13.7) (3.2) (4.6) (9.4)
Bacterial contamination of the waterpipe
meningitides and N. gonorrhoeae are the two species that may cause symptomatic infections.17 More pathogenic bacteria, such as P. aeroginosa, Klebsiella spp and E. coli, were predominantly seen in bowl water (Table 1). Klebsiella is considered one of the causes of community-acquired bacterial pneumonia and can cause severe pulmonary infections in immunocompromised patients, such as diabetics and those with chronic obstructive pulmonary disease.18 Klebsiella grows in humid environments, such as surface water, sewage and human mucosal surfaces. The detection rates in nasopharynx and stool samples range from 1% to 6% and from 5% to 38%, respectively.18 In the present study, bowl water contamination with Klebsiella was 33.3%, which is in the upper range limit of the stool detection rate.18 The high detection rate of E. coli also confirms the bacterial contamination of bowl water with humanorigin bacteria. No case of P. aeruginosa was found in the mouthpiece, but its detection rate in bowl water was 14.4%. P. aeruginosa is an opportunistic pathogen that could be an important cause of infection in immunocompromised individuals and in those with chronic respiratory problems.19 Overall, 73% of studied samples were heavily contaminated (Table 2). The highest rate of contamination (96%) was observed in bowl water (Figure 1), which might be due to the frequent use of bowl water and its humid environment. Although we did not study the effect of changing the bowl water and rinsing the hose on the risk and pattern of contamination to reduce the risk of contamination of waterpipe devices, sharing habits should be avoided. As a general rule, in roughly similar contexts, rinsing the hose and bowl after each session and allowing them to dry should be considered.20
CONCLUSIONS The present study showed that the mouthpieces and bowl water of waterpipes are contaminated with several bacteria that can act as a source of respiratory system colonisation. Although most of these bacteria are not pathogenic in normal conditions, due to the high prevalence of waterpipe smoking, immunedeficient people and those with predisposing factors for lung disease should be warned about the potential risk of infection transfer via the common use of waterpipes. More studies on quantitative risk assessment of pathogenicity of detected bacteria are warranted. Acknowledgement We thank Ms. Azizian for providing us with the photograph. Conflict of interest: none declared.
991
References 1 Jawad M, McEwen A, McNeill A, Shahab L. To what extent should waterpipe tobacco smoking become a public health priority? Addiction 2013; 108: 1873–1884. 2 Knishkowy B, Amitai Y. Water-pipe (narghile) smoking: an emerging health risk behavior. Pediatrics 2005; 116: e113– e119. 3 Maziak W. The global epidemic of waterpipe smoking. Addict Behav 2011; 36: 1–5. 4 Maziak W, Ward K D, Afifi Soweid R A, Eissenberg T. Tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic. Tob Control 2004; 13: 327–333. 5 Akl E A, Gaddam S, Gunukula S K, Honeine R, Abou Jaoude P, Irani J. The effects of waterpipe tobacco smoking on health outcomes: a systematic review. Int J Epidemiol 2010; 39: 834– 857. 6 Jawad M, Jawad S, Mehdi A, Sardar A, Jawad A M, Hamilton F L. A qualitative analysis among regular waterpipe tobacco smokers in London universities. Int J Tuberc Lung Dis 2013; 17: 1364–1369. 7 Sabahy A R, Divsalar K, Bahreinifar S, Marzban M, Nakhaee N. Waterpipe tobacco use among Iranian university students: correlates and perceived reasons for use. Int J Tub Lung Dis 2011; 15: 844–847. 8 Raad D, Gaddam S, Schunemann H J, et al. Effects of waterpipe tobacco smoking on lung function: a systematic review and meta-analysis. Chest 2011; 139: 764–774. 9 Hawari F I, Obeidat N A, Ayub H, et al. The acute effects of waterpipe smoking on lung function and exercise capacity in a pilot study of healthy participants. Inhal Toxicol 2013; 25: 492–497. 10 Salameh P, Waked M, Khoury F, et al. Waterpipe smoking and dependence are associated with chronic bronchitis: a casecontrol study in Lebanon. East Mediterr Health J 2012; 18: 996–1004. 11 El-Barrawy M A, Morad M I, Gaber M. Role of Helicobacter pylori in the genesis of gastric ulcerations among smokers and non-smokers. East Mediterr Health J 1997; 3: 31–321. (check AU – the page numbers don’t look right, but I can’t find this in PubMed) 12 Szyper-Kravitz M, Lang R, Manor Y, Lahav M. Early invasive pulmonary aspergillosis in a leukemia patient linked to Aspergillus contaminated marijuana smoking. Leuk Lymphoma 2001; 42: 1433–1437. 13 Munckhof W J, Konstantinos A, Wamsley M, Mortlock M, Gilpin C. A cluster of tuberculosis associated with use of a marijuana water pipe. Int J Tuberc Lung Dis 2003; 7: 860–865. 14 Cohen H A, Kahan E, Cohen Z, et al. Microbial colonization of nebulizers used by asthmatic children. Pediatr Int 2006; 48: 454–458. 15 Huebner J, Goldmann D A. Coagulase-negative staphylococci: role as pathogens. Annu Rev Med 1999; 50: 223–236. 16 Facklam R. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev 2002; 15: 613–630. 17 Saez Nieto J A, Marcos C, Vindel A. Multicolonization of human nasopharynx due to Neisseria spp. Int Microbiol 1998; 1: 59–63. 18 Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11: 589–603. 19 Tummler B, Wiehlmann L, Klockgether J, Cramer N. Advances ¨ in understanding Pseudomonas. F1000Prime Rep 2014; 6: 9. 20 Brzezinski L X, Riedi C A, Kussek P, Souza H H, Rosa´rio N. Nebulizers in cystic fibrosis: a source of bacterial contamination in cystic fibrosis patients? J Bras Pneumol 2011; 37: 341– 347.
Bacterial contamination of the waterpipe
i
RESUME C O N T E X T E : La fum´ee de pipe a` eau est un sujet e´ mergent en mati`ere de recherche li´ee au tabac, car elle pourrait avoir des risques sanitaires non encore reconnus. O B J E C T I F : D´eterminer si les pipes a` eau sont une source de contamination bact´erienne. M E´ T H O D E S : Un ensemble de 15 restaurants et caf´es offrant des pipes a` eau ont e´ t´e s´electionn´es de fa¸con al´eatoire sur la liste des endroits proposant de fumer la pipe orientale dans la ville de Kerman, Iran. L’´echantillonnage a e´ t´e r´ealis´e sur diff´erents e´ l´ements des pipes, notamment l’embout buccal a` usage unique, l’embout du tuyau d’aspiration et l’eau dans le bol de la pipe. Les e´ chantillons ont e´ t´e inocul´es dans des milieux de culture bact´eriens, y inclus le bleu de m´ethyl`ene a` l’´eosine, l’agar au sang et le milieu de croissance sur agar
de MacConkey, et ont e´ t´e incub´es a` 378C. Apr`es 24 a` 48 h, on a examin´e la croissance des colonies. R E´ S U L T A T S : Sur 285 e´ chantillons des diff e´ rents e´ l´ements des pipes a` eau, 236 (82,8%) ont mis en e´ vidence une culture positive et le taux de contamination variait de 69% dans les embouts buccaux fixes a` 96% dans le bol d’eau. Les contaminants les plus fr´equents e´ taient respectivement des staphylocoques coagulasen´egatifs (32,9%), des streptocoques (26,9%), Neisseria (13,7%) et Escherichia coli (9,4%). C O N C L U S I O N : Les pipes a` eau sont fr´equemment contamin´ees par des microorganismes. Cette e´ tude a r´ev´el´e les risques bact´eriens potentiels des pipes a` eau qui pourraient contribuer a` la colonisation de l’arbre respiratoire.
RESUMEN M A R C O D E R E F E R E N C I A: El consumo de tabaco en narguile representa un nuevo aspecto de la investigacion ´ sobre el tabaquismo cuyos riesgos sanitarios se han desconocido. O B J E T I V O: Determinar si el uso del narguile constituye una fuente de contaminacion ´ bacteriana. M E´ T O D O S: Se escogieron de manera aleatoria 15 restaurantes y caf´es donde se utiliza el narguile, a partir de una lista de locales que ofrecen consumo de este tipo de tabaco en la ciudad de Kerman en Ira´n. Se tomaron muestras de diferentes partes de la pipa de agua, como las boquillas desechables, la manguera de la boquilla y el agua en el recipiente. Las muestras se sembraron en medios de cultivo de bacterias, entre ellos el medio con eosina y azul de metileno, el agar sangre y el agar de MacConkey, se incubaron a 378C y se
examinaron 24 a 48 horas despue´ s en busca de formacion ´ de colonias. R E S U L T A D O S: De las 285 muestras tomadas de diferentes partes de los narguiles, 236 dieron cultivos positivos (82,8%) y la tasa de contaminacion ´ oscilo´ entre 69% en las boquillas fijas y 96% en el agua del recipiente. Los contaminantes ma´s frecuentes fueron estafilococos coagulasa positiva (32,9%), estreptococos (269%), el g´enero Neisseria (13,7%) y Escherichia coli (9,4%). ´ N: La contaminacion CONCLUSIO ´ bacteriana de los narguiles es frecuente. Los resultados del presente estudio revelaron los riesgos microbianos que plantean las pipas de agua y que pueden contribuir a la colonizacion ´ bacteriana de las v´ıas a´ereas.
Bacterial contamination of different components of the waterpipe H. Safizadeh,* M. Moradi,† M. Rostami Rad,‡ N. Nakhaee§ *Research Center for Social Determinants of Health, Institute for Futures Studies in Health, †Research Center for Tropical and Infectious Diseases, ‡Medical Student Research Committee, and §Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran SUMMARY S E T T I N G : Waterpipe smoking is an emerging topic in tobacco research that may have unrecognised health hazards. O B J E C T I V E : To determine whether waterpipes are a source of bacterial contamination. M E T H O D S : A total of 15 restaurants and waterpipe caf´es were randomly selected from the list of locations serving waterpipe tobacco in Kerman city, Iran. Different parts of the waterpipe devices were sampled, including the disposable mouthpiece, the mouthpiece of the hose and the water in the bowl of the waterpipe. The samples were smeared onto bacterial culture media, including eosin methylene blue, blood agar and MacConkey agar growth media, and incubated at 378C. After 24–48 h, they were examined for colony growth.
Of 285 samples from different parts of the waterpipes, 236 (82.8%) showed positive cultures; the rate of contamination ranged from 69% in the fixed mouthpiece to 96% in bowl water. Coagulase-negative staphylococci (32.9%), Streptococcus spp (26.9%), Neisseria spp (13.7%) and Escherichia coli (9.4%) were the most frequent contaminants. C O N C L U S I O N : Waterpipes are frequently contaminated with microorganisms. This study revealed potential microbial hazards in waterpipes that may contribute to respiratory tract colonisation. K E Y W O R D S : bacterial contamination; hookah; lung infection
THE ADVERSE HEALTH EFFECTS of waterpipe smoking have been more evident in the past few decades than ever before.1 The history of waterpipe smoking goes back more than 400 years.2 Although until recently waterpipe smoking was limited to elderly and retired men in traditional coffee shops, mainly in the Mediterranean region, we are now facing an increasing rate of consumption, especially among adolescents and youths in different regions around the world, including Western countries such as the United Kingdom, France, Russia, Canada, Australia and America.2,3 Waterpipes come in various shapes, but generally have the following components: a head, where tobacco and coal are placed, isolated by a perforated aluminum foil; a body that fixes the head to a glass bottle (bowl); a bowl that contains water through which the smoke passes; and a hose, a flexible connecting tube with a fixed mouthpiece.2 A disposable plastic mouthpiece may be attached to the fixed mouthpiece at the end of the hose (Figure 1). The average length of each session of waterpipe
smoking is 45–60 min,4,5 but can last up to 6 h.6 The results of recent studies have shown significant harmful effects from waterpipe smoking, as well as potential for addiction.1,3–5 Due to waterpipe smoking in social situations and misconceptions about their safety,6,7 waterpipe sharing has become such a common custom that in some countries use of a shared mouthpiece has been reported among 90– 100% of consumers.4 In some cultures, refusal to share a waterpipe is even regarded as a disrespectful act.4 Present evidence indicates that waterpipe smoking increases the risk of lung cancer, respiratory illness, low birth weight and periodontal disease,5 and there is particular emphasis on the role of waterpipe smoking in pulmonary disease.8–10 In a systematic review, waterpipe smokers showed poorer spirometry indices and lung function than non-smokers, while they showed no significant difference with cigarette smokers in this regard.8 It has been observed in a case-control study that waterpipe dependence significantly (odds ratio [OR] 3.7) increases the risk of
R E S U LT S :
Correspondence to: Nouzar Nakhaee, Professor of Community Medicine, Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Somayeh Cross, PO Box 76175-113, Kerman, Iran. Tel: (þ98) 341 226 4196. Fax: (þ98) 341 226 4198. e-mail: [email protected]; [email protected] Article submitted 10 January 2014. Final version accepted 16 April 2014.
Bacterial contamination of the waterpipe
Coal Tobacco
Fixed mouthpiece
Body
Hose
Disposable mouthpiece
Bowl
Figure 1 Different parts of a waterpipe device. This image can be viewed online in colour at http://www.ingentaconnect.com/ content/iuatld/ijtld/2014/00000018/00000008/art000020
chronic bronchitis.10 In addition to non-infectious diseases attributed to waterpipe smoking, there is a potential risk of transfer of infectious diseases, because a waterpipe is commonly used in small groups of people using the same mouthpiece and hose.4 Some review articles have mentioned an increased risk of infection transfer due both to using common waterpipes and to the limited space in restaurants and coffee shops where they are used.2,4 Three studies have reported a risk of Helicobacter pylori infection,11 pulmonary aspergillosis in immunocompromised patients12 and tuberculosis transmission13 due to waterpipe smoking. Despite the probability of increased risk of infection transmission due to using a common waterpipe, to our knowledge no studies have been published on bacterial contamination of waterpipes. The present study therefore aimed to investigate bacterial contamination of waterpipes used in restaurants and coffee shops.
989
selected places and explaining the aim of the study, informed consent was obtained from the owners and the waterpipes were sampled. The owners were assured of the confidentiality of the results. The research protocol was approved by the Ethical Committee of the Kerman Neuroscience Research Center, Kerman University of Medical Sciences. Sampling was performed from the disposable mouthpiece, the mouthpiece of the hose and the water in the bowl of the waterpipe (Figure 1). In the 15 restaurants and coffee shops studied, the water in the bowl was reported to be changed from ‘at least once a day’ to ‘after every session’ (i.e., 3–4 times/ day). The fixed mouthpiece and the hose were rinsed once daily to once monthly. Disposable mouthpieces were replaced after each session. Each waterpipe device was used one to four times a day. Samples were collected from the disposable and fixed mouthpieces of the hose using a wet sterile swab and cultured in bacteriological growth media, including eosin methylene blue (EMB), blood agar and MacConkey agar. For collecting samples of the bowl content, 5 ml was taken with a sterile syringe and transferred to the laboratory. Aseptic practices were followed in all sampling processes. In the laboratory, the collected samples were grown on EMB, blood agar and MacConkey agar growth media and incubated at 378C. After 24–48 h, the colonies were studied and identified. A colony count was performed and the samples were investigated further for the presence and number of pathogenic bacteria, including Staphylococcus aureus, Pseudomonas and Enterobacteriaceae. Samples taken from the inside of the hose were centrifuged and 0.1 ml of the concentrated sample was cultured in each of blood agar, EMB and MacConkey media. After 24–48 h incubation, colonies were counted and identified. To identify the isolated bacteria, first one smear was prepared from each sample and Gram staining was performed. For the identification of staphylococci, catalase and coagulase tests, and for susceptible colonies, growth in Manitol salt agar, were performed; all three tests are positive for S. aureus. To identify intestinal gram-negative bacillus, for each gramnegative bacillus with negative oxidase test, biochemical tests such as growth in triple sugar iron, Simmon citrate, mobility, indole, urea, lysine decarboxylase and methyl red and Voges-Proskauer (MR&VP) were used. We categorised sites as clean (,10 colony-forming units [cfu] per ml), mildly contaminated (10–100 cfu) and heavily contaminated (.100 cfu).
MATERIALS AND METHODS This cross-sectional study was performed in Kerman, the centre of the largest province in Iran. A total of 15 restaurants and coffee shops in different parts of the city were randomly selected. After contacting the
RESULTS A total of 285 samples from different waterpipe parts were microbiologically investigated. The fixed mouthpiece had the highest number of samples
990
The International Journal of Tuberculosis and Lung Disease
Table 2 Number of colony-forming units per cultured specimen Colony-forming units n ¼ 285 Specimen Fixed mouthpiece Disposable mouthpiece Water in bowl Overall
Figure 2 Microbial colonisation of different segments of a waterpipe.
(131, 46.0%), 96 (33.7%) were from disposable mouthpieces and 58 (20.3%) were from bowl water (Figure 2). In total, 236 samples (82.8%) were positive on culture; the rate of contamination ranged from 69% in fixed mouthpieces to 96% in bowl water (Figure 2). A total of 438 strains were isolated from 236 samples with positive cultures (Table 1). The highest numbers of isolates were related to coagulase-negative staphylococci (32.9%), followed by Streptococcus (26.9%) and Neisseria spp (13.7%). In fixed and disposable mouthpieces, the highest numbers of isolates were coagulase-negative Staphylococcus and Streptococcus, but in bowl water Escherichia coli and Klebsiella had the highest prevalence rates (Table 1). More than two thirds (64.8%) of culture-positive samples showed multiple contamination. The highest rate was related to the disposable mouthpieces (76.4%), followed by bowl water (60.0%) and fixed mouthpieces (56.0%). To determine the degree of contamination, the total number of microorganisms in each milliliter of sample was determined, and accordingly the highest frequency of contamination was related to samples with a count of .102 cfu/ml (Table 2).
DISCUSSION The present study showed bacterial contamination of Table 1
,10 n (%) 51 17 7 75
(17.9) (6.0) (2.5) (26.3)
10–100 n (%) 2 (0.7) 0 0 2 (0.7)
.100 n (%) 78 79 51 208
(27.4) (27.7) (17.9) (73.0)
the mouthpiece (either fixed or disposable) and/or bowl in 83% of cases. Many of the isolates, such as P. aeruginosa, Klebsiella spp and Streptococcus spp, are included amongst the potential causes of bacterial pneumonia. Coagulase-negative staphylococci (32.9%), Streptococcus spp (26.9%) and Neisseria spp (13.7%) were the most prevalent bacteria. The most prevalent contaminant in the bowl water was E. coli (42.3%). The contamination pattern of the mouthpieces, regardless of fixed or disposable type, was similar in that while no case of E. coli contamination was found in the culture medium of samples from these two sources, coagulase-negative staphylococci and Streptococcus spp were found in both samples (Table 1). Bacteria such as P. aeruginosa and Klebsiella spp, which are relatively uncommon human pathogens,14 were found in almost none of the samples from the fixed and disposable mouthpieces (Table 1). Although coagulase-negative staphylococci commonly inhabit the skin of humans and mucous membranes, they are considered important pathogens, as this group of organisms is known as one of the main causative agents of nosocomial infections.15 In out-patient settings, coagulase-negative staphylococci have been identified as one of the major causes of prosthetic cardiac valve infection and of urinary tract infections in women.15 Some Streptococcus species are capable of causing streptococcal pharyngitis, bacterial pneumonia, meningitis and skin infections in human.16 Neisseria species usually colonise the mucosal surface of the nasopharynx; N.
Distribution of bacterial isolates from waterpipe
Organism Coagulase-negative staphylococci Streptococcus spp Staphylococcus aureus Klebsiella spp Neisseria spp Pseudomonas aeruginosa Bacillus spp. Escherichia coli
Fixed mouthpiece n ¼ 160 n (%) 70 50 4 0 25 0 11 0
(43.8) (31.2) (2.5) (0) (15.6) (0) (6.9) (0)
Disposable mouthpiece n ¼ 181 n (%) 68 67 3 2 34 0 7 0
(37.6) (37.0) (1.6) (1.1) (18.8) (0) (3.9) (0)
Water in bowl n ¼ 97 n (%) 6 1 0 32 1 14 2 41
(6.2) (1.0) (0) (33.0) (1.0) (14.4) (2.1) (42.3)
Overall n ¼ 438 n (%) 144 118 7 34 60 14 20 41
(32.9) (26.9) (1.6) (7.7) (13.7) (3.2) (4.6) (9.4)
Bacterial contamination of the waterpipe
meningitides and N. gonorrhoeae are the two species that may cause symptomatic infections.17 More pathogenic bacteria, such as P. aeroginosa, Klebsiella spp and E. coli, were predominantly seen in bowl water (Table 1). Klebsiella is considered one of the causes of community-acquired bacterial pneumonia and can cause severe pulmonary infections in immunocompromised patients, such as diabetics and those with chronic obstructive pulmonary disease.18 Klebsiella grows in humid environments, such as surface water, sewage and human mucosal surfaces. The detection rates in nasopharynx and stool samples range from 1% to 6% and from 5% to 38%, respectively.18 In the present study, bowl water contamination with Klebsiella was 33.3%, which is in the upper range limit of the stool detection rate.18 The high detection rate of E. coli also confirms the bacterial contamination of bowl water with humanorigin bacteria. No case of P. aeruginosa was found in the mouthpiece, but its detection rate in bowl water was 14.4%. P. aeruginosa is an opportunistic pathogen that could be an important cause of infection in immunocompromised individuals and in those with chronic respiratory problems.19 Overall, 73% of studied samples were heavily contaminated (Table 2). The highest rate of contamination (96%) was observed in bowl water (Figure 1), which might be due to the frequent use of bowl water and its humid environment. Although we did not study the effect of changing the bowl water and rinsing the hose on the risk and pattern of contamination to reduce the risk of contamination of waterpipe devices, sharing habits should be avoided. As a general rule, in roughly similar contexts, rinsing the hose and bowl after each session and allowing them to dry should be considered.20
CONCLUSIONS The present study showed that the mouthpieces and bowl water of waterpipes are contaminated with several bacteria that can act as a source of respiratory system colonisation. Although most of these bacteria are not pathogenic in normal conditions, due to the high prevalence of waterpipe smoking, immunedeficient people and those with predisposing factors for lung disease should be warned about the potential risk of infection transfer via the common use of waterpipes. More studies on quantitative risk assessment of pathogenicity of detected bacteria are warranted. Acknowledgement We thank Ms. Azizian for providing us with the photograph. Conflict of interest: none declared.
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References 1 Jawad M, McEwen A, McNeill A, Shahab L. To what extent should waterpipe tobacco smoking become a public health priority? Addiction 2013; 108: 1873–1884. 2 Knishkowy B, Amitai Y. Water-pipe (narghile) smoking: an emerging health risk behavior. Pediatrics 2005; 116: e113– e119. 3 Maziak W. The global epidemic of waterpipe smoking. Addict Behav 2011; 36: 1–5. 4 Maziak W, Ward K D, Afifi Soweid R A, Eissenberg T. Tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic. Tob Control 2004; 13: 327–333. 5 Akl E A, Gaddam S, Gunukula S K, Honeine R, Abou Jaoude P, Irani J. The effects of waterpipe tobacco smoking on health outcomes: a systematic review. Int J Epidemiol 2010; 39: 834– 857. 6 Jawad M, Jawad S, Mehdi A, Sardar A, Jawad A M, Hamilton F L. A qualitative analysis among regular waterpipe tobacco smokers in London universities. Int J Tuberc Lung Dis 2013; 17: 1364–1369. 7 Sabahy A R, Divsalar K, Bahreinifar S, Marzban M, Nakhaee N. Waterpipe tobacco use among Iranian university students: correlates and perceived reasons for use. Int J Tub Lung Dis 2011; 15: 844–847. 8 Raad D, Gaddam S, Schunemann H J, et al. Effects of waterpipe tobacco smoking on lung function: a systematic review and meta-analysis. Chest 2011; 139: 764–774. 9 Hawari F I, Obeidat N A, Ayub H, et al. The acute effects of waterpipe smoking on lung function and exercise capacity in a pilot study of healthy participants. Inhal Toxicol 2013; 25: 492–497. 10 Salameh P, Waked M, Khoury F, et al. Waterpipe smoking and dependence are associated with chronic bronchitis: a casecontrol study in Lebanon. East Mediterr Health J 2012; 18: 996–1004. 11 El-Barrawy M A, Morad M I, Gaber M. Role of Helicobacter pylori in the genesis of gastric ulcerations among smokers and non-smokers. East Mediterr Health J 1997; 3: 31–321. (check AU – the page numbers don’t look right, but I can’t find this in PubMed) 12 Szyper-Kravitz M, Lang R, Manor Y, Lahav M. Early invasive pulmonary aspergillosis in a leukemia patient linked to Aspergillus contaminated marijuana smoking. Leuk Lymphoma 2001; 42: 1433–1437. 13 Munckhof W J, Konstantinos A, Wamsley M, Mortlock M, Gilpin C. A cluster of tuberculosis associated with use of a marijuana water pipe. Int J Tuberc Lung Dis 2003; 7: 860–865. 14 Cohen H A, Kahan E, Cohen Z, et al. Microbial colonization of nebulizers used by asthmatic children. Pediatr Int 2006; 48: 454–458. 15 Huebner J, Goldmann D A. Coagulase-negative staphylococci: role as pathogens. Annu Rev Med 1999; 50: 223–236. 16 Facklam R. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev 2002; 15: 613–630. 17 Saez Nieto J A, Marcos C, Vindel A. Multicolonization of human nasopharynx due to Neisseria spp. Int Microbiol 1998; 1: 59–63. 18 Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11: 589–603. 19 Tummler B, Wiehlmann L, Klockgether J, Cramer N. Advances ¨ in understanding Pseudomonas. F1000Prime Rep 2014; 6: 9. 20 Brzezinski L X, Riedi C A, Kussek P, Souza H H, Rosa´rio N. Nebulizers in cystic fibrosis: a source of bacterial contamination in cystic fibrosis patients? J Bras Pneumol 2011; 37: 341– 347.
Bacterial contamination of the waterpipe
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RESUME C O N T E X T E : La fum´ee de pipe a` eau est un sujet e´ mergent en mati`ere de recherche li´ee au tabac, car elle pourrait avoir des risques sanitaires non encore reconnus. O B J E C T I F : D´eterminer si les pipes a` eau sont une source de contamination bact´erienne. M E´ T H O D E S : Un ensemble de 15 restaurants et caf´es offrant des pipes a` eau ont e´ t´e s´electionn´es de fa¸con al´eatoire sur la liste des endroits proposant de fumer la pipe orientale dans la ville de Kerman, Iran. L’´echantillonnage a e´ t´e r´ealis´e sur diff´erents e´ l´ements des pipes, notamment l’embout buccal a` usage unique, l’embout du tuyau d’aspiration et l’eau dans le bol de la pipe. Les e´ chantillons ont e´ t´e inocul´es dans des milieux de culture bact´eriens, y inclus le bleu de m´ethyl`ene a` l’´eosine, l’agar au sang et le milieu de croissance sur agar
de MacConkey, et ont e´ t´e incub´es a` 378C. Apr`es 24 a` 48 h, on a examin´e la croissance des colonies. R E´ S U L T A T S : Sur 285 e´ chantillons des diff e´ rents e´ l´ements des pipes a` eau, 236 (82,8%) ont mis en e´ vidence une culture positive et le taux de contamination variait de 69% dans les embouts buccaux fixes a` 96% dans le bol d’eau. Les contaminants les plus fr´equents e´ taient respectivement des staphylocoques coagulasen´egatifs (32,9%), des streptocoques (26,9%), Neisseria (13,7%) et Escherichia coli (9,4%). C O N C L U S I O N : Les pipes a` eau sont fr´equemment contamin´ees par des microorganismes. Cette e´ tude a r´ev´el´e les risques bact´eriens potentiels des pipes a` eau qui pourraient contribuer a` la colonisation de l’arbre respiratoire.
RESUMEN M A R C O D E R E F E R E N C I A: El consumo de tabaco en narguile representa un nuevo aspecto de la investigacion ´ sobre el tabaquismo cuyos riesgos sanitarios se han desconocido. O B J E T I V O: Determinar si el uso del narguile constituye una fuente de contaminacion ´ bacteriana. M E´ T O D O S: Se escogieron de manera aleatoria 15 restaurantes y caf´es donde se utiliza el narguile, a partir de una lista de locales que ofrecen consumo de este tipo de tabaco en la ciudad de Kerman en Ira´n. Se tomaron muestras de diferentes partes de la pipa de agua, como las boquillas desechables, la manguera de la boquilla y el agua en el recipiente. Las muestras se sembraron en medios de cultivo de bacterias, entre ellos el medio con eosina y azul de metileno, el agar sangre y el agar de MacConkey, se incubaron a 378C y se
examinaron 24 a 48 horas despue´ s en busca de formacion ´ de colonias. R E S U L T A D O S: De las 285 muestras tomadas de diferentes partes de los narguiles, 236 dieron cultivos positivos (82,8%) y la tasa de contaminacion ´ oscilo´ entre 69% en las boquillas fijas y 96% en el agua del recipiente. Los contaminantes ma´s frecuentes fueron estafilococos coagulasa positiva (32,9%), estreptococos (269%), el g´enero Neisseria (13,7%) y Escherichia coli (9,4%). ´ N: La contaminacion CONCLUSIO ´ bacteriana de los narguiles es frecuente. Los resultados del presente estudio revelaron los riesgos microbianos que plantean las pipas de agua y que pueden contribuir a la colonizacion ´ bacteriana de las v´ıas a´ereas.
