Contamination by pathogenic multidrug resistant bacteria on interior surfaces of ambulances

Pre-hospital emergency care is a healthcare delivery service to victims of illnesses or accidents with a wide variety of health profiles. Ambulances may be a source of pathogenic microorganisms if not sanitized properly. Considering this, the present study aimed to evaluate the bacterial community present in different equipment and surfaces from ambulances used on pre-hospital care and verify the antibiotic resistance profile of the isolates. For that, three ambulances were chosen in unannounced visits, and eight sampling points were selected: wall inside the vehicle, cervical collar, spinal boards, bedsheets, masks, earplugs, head blocks and steering wheels. Pathogenic bacteria were isolated, identified and submitted to antimicrobial assays. A total of 144 samples were collected, and 33 different bacteria species were isolated. Among them, pathogenic genera Staphylococcus, Acinetobacter, Listeria and Ewingella. Staphylococcus, Acinetobacter, Listeria presented strains resistant to oxacilin, isolated from earplugs, spinal boards and masks which are in frequent contact with patients and rescuers. The results highlight the presence of pathogenic bacteria on surfaces and equipment that had been cleaned and deemed free of contagion.


Introduction
Pre-hospital emergency care is a coordinated and timely delivery of healthcare services to victims of illnesses or accidents (Hudson et al., 2017). The service provides 24 hours care for urgent and emergency health problems, helping everyday thousands of patient in different situations outside the hospital, transporting them with ambulances. Patients transported to hospitals or other health facilities by this system may have infections; therefore, after each run, ambulances must be cleaned and decontaminated to be ready for its next use (Sheahan et al., 2019).
The relevance of this cleaning and decontamination process lies in the fact that uncleaned ambulances can be a source of pathogenic organisms such as influenza virus, severe acute respiratory syndrome (SARS), coronavirus, meningococcal meningitis, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, and vancomycin-resistant enterococci (Kampf et al., 2020;Miramonti et al., 2013). Patients and rescuers can be contaminated during the assistance by direct contact with the surfaces or equipment (Varona-Barquin et al., 2017), and become a source of cross-contamination between ambulances and hospital environments (Russotto et al., 2015). Nonetheless, infection control of pre-hospital emergency care services has not yet been recognized as an essential part of public health (Noh et al., 2011), which could become a critical problem during a pandemic event.
Once that evidence of contamination of surfaces and equipment in pre-hospital emergency care services is little known, and the prevalence and virulence of multiple-drug-resistant organisms has increased, it becomes important to limit exposure conditions, identify the most contaminated sites and which microorganisms are present, and eradicate contamination by using appropriate disinfection and sanitation procedures (Russotto et al., 2015).
Considering the lack of research on these services, the present study aims to evaluate the bacterial community present in different equipments and surfaces from emergency ambulances ready for service used on pre-hospital care and verify the antimicrobial resistance profiles of the isolates of clinical importance.

Study design and setting
The study was developed on a rescue station, in unannounced visits. Three ambulances were chosen, and eight sampling points inside each ambulance were selected based on their frequency of contact with rescuers and patients, and then divided into: a) direct contact: cervical collar, spinal boards, bedsheets, masks, earplugs, head blocks; b) indirect contact: wall inside the vehicle and steering wheel. Samples were collected from August 2017 to April 2018, totalizing 144 samples.

Bacteria sampling and identification
Surfaces were sampled using swabs moistened with sterile saline solution (0.9% NaCl). During the sampling process, swabs were rubbed over the entire sampling point and rotated to collect as much material as possible. Swabs were inserted into sterile tubes containing BHI broth (Becton Dickinson GmbH, Heidelberg, Germany) and incubated for 24 h at 37°C.
Subsequently, the enriched cultures were streaked onto mannitol salt agar and MacConkey's agar, and incubated for 24 h at 37°C. Isolated colonies were regrown on nutrient agar, and then biochemically identified by the following tests: motility; indole production; hydrogen sulphide production; lactose, glucose and sucrose fermentation; use of citrate as sole carbon source; urease and catalase enzyme production; and decarboxylation of lysine and ornithine. Bacteria were identified with the Advanced Bacterial Identification Software (ABIS Online), where only scores above 95% of identification were considered.

Statistical analysis
The results were statistically analyzed by the Pearson's chi-squared test. The chi-squared test was used to evaluate the correlation between contamination by bacteria (considering all species identified), sampled sites and the type of contact (direct or indirect) with the surface. Then it was applied to evaluate the association between the frequency of pathogenic bacteria and the surfaces studied. The dummy variable was established as 1 (for pathogenic bacteria) and 0 (for non-pathogenic bacteria).
All tests were applied with 95% of confidence (p< 0.05). All test were performed on the Stata ® software.

Results
Of the 144 samples collected, 121 (84%) tested positive for bacterial contamination (Figure 1). Each ambulance presented an average of 40± 4.7 positive samples. A total of 33 bacteria species were isolated and identified, and another 19 could not be identified. However, the chi-square test indicated that there is no association between the positive sample and the site where they were found (chi-square = 173.3, p >0.05).  (Table 1), but there was no association with direct or indirect contacts (chi-square = 0.689, p> 0.05).  Source: Authors.
The isolates classified as of clinical importance were submitted to antibiotic susceptibility test and the results are presented on Table 2.

Discussion
The genera Staphylococcus is an opportunistic pathogen known for its ability to evade the immune system and cause a variety of different infections (Pollitt et al., 2018), including bacteremia, endocarditis, and skin and pleuro-pulmonary infections (Gordon et al., 2019). Staphylococcus aureus is of special concern, once that the World Health Organization (WHO) recently categorized them as of high priority threat in terms of resistance on global a level (Haaber et al., 2017). Methicillinresistant Staphylococcus aureus (MRSA) has spread globally and account for approximately 59% of skin infections (Haaber et al., 2017). It is estimated that up to half of the world's adult population carries S. aureus in mucous membranes; healthcare professionals, people who regularly use needles (diabetics and intravenous drug users), hospitalized patients and immunocompromised individuals tend to have higher colonization rates (up to 80%). S. aureus can be transmitted from person to person by direct or indirect contact (Tong et al. 2015), as by touching a steering wheel (Eibicht & Vogel, 2011).
Additionally, ambulance contamination by MRSA strains has been reported even in transportations as short as 20 min (Eibicht & Vogel, 2011).
Acinetobacter spp. have emerged in recent years as a multidrug or extensively drug resistant strain, and it is a major cause of healthcare-associated infections and hospital outbreaks, especially in intensive care unit patients (Zarrilli et al., 2018), where mortality rates reach 43% (Asif et al., 2018). Acinetobacter baumannii (A. baumannii) infections account for ~ 2% of all healthcare-associated infections in the United States and Europe; however, these rates are twice higher in Asia and in the Middle East (Almasaudi, 2018). Additionally, out of all A. baumannii isolated globally, 45% are considered multidrug resistant (Zarrilli et al., 2013). This is aggravated by the bacteria's ability to survive on dry surfaces with limited nutrients available for up to 4 months (Almasaudi, 2018).
The Listeria genus are intracellular pathogens often associated with foodborne infections, and it can be fatal for children, pregnant women, elderly and immunocompromised individuals (Caldron et al., 2015). In addition, L. monocytogenes causes severe food poisoning in humans, systemic sepsis, liver abscess, and acute meningitis, which can increase the mortality rate by about 30% for each hour that correct treatment is delayed. In pregnant women, it can cross the placental barrier and lead to abortion or neonatal infection (Caldron et al., 2015). Its high pathogenicity, biofilm-forming capacity and omnipresence qualify L. monocytogenes as a huge risk to human health (Santos et al., 2019;Olaimat et al., 2018). Listeriosis has been described as the third cause of death from foodborne contamination (Wadhwa et al., 2017).
The Ewingella genus has been associated with peritonitis, bacteremia, fatal cases of nosocomial pneumonia and Waterhouse-Friderichsen syndrome (Spagnolo et al., 2019). Ewingella americana is considered resistant to several βlactamases, mainly those from cefalosporins of first and second generation, but sensitive to third and fourth generation drugs from the same group (Esposito et al., 2019).
The results highlight the need for careful disinfection procedures, once that these sites have the highest degree of contact with the body of the patients; thus, they are exposed to pathogens capable of initiating an infection before they reach the hospital (Farhadloo et al., 2018). Additionally, there is huge concern about rescuers, as they stay for extended periods of time in ambulances and some pathogens can have multiple entrance pathways, such as mucous membranes from the respiratory tract, through the skin or another parenteral route (Esposito et al., 2019).
Bacterial contamination on surfaces and equipment of ambulance vehicles is a health problem that happens around the world, because it can play a role in cross-transmitting pathogens that can lead to infection or colonization, and this colonization can generate a possible reservoir of multidrug-resistant pathogens (Russotto et al., 2017). Additionally, the pathogenic genera isolated from these places are capable to form biofilms, and this is one of the key factors that allow bacteria to resist to cleaning products and survive in the environment for a long time (Qi et al., 2016).
Antibiotic resistance is considered a major threat to global health, resulting in increased mortality rates, longer hospital recovery time, and higher medical costs (Shrestha et al., 2018). Therefore, as patients transported by ambulances are often in critical condition, care must be taken to avoid the presence of antibiotic resistant contaminants (El-Mokhtar & Hetta, 2018).
Each new emergency service can become a new source of contamination, and considering that ambulances cannot be a sterile environment, continuous pathogens monitoring becomes necessary (Farhadloo et al., 2018). Although it is not possible to associate cases of infections with their onset in ambulances, the present study results highlight the pathogenic bacteria presence on already cleaned surfaces and equipment, showing the vulnerability of users and rescuers in these environments.

Conclusion
The present study evaluated the bacterial community present in ambulances, and the antibiotic resistance profile from the isolates of clinical importance. The results showed that 84% from the samples presented positive contamination, with 33 bacteria species, and four pathogenic genera: Staphylococcus, Acinetobacter, Listeria and Ewingella. There was a positive correlation between the presence of pathogenic strains with the earplugs, spinal board and mask surfaces. Staphylococcus, Acinetobacter, Listeria presented strains resistant to oxacilin.
This scenario of contamination has been observed in ambulances worldwide and in different surfaces inside de vehicle, suggesting that in a near future the effectiveness of cleaning protocols need to be improved. Therefore, we recommend training and education programs to rescuers and professionals that work on frontline healthcare, for cleaning and disinfection procedures implementation, once ambulances are key points of the infection control strategies.