Possibility of a case of pneumonia caused by green tea contaminated with Pseudomonas aeruginosa and countermeasures against it

Pseudomonas aeruginosa is widely distributed in moist environments and causes serious infection in compromised hosts. We report a patient (82 years, male, immunological background; cancer) who sprayed green tea contained in a spray bottle (nozzle-type container) that was contaminated with P. aeruginosa in their oral cavity, causing pneumonia. We discuss the risk of contamination of a spray bottle.

When infection prevention team members entered the patient's room for environmental surveillance, a spray bottle was placed in the room. We asked the patient ‘What do you use this spray bottle for?’ and the patient who was recovering from surgery for esophageal cancer answered ‘Since I can't help being thirsty, I spray green tea in the oral cavity using this spray bottle. I spray it about 20 times a day. Green tea contained in this spray bottle is delivered at meal time and I exchange it in the spray bottle 3 times a day’. As contamination by microorganisms was of concern due to the difficulties of consistent and thorough washing and drying of the spray bottles, contamination of green tea in the spray bottle was investigated.

Approximately 100 mL of ethanol for disinfection (76.9–81.4 vol% ethanol, Kenei Pharm. Co., Osaka) was added into the spray bottle. The inner wall was in contact with ethanol for 10 min and ethanol was sprayed 10 times to disinfect the inside of the spray bottle, including the nozzle region.

The P. aeruginosa isolated from the green tea in the spray bottle (henceforth referred to as P. aeruginosa GT), P. aeruginosa IFO3919, and other common pathogens: Escherichia coli NIHJ-JC2, Serratia marcescens IFO3936, Staphylococcus aureus 209P, Candida albicans IFO1594, and the clinical isolate methicillin-resistant Staphylococcus aureus (MRSA) were used.

Green tea prepared by the nutritionist in the hospital kitchen, green tea prepared by an automated machine in the hospital rest room, and seven commercially available brands of green tea in plastic bottles were tested: Oi Ocha Original (green tea A), Oi Ocha Dark (green tea B, Ito En Co., Tokyo, Japan), Ayataka (green tea C, Japan Coca-Cola Co., Tokyo), Iyemon (green tea D), Iyemon Green Espresso (green tea E, Suntory Co., Osaka, Japan), Sencha (green tea F, Yamazaki Baking Co., Tokyo), and Namacha (green tea G, Kirin Co., Tokyo). Three commercially available brands of black tea containing no sugar were tested: GoGo no Koucha unsweetened (black tea A), GoGo no Koucha straight (black tea B, Kirin Co., Tokyo), and Teo (black tea C, Asahi Soft Drinks Co., Tokyo). Three coffee beverages containing no sugar were also tested: BOSS (coffee A, Suntory Co.), UCC unsweetened (coffee B, UCC Ueshima Coffee Co., Kobe, Japan), and FIRE (coffee C, Kirin Co.). In addition, one mineral water product, I LOHAS (Japan Coca-Cola Co.), was included in the study. All of the beverages tested for microbial viability were purchased within the hospital. For the control, saline and distilled water for injection (Hikari Pharm. Co., Tokyo) were used.

The samples of green tea contaminated by P. aeruginosa GT in the spray bottle were diluted in sterile distilled water, and diluted samples were added to the beverages, sterile distilled water, or sterile saline, yielding concentrations of approximately 10³ colony forming units (cfu) of P. aeruginosa per mL. All other microbes used in the experiment were cultured on trypticase soy agar (TSA; Eiken Chemical, Tokyo) for 1–4 days at 30 °C, scraped into sterile phosphate-buffered saline (PBS), and centrifuged three times at 3,000 rpm for 10 min to remove the growth medium. Resuspension was carried out in PBS, yielding concentration of approximately 10³ cfu. Next, 0.05 mL of the resuspension was added to 4.95 mL of each beverage tested. The test solutions were incubated at 30 °C, and plate counts were performed at 6 h, and 1, 2, and 7 days. Each experiment was repeated three times and the mean of the three repeats was calculated.

P. aeruginosa isolated from green tea in the spray bottle, P. aeruginosa isolated from sputum of the patient, and P. aeruginosa IFO3919 were subjected to pulsed-field gel electrophoresis. The high-molecular-weight chromosomal DNA was prepared according to the method of Murray et al. (1991), and the DNA sample in a small slice of an agarose plug in 200 μL of reaction buffer was digested with 30 U of SpeI (New England Bio Labs, USA). Pulsed-field gel electrophoresis was carried out with the Bio-Rad Gene Path system (Bio-Rad, USA) in 1% agarose gel in 0.5 × TBE buffer at 14 °C with a linear ramp time of 1–23 s over a period of 18.5 h. Thereafter, the gels were stained with ethidium bromide and photographed.

Disinfection of the inside of the spray bottle was not possible even though ethanol for disinfection was used, i.e., P. aeruginosa was detected even after disinfection with ethanol for disinfection.

P. aeruginosa can proliferate using a small amount of nutrients in humid conditions, and therefore proliferates robustly in kitchen sponges, bath chair padding, shaving brushes, antiseptic-soaking cotton, enteral feeding solution, and fruits and vegetables (Oie & Kamiya 1992, 1996, 2001a, 2001b; Oie et al. 2005, 2008). It is also known that intravenous infusion and medical equipment and devices that are contaminated by P. aeruginosa become sources of nosocomial infection (Archibald et al. 1998; Prospero et al. 2006; Kayabas et al. 2008). Therefore, it is imperative that contamination of P. aeruginosa in medicine, food, and equipment be prevented, especially in the environment of compromised hosts.

We reported a patient who kept green tea in a spray bottle, sprayed it in the oral cavity, developed pneumonia caused by P. aeruginosa, and clinical course is not favorable as usual treatment (third-generation cephems) for pneumonia caused by P. aeruginosa. However, the patient recovered from pneumonia by discontinuing spraying the green tea in the oral cavity and by continuing administering the antibiotics. As P. aeruginosa isolated from the sputum of the patient was identical to that isolated from the green tea on pulsed-field gel electrophoresis fingerprinting, contamination of the green tea with P. aeruginosa was considered the possible cause of infection. Infection does not usually occur just by drinking green tea contaminated with P. aeruginosa, but the patient frequently sprayed this green tea in the oral cavity, which may have caused infection.

As this patient wanted to continue spraying green tea in the oral cavity to hydrate, this request was investigated. First, the spray bottle contaminated with P. aeruginosa was disinfected with alcohol but was ineffective to suppress microbial growth. We hypothesized that disinfection was not possible due to a possibility of P. aeruginosa forming biofilm in the nozzle of the spray bottle. As it is not structurally feasible to wash or dry the nozzle region of a spray bottle easily, it is a region that is readily contaminated by microorganisms. Next, the growth of the bacteria was investigated by adding the contents of the spray bottle containing P. aeruginosa into four green tea products, one sugar-free black tea product, and one sugar-free coffee product. No growth of P. aeruginosa was noted in sugar-free black tea or sugar-free coffee, but growth was detected in the four green tea products (Figure 3). The green tea contains catechins, coffee contains caffeine, and the black tea contains theaflavin, all of which have antibacterial activity (Toda et al. 1989; Daglia et al. 1994, 2007; Hamiton-miller & Shah 2000; Hu et al. 2002; Cantatore et al. 2013; Kaur et al. 2019). However, green tea contains abundant nutrients, such as electrolytes, and these nutrients may counteract the antibacterial effects of catechin and enable P. aeruginosa to grow in the green tea. On the other hand, black tea and coffee contain less nutrients, such as electrolytes compared to green tea, and the antibacterial effects of theaflavin and caffeine may have fuller effect.

As the catechin content varies among commercial green tea products, the viability of P. aeruginosa IFO3919 in seven green tea products was investigated (Figure 4). P. aeruginosa grew in all products. In contrast, P. aeruginosa was unable to grow in any of three sugar-free black tea products or the three sugar-free coffee products (data not shown). In addition, the viability of six species of microorganism in a green tea product were investigated. S. aureus and MRSA did not grow, but E. coli, S. marcescens, and C. albicans did (Figure 5), confirming that not only P. aeruginosa, but also other bacteria and fungi can grow in green tea. A spray bottle is easy to be contaminated and may be a possible source of pneumonia in an immunodeficient host. Considering that some patient care settings and personal preferences may opt for the use of green tea for hydration or any medical uses that presumes antibacterial effect of catechins contained in green tea, it is necessary to be aware that green tea can also be a medium that is readily contaminated by opportunistic microorganisms.

Green tea in a spray bottle should not be used because pathogenic microorganisms such as P. aeruginosa rapidly grow green tea, and a spray bottle is not structurally possible to wash or dry the nozzle region.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.