ESKAPE pathogens are the causative agents of most nosocomial infections worldwide. The abbreviation stands for the species Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium/faecalis. Those organisms can be highly virulent and carry or transfer antibiotic resistances (20, 21).

Antibiotic resistance and the spread of multidrug-resistant bacteria (AMR) is a problem that was associated with about 4.9 million deaths worldwide in 2019 (22). However, the spread of these organisms does not occur in hospitals alone, but also in places with a high frequency of people, such as on public transportation. Notably, the following studies were focused on the detection of pathogenic species, mostly based on (selective) cultivation followed by PCR of pathogen-associated marker genes, e.g., the mcr-1 gene for E. coli that mediates colistin resistance.

The most prominent ESKAPE species within the public transport studies is the (opportunistic) pathogen Staphylococcus aureus. In general, due to the natural passenger’s microbiome, the skin-associated species are highly abundant in busses and subways. In a bus, serving both hospital and community routes, methicillin-resistant S. aureus (MRSA) was found (community-associated SCCmec type IV and healthcare-associated SCCmec type II). Of the detected MRSA strains, 65% were multidrug resistant (23). Within this study, seats and seat rails were most contaminated. In subways, S. aureus containing the mecA gene was detected, alongside natural skin-associated species of S. aureus (11). mecA is associated with methicillin-resistant S. aureus (MRSA) and nosocomial infections, but the study concludes no strong evidence for pathogenicity based on the obtained sequences. Other studies showed the prevalence of MRSA in public transport (24–27).

Escherichia coli with a multidrug resistance, including mcr-1 which mediates colistin resistance, was found in public transportation in Guangzhou, China (28). Twenty-three isolates of 737 samples with bacterial growth were positive for mcr-1, most of them were resistant against ampicillin, cefotaxime, fosfomycin, and gentamicin.

For Klebsiella pneumoniae, there were less findings of drug resistant isolates. In the Beijing (China) subway environment, highly touched surfaces were sampled and from a total of 603 samples across 15 metro lines, 11 carbapenem-resistant K. pneumoniae isolates were detected (29).

Enterobacter species were also found in public transport studies. E. faecium was abundant throughout the subway in New York City, United States (11), and multidrug resistant E. faecalis was observed on shared bicycles in Chengdu, China (30).

No studies have been found on the occurrence of multidrug resistant Acinetobacter baumannii and Pseudomonas aeruginosa in public transport.

Nowadays, next-generation-sequencing (NGS) is the most used technology for sequencing. With this approach, high throughput analysis is possible and enables the identification of microbes within a high sample size in a cost-effective manner, generating high amounts of data (32, 33). There is a big variation within the NGS DNA sequencing technologies, varying in amplification method, sequencing chemistry, sequencing speed, etc. (32, 33). The most established NGS platform was created by Illumina, followed by Oxford Nanopore, which revolutionized the field by releasing a first portable nanopore sequencing device in 2014. Each technology differs in its output, advantages and limitations. With NGS, not only the microbial identity can be detected, also the diversity within or of all samples can be determined, outshining the limited information obtained by cultivation. The possibilities within NGS and the bioinformatical analysis are rapidly evolving more and more. Nevertheless, there are some shortcomings when it comes to profiling uncharacterized species in environmental microbiomes, as strain-level analyses are usually tested for human metagenomes and the tools are tailored to human metagenomes (34).

Metagenomics is a powerful tool to identify the microbiome of a sample. If specific organisms are to be screened for, such as potential pathogens, there are methods such as loop-mediated isothermal amplification (LAMP) that allow the targeted detection of species. LAMP is a fast, cost effective, and easy tool to detect specific organisms and requires only a few devices, while the evaluation occurs after 30 min.

Using marker genes, which differ for every organism, fast and detailed detection with a high specificity and sensitivity are possible (35). The potential of LAMP has already been established in relation to the detection of pathogens in the food industry (36). It is also useful for hospitals or in human high traffic environments to monitor microbial threats. During the SARS-CoV-2 pandemic, several publications showed the successful application of reverse transcription LAMP for this pathogen (37–39).

To this date and to our knowledge, there is no publication on the use of LAMP for pathogen detection in public transport as microbial monitoring measure. The detection of drug-resistant organisms is an important factor in monitoring the spread of pathogens and has yet to be implemented.

The transmission of pathogens is especially meaningful when it occurs through surfaces in epidemic and endemic scenarios (42). Although the transmission of pathogens through contact surfaces can be reduced by antimicrobial surfaces, the long-term usage and consequences have to be evaluated. One important factor is the increased risk of the development and transmission of antibiotic resistances between bacteria, when such materials are overused (43).

One of the best investigated antimicrobial material is copper. It causes cell damage by releasing copper ions which causes the cell membrane to rupture, leading to a membrane potential loss and depletion of cytoplasmic subtances (44). Further, copper ions induce reactive oxygen species (ROS), which in turn cause DNA damage (45). While copper as a material is costly, surfaces using the antibacterial effect of copper and integrating it as metal nanoparticles within a polymer matrix makes it cost effective, as reviewed by Tamayo et al. (46), and therefore could be suitable for a broad use.

While the antimicrobial properties of copper have long been known and researched, there are many different antimicrobial surfaces available (47, 48). For example, anti-biofouling surfaces can reduce microbial adhesion to the surfaces, biocidal nanocomposites kill microbes using biocidal species. Physical mechanisms as nanostructured surfaces can rupture bacterial cells, others can even prevent the attachment on the surfaces (49).

Some innovative antimicrobial materials were already tested in public transportation, such as antimicrobial photodynamic coatings, showing an absolute risk reduction of 22.6% for high bacterial counts (50). Other tested materials showed no significant reduction of the microbial burden, using photocatalyst-coated and uncoated hand-contact surfaces (51).

In the process of fumigation, an antimicrobial solution is nebulized in an enclosed environment with the aim to reduce the microbial burden in the air and on surfaces. The nebulization of chemicals, e.g., hydrogen peroxide has been in use (52, 53). There are also different forms of fumigation, that even consider the application in public transport (54). Here, peracetic acid stabilized with acetic acid and hydrogen peroxide showed an effectiveness of disinfection of 81.7% in busses, and even worked against highly resistant spores. Hydrogen peroxide facilitates the penetration of peracetic acid, which contributes to a fortified sporicidal activity of the agents, as tested with Bacillus subtilis spores (55).

The effectiveness of fumigation is highly dependent on the materials to be disinfected (54, 56), e.g., the effect of fogged peracetic acid and hydrogen peroxide was shown to be particularly high on glass windows and doors, and low on fabric materials (56). Further, the efficacy depends on the type of microorganism, the fumigation device and technology and the substance (57–59). One downfall of the fumigation of chemicals is the safety measures, that have to be ensured. Therefore, the usage of fumigation can only occur while the passenger cabins are not in service, but could be performed during night times. Considering the costs of fumigation, it depends on the device and fumigation technologies used. Costs for consumables are low, e.g., ~2 € / L of hydrogen peroxide.

Another method for disinfection in public transport, but more commonly employed in hospital settings, is UV-C disinfection. UV radiation causes DNA damage (60), which is mediated by the generation of ROS (61). UV-C operates in a spectrum of 200–280 nm. Because UV-C is also harmful to humans, some efforts have been made to employ mobile robots for disinfection with UV-C radiation (62, 63). In hospitals, there have been several systems using and testing UV-C disinfection, that are combined with disinfectant chemical agents (64, 65). One disadvantage of this approach is the material damage (66) and the incomplete light contact in all areas in a room or cabin. An advantage of UV-C disinfection is the economical aspect. Some low-cost UV-C light devices can be purchased with high efficacy against strains of Candida auris, MRSA, and bacteriophage Phi6 (67). Although UV-C disinfection shows effectiveness against some pathogens, it can cause bacterial mutations (68). A new, LED-based UV-irradiation technology has shown to be effective against some bacteria and viruses, but it is connected to high costs (69), which is uneconomical for use in public transport.

A tool designed to provide both air purification and surface disinfection is plasma. Plasma is also known as the fourth state of matter, which is a particle mix with a high electrical conductivity and is chemically reactive. The use of plasma is well established in the food industry (70) and in the medical field (71, 72), but it may be useful for the application in public transport.

The antimicrobial effect of plasma has been long known and is created by the combination or single effect of charged particles (ions, electrons), reactive species (e.g., ozone, ROS), radiation of UV-C/Vacuum-UV (VUV) as well as heating (73–75).

There are different types of plasma that can be used for disinfection. In a study conducted by Liang and Wu (76), culturable bacterial aerosol diversity loss was observed after using non-thermal plasma. Tested with Aspergillus niger, Bacillus subtilis, and Pseudomonas fluorescens as test organisms, it was described as a highly efficient air decontamination method.

To date, no study has used plasma as a system to reduce the microbial load in public transport. Only plasma related methods, such as a needle-point bipolar ionization system was tested in trams to investigate the reduction of bioaerosols (77). It was shown that environmental bioaerosols were reduced with this method, but it was not sufficient for surfaces. Therefore, more research is needed to test the feasibility of plasma technologies in the public transport context. Regarding cost-efficiency, only publications are available on the use of plasma in water treatment plants or in food industry (78), using plasma activated water (79). But in general, the formation of non-thermal plasma is connected to low energy input, unlike thermal plasma (80).

All approaches that were introduced in this section have their advantages and disadvantages. Different factors have to be considered when finding a best suiting method for a specific environment, such as passenger cabins. These include cost-effectiveness, service life, operation of devices, combined with the effectiveness of microbial reduction. In the end, the aim to apply countermeasures within the passenger cabins is to reduce the microbial load and therefore decrease the spread of potential pathogens, and a combination of some methods may bring all advantages together and ensure passenger safety and comfort.