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- Materials and methods
Aims: To examine bacterial contamination of passenger aircraft and to identify aeroplane environments posing the greatest potential health risk.
Methods and Results: DNA was extracted from ten environmental samples collected on four different flights (three domestic, one international) from a variety of surfaces frequently touched by passengers. PCR clone libraries were made from the DNA samples using bacterial-specific 16S ribosomal DNA (rDNA) primers. A total of 271 bacterial rDNA sequences were obtained. We used BLAST analysis of the rDNA clone sequences to identify sequences in Genbank with the highest sequence similarity. The majority of the rDNA clones obtained from aeroplane environments were more than 97% identical to rDNA sequences from cultured bacterial species. Samples collected from the cabin surfaces (e.g., tray tables and arm rests) had undetectable levels of DNA and produced no PCR products. Bacterial diversity was highest on lavatory surfaces, including door handles, toilet handles, and sink faucets. Sequence data from these surfaces detected species from 58 different bacterial genera, and many of the best BLAST hits matched rDNA sequences of cultured species known to be opportunistic pathogens. The most frequently observed species came from five genera commonly associated with humans: Streptococcus, Staphylococcus, Cornybacterium, Proprionibacterium and Kocuria.
Conclusions: The results show that there is a large diversity of bacterial contamination on aeroplanes, including organisms known to be opportunistic pathogens.
Significance and Impact of the Study: Our results indicate that aeroplanes have the potential to spread an enormous diversity of bacterial species among passengers and destinations. Aeroplane lavatories present an especially significant concern to public health.
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- Materials and methods
Over the past five centuries, Europeans travelling to the New World introduced measles, small pox and tuberculosis with devastating consequences (Crosby 1972). In modern times, commercial travel has spread viral diseases such as dengue fever, SARS, and HIV, as well as bacterial pathogens such as Staphyloccus, Shigella and Salmonella (Wilson 2003). Aeroplane travel, in particular, is a major reason these emerging and reemerging diseases have spread so widely and so rapidly (Wilson 2003). Aeroplanes pack a diversity of passengers, including some who may be symptomatic or asymptomatic carriers of disease as well as others who may be immunocompromised, into a confined space for long periods of time. The close seating arrangement combined with the low humidity in the pressurized cabin appears to aid the spread of droplet nuclei 1–5 μm in diameter, increasing the likelihood of spreading respiratory infections (Wilson 2003). In the late 1990s, a single passenger carrying antibiotic resistant tuberculosis infected 30% of passengers seated within two rows and an additional 3% of passengers on the flight (Kenyon et al. 1996).
Culturing studies of aeroplane environments have revealed high levels of bacterial contamination in aeroplane water supplies (http://www.epa.gov/airlinewater/summarytable.html, 9/22/04). Although culturing methods are useful for detecting microbes with known growth conditions, studies of environmental samples have shown that <1% of bacterial species are cultivatable using current methods (Pace et al. 1985). As a result, culturing methods routinely miss the vast majority of microbial diversity present in any given environment (Amann et al. 1995; Torsvik et al. 1998). The development of culture-free molecular methods based on PCR technology has revolutionized the field of microbiology and exposed an enormous breadth of microbial diversity (Amann et al. 1995; Kelley et al. 2001). Culture-free methods are just beginning to be used in the arena of public health and preliminary studies suggest they will prove extremely valuable in identifying microbial diversity and potential pathogenic organisms present in human environments (Kelley et al. 2004).
To determine the composition and location of bacterial contamination on aeroplanes, we used a PCR-based molecular approach to examine environmental DNA samples collected from aeroplane environments. Using evolutionarily conserved bacterial small subunit ribosomal DNA (rDNA) primers, we created PCR clone libraries from 10 environmental samples collected from four flights: Honolulu to San Francisco/Denver (HO–SFO/DEN), Mexico to San Diego (MEX–SAN), Oakland to San Francisco (OAK–SFO) and Chicago to Washington DC (CHI–WAS). The samples included a variety of potentially contaminated sites frequently touched by passengers, and the results of this study provide insight on the latent health risks involved in aircraft transportation.
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- Materials and methods
Figure 1 shows the results of a typical series of PCR amplifications using DNA isolated from aeroplane samples. The DNA extraction and PCR negative controls had no visible products under any reaction conditions (Fig. 1). With the exception of the tray table and armrest, all the sampled aeroplane environments produced PCR product after adjusting the DNA concentration and the PCR cycling parameters. Tray table and armrest samples from two different flights (four DNAs total) did not produce any PCR amplification under any conditions. In fact, spectrophotometer analysis was not able to detect any DNA in these samples, suggesting an absence of bacteria. Figure 2 shows the results of clone insert screening for one of the environmental samples positive for bacterial DNA. The cloning efficiency ranged from 85 to 95%.
Figure 1. The PCR products from reactions containing 5 μl of sample. Lanes 3 and 5 are samples from Mexico to San Diego (MEX–SAN) flight, and the rest are from the Honolulu to San Francisco/Denver (HO–SFO–DEN) flight (see Materials and methods). Lane 1, toilet seat; lane 2, doorknob; lane 3, paper towel; lane 4, floor; lane 5, bathroom sample; lane 6, handle; lane 7, sink; lane 8 was a PCR amplification with a negative extraction control on a clean kimwipe; Escherichia coli DNA was used as a positive control in lane 9 and lane 10 was the negative PCR control
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Figure 2. Results of PCR screening using M13 primers for 24 clones inserts from a doorknob sample from the Honolulu to San Francisco/Denver (HO–SFO–DEN) flight
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Single pass sequencing of the clone inserts using the 8F primer provided c. 600 bp of high quality sequence for BLAST analysis. Most of the BLAST ‘hits’ were >97% identical to the 16S sequences in Genbank and gave us a high level of confidence concerning the phylogenetic relationships of the determined sequences. Collectively, the diversity proved considerable; based on rDNA identifications, we found 58 genera of bacteria from numerous divisions (Table 1). Calculations of the Chao1 estimation based on dereplicated sequence information at the 97% level estimated that the samples contained anywhere from 50 to 200 bacterial species. As a comparison, we note that this level of diversity is similar to that detected in the human mouth and gut (Hughes et al. 2001). The dereplicated rDNA sequences have been deposited in Genbank under accession numbers AY957663–AY957771.
Table 1. The genus and species of the bacteria in which the cloning sequences matched 97% and above, and the amount of times that sequence was discovered
|Genus and species||Door knob*|| Floor*|| Handle*||Paper towel†|| Sink*||Toilet seat*|
|Abiotrophia sp.||1|| || || || || |
|Achromobacter xylosoxidans|| || || ||1|| || |
|Acidithiobacillus thiooxidans|| || || ||1|| || |
|Aerococcus viridans|| || || || ||1|| |
|Agrobacterium tumefaciens||1|| || || || || |
|Anaerococcus octavius|| ||1|| || || ||1|
|Anaerococcus vaginalis|| || ||1|| || ||1|
|Bacillus psychrosaccharolyticus|| || || || || ||1|
|Bacteroides ureolyticus|| || ||1|| || || |
|Brachybacterium paraconglomeratum||1|| || ||1|| || |
|Brachybacterium sp.||1|| || || ||1|| |
|Bradyrhizobium sp.|| || || || ||1|| |
|Brevibacterium aurantiacum|| || || || ||1|| |
|Comamonas aquatica|| || || || || ||1|
|Comamonas terrigena|| || ||3||1|| || |
|Comamonas testosteroni|| ||3||1|| || || |
|Corynebacterium imitans|| || || || || ||2|
|Corynebacterium sp. 61720|| ||10||8|| ||3||2|
|Corynebacterium sp. 96447|| ||4||1|| ||2|| |
|Corynebacterium sp. CIP107067|| ||1|| || || || |
|Delftia tsuruhatensis strain ARI_7|| ||3||1|| || || |
|Dermabacter hominis|| || ||1|| || || |
|Dietzia psychralcaliphila||2|| || || || || |
|Dietzia sp.|| || || || || || |
|Finegoldia magna|| || ||1|| ||1||1|
|Flavobacterium sp.|| || || || ||1|| |
|Fusobacterium periodonticum|| || || ||1|| || |
|Gemella haemolysans|| || || ||1|| || |
|Granulicatella elegans|| || || ||1|| || |
|Haemophilus parainfluenzae|| || || ||2|| || |
|Kocuria carniphila||11|| || || || || |
|Kocuria palustris||3|| || ||1||1|| |
|Lactobacillus iners|| ||2||5|| || || |
|Leptotrichia genomosp. C1|| || || || || ||1|
|Leuconostoc mesenteroides|| || || || || ||1|
|Micrococcus sp.||5|| || || || || |
|Moraxella osloensis||3|| || || ||1||1|
|Mycobacterium sp.||1|| || || || || |
|Neisseria elongata|| || ||1|| || || |
|Neisseria flava|| || ||2|| || || |
|Neisseria subflava|| || || ||1|| || |
|Paracoccus sp.|| || || || ||1|| |
|Peptoniphilus sp.||1|| || || || || |
|Prevotella genomosp.|| || || || || ||1|
|Prevotella sp.||1|| || ||1|| || |
|Propionibacterium acnes|| ||1||2||3|| ||14|
|Pseudomonas sp.||1|| || || || || |
|Psychrobacter ikaite||2|| || || || || |
|Sphingobacterium antarcticum|| || ||1|| || || |
|Sphingomonas sp.|| || || || || ||1|
|Staphylococcus epidermidis|| || ||2|| || || |
|Staphylococcus equorum|| || || || ||3|| |
|Staphylococcus hominis|| ||3|| || ||1||1|
|Staphylococcus sp.|| || || || ||1|| |
|Stenotrophomonas maltophilia|| ||2||1||1||1|| |
|Stenotrophomonas sp.|| ||1|| || || || |
|Streptococcus gordonii|| || ||1|| || || |
|Streptococcus mitis||1||1|| ||7|| || |
|Streptococcus oralis|| || || || || ||1|
|Streptococcus sanguinis||1|| || ||1|| ||1|
|Streptococcus thermophilus|| || || || || ||1|
|Sulfobacillus disulfidooxidans|| || || || ||2|| |
|Weissella minor|| || || || ||1|| |
|Uncultured bacteria|| ||2||3|| ||7||2|
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- Materials and methods
The organismal diversity present in the various aeroplane samples was surprisingly complex. However, we found some consistent patterns in terms of the type and abundance of bacteria. In each area, specific-bacterial genera represented a significant proportion of the sampled diversity (Fig. 3). The most frequent types of organisms present in the samples are commonly associated with humans: Streptococcus, Staphylococcus, Corynebacterium, Propionibacterium and Kocuria. In some instances, these bacteria represented >30% of the sample (Fig. 3).
The organisms that we detected with the greatest known potential for harm were the ones related to Staphylococcus spp. Staphylococcus epidermis and Staphylococcus hominis were both detected in several samples (Table 1) and these species have been associated with serious infections (Pfaller and Herwaldt 1988). Staphylococcus epidermis is of particular concern. An organism that was once dismissed as a laboratory contaminant is now considered an opportunistic pathogen that can cause bacteraemia, mediastinitis, meningitis and endocarditis in patients with prosthetic devices, and general infections in immunocompromised patients (Pfaller and Herwaldt 1988; Tanner et al. 1999). Our results suggest that the potential for the spread of these organisms via aeroplanes is extremely high. Although we did not detect the medically challenging antibiotic resistant Staphylococcus aureus in our samples, we see no reason why this and other disease threats could not spread in aeroplanes in the same manner as these other Staphylococcus.
The next most abundant organisms of concern were the streptococci. Many species of Streptococcus are known pathogens, especially Streptococcus pneumoniae. Although we did not find sequences from this particular organism in our samples, we did find a number of other Streptococcus spp. known to be opportunistic pathogens, such as Streptococcus mitis and Streptococcus oralis (Table 1). Streptococcus mitis is frequently associated with respiratory disease, and both S. mitis and S. oralis cause infections in immunocompromised patients (Whatmore et al. 2000).
Other common genera found in this study included Corynebacterium, Kocuria and Propionibacterium (Table 1; Fig. 3). Corynebacterium is very difficult to culture and, as a result are not well understood (Tanner et al. 1999). However, Corynebacterium has been identified in patients with blood infections, urethritis, renal failure and prostatitis (Funke et al. 1997). Kocuria is in the family Micrococcaceae and in humans they can be found living on the skin, mucosae and oropharynx. These bacteria are thought to be associated with prosthetic valve endocarditis and catheter-related bacteremias (Basaglia et al. 2002). Propionibacterium is most popularly known as the bacteria that cause acne and are part of the normal skin flora. Propionibacterium sp. is rarely life-threatening but can affect the psychological well-being of highly infected individuals (Kim et al. 2002).
In addition to exposing the diversity of bacterial species that may be found in aeroplane environments, our results also shed light on the most troublesome aeroplane environments. The substantial diversity of human-associated bacterial species found on numerous lavatory surfaces on every flight tested, including a paper towel, suggests that lavatories represent the biggest potential hazard for passengers and crew (Table 1; Fig. 3). This makes sense given the purpose of the lavatory and the fact that it is the most commonly visited area of the aeroplane. Interestingly, a significant number of the species that we identified appeared to typically inhabit human oral cavities. The passengers possibly spread these organisms around the confined space of the lavatory via aerosols by activities such as coughing or blowing their noses.
Although culture-free molecular techniques are highly useful for determining microbial diversity, they cannot by themselves assess whether the organisms detected are still viable. However, other research studies indicate that bacteria can stay viable for some time on similar artificial surfaces. Scott and Bloomfield (1990) found that coliform bacteria can survive for up to 24 h on a laminate surface. In another study, Rusin et al. (1998) were able to culture a variety of different bacterial species by swabbing household bathroom and kitchen surfaces (Scott and Bloomfield 1990; Rusin et al. 1998). Thus, it appears that many, if not most, of the human-associated bacteria species we discovered in aeroplane environments could potentially be viable for the duration of the flight and certainly long enough to transfer among passengers.
With the exception of the rising numbers of immunocompromised patients, the organisms we detected are not especially threatening to the general public. However, we see serious potential for ill persons with more threatening conditions to spread disease to other passengers on planes. Based on the findings of our study and the conclusions of other aeroplane environment research, we suggest the aeroplane lavatories pose a particular threat to public health. We recommend that the airlines take this matter very seriously and institute periodic in-flight cleanings of lavatory door, toilet and sink handles using a 10% bleach solution to reduce contamination.
Future studies in this area would also be enhanced by using more rigorous sampling methods. For instance, the use of sterile latex gloves during our sampling procedure would have been preferable, but we avoided using gloves so as not to alarm fellow passengers unnecessarily. We decided to risk the potential for cross-contamination by avoiding the use of laboratory gloves and relied on thorough hand-washing instead. The fact that our negative kimwipe extraction controls, which had been handled by samplers, and armrest and tray table samples were negative for DNA and PCR products indicates that we successfully minimized cross-contamination. Also, as our principle aim in this study was to prove the utility of culture-free methods and make an initial survey of diversity, we sampled as much of each surface as possible. Future studies should also make greater attempts to quantify the amount of bacteria on surface areas and standardize the amount of surface area sampled. Finally, the sampling method could be improved by standardizing the time from sampling to storing the DNA on ice.
In addition to improvements in the sampling methods, future research in this area should also pursue more thorough investigation of the bacterial and viral diversity in aeroplane environments. As a preliminary survey of just four flights, this study exposed a remarkable diversity of human-associated bacteria (even discovering a few species without cultured relatives; Table 1) and suggested that many more species may be found commonly on aeroplanes. Future studies should investigate the types and frequency of cleaning regiments effective for minimizing exposure and transmission of microbes. Other questions to address include: what is the importance of biofilms in maintaining bacterial viability on aeroplanes? How many bacterial cells are aerosolized at any given time? How do the number of passengers, the destination and duration of flights impact bacterial contamination? Is the risk greater on international flights compared with domestic flights? What physical factors (e.g. toilet flushing) are the most important for spreading microbes? There is also a need to quantify the viability of many of the microbial species we discovered. The answers to these questions should prove useful for minimizing the spread of bacteria and potential diseases.