ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species

Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.


Introduction
Antimicrobial resistance (AMR) is a major global health threat, resulting in millions of deaths each year from antibiotic-resistant infections [1,2]. The most common causes of these infections are a group of bacteria known as ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli). As patients with weakened immune systems and antibiotic-induced dysbiosis are at increased risk of developing these infections, hospitals are particularly vulnerable to the spread of ESKAPEE pathogens [3,4]. Reducing the transmission of these infections in hospitals is critical to addressing the overall public health threat posed by AMR [3,5,6].
To reduce the risk of hospital-acquired infections (HAIs) in hospitals and other healthcare facilities, commonly, disinfectants are used. However, the overuse and improper use of disinfectants favor the development and spread of AMR organisms through the selection pressure exerted on microbial populations [7][8][9][10][11]. The reduction in susceptible microorganisms facilitates multiplication and overgrowth of the resistant ones. In addition, the widespread use of disinfectants can also lead to the emergence of cross-resistance [12,13].
Unlike traditional disinfectants, which unselectively eliminate microorganisms, probiotics used in this context are more selective and might only target specific microorganisms by directly eliminating or suppressing growth. This means that they are less likely to lead to the development of AMR strains of bacteria. There is some evidence to suggest that probiotic-based cleaning formulations may be effective at reducing the risk of HAIs in hospitals [8,[14][15][16]. However, more research is needed to confirm the effectiveness of probiotics as a standalone cleaning agent in the hospital setting. The effectiveness of probiotics in reducing the risk of HAIs depends on various factors, including the specific strain or strains of bacteria used, the dose and mode of application of the probiotic, and the patient population under study.
In-depth analysis is needed to identify which specific probiotic bacterial strains are most effective in reducing the risk of hospital-acquired infections. While Lactobacillaceae and Bacilli have demonstrated their potential in some studies, it is crucial to consider the unique properties of different probiotic strains and the specific conditions in which they are used. It is also essential to consider the ways in which probiotic strains may interact with each other, conventional antimicrobials, and the potential for adverse effects in certain patient populations. In this review, we focus on the effect of probiotic Lactobacillaceae and Bacilli as well as their products in the direct and indirect elimination of ESKAPEE pathogens.

Nature of Conducted Experiments
The vast majority of studies were based on in vitro experiments (132 times) in which the effects of probiotic bacteria against pathogens were mainly evaluated by agar spot tests, agar well tests, co-culture in liquid media and on the surface of human cell lines. The latter included Caco-2 [44,50, 89,103], HT29 [24,89,101], HeLa 229 [65,83], vaginal epithelial cells [71], and uroepithelial cells [143]. Some of the in vitro experiments tested the antibiofilm properties of the probiotic bacteria on inert surfaces [81,93,125], most importantly on metals such as stainless steel [120] or titanium [28]. Other surfaces included those made of silicon [32], ceramics, and linoleum [144]. Six studies were based exclusively on in vivo experiments. These included three rat disease models, two of which were for wounds [53,54] and one was for surgical implants [55], two murine disease models [145], one of which also included a bovine disease model [130], one rabbit model knee implant infection [76] and one bee model [123]. Three studies were based on combined in vitro and in vivo experiments and exclusively included murine disease models, two intestinal colonization models [36,52] and one urinary tract infection model [113]. In one of the latter, the anti-pathogenic properties demonstrated in vitro could not be replicated in vivo [52].

Suppression of ESKAPEE Pathogens
The ESKAPEE pathogens that were most frequently reported to be displaced by the probiotic bacteria were by far Staphylococcus aureus (77 times) and Escherichia coli (73 times). The next most common ESKAPEE pathogen to be described as having been displaced was Pseudomonas aeruginosa (30 times). These three were also by far the most frequently tested ESKAPEE pathogens. Table 1 displays the number of times each probiotic species was reported to displace these three pathogens. Less frequently tested and displaced were Klebsiella pneumoniae (nine times) [26,43,52,56,65,79,132,136,146], Enterococci (five times) [85,103,107,141,147], Enterobacter species (two times) [26,61] and Acinetobacter baumannii (once) [68]. Of the Enterococcus isolates, four were E. faecalis and one E. faecium. Nine reports described pathogen inhibition to be caused by direct competition between probiotic and pathogenic bacterial cells [24,29,32,40,102,108,135,143,148]. Nineteen studies pinned the bactericidal properties down on one or more specific cell products [46,49,51,56,57,59, 73,76,99,100,117,125,127,129,133,136,[149][150][151]. Eleven studies ascribed the suppression of pathogens to be attributable to both, direct competition and cell products [27,55,61,64,72,75,77,104,116,126,152]. Five studies linked attributed bactericidal activity exclusively to lactic acid and pH reduction [98,119,137,138,153]. The remaining 52 studies that identified the inhibition mechanism narrowed it down to be the cell-free supernatant (CFS) or exclusively investigated the CFS of probiotic bacteria. Nigatu and Gashe (1994) reported inhibitory effects to be independent of pH [136], Kang et al. (2017) discovered that pH neutralized CFS was still efficient in killing pathogenic bacteria as long as it was not heat inactivated or proteinase K treated [111]. Jeyanathan et al. (2021) described whole cell cultures to be ineffective in preventing P. aeruginosa growth, while CFS significantly reduced adherence and viability [28].

Discussion
The protective effects of colonizing the human body [159][160][161][162][163][164][165] or food [159,166,167] with probiotics have been well studied. However, applying this concept to infection vectortransmitting surfaces such as those found in hospitals through protective probiotic films is a novel approach that challenges traditional infection prevention strategies centered on maintaining a sterile hospital environment. Probiotic cleaning formulations show promise as a potential solution to the proliferation of antimicrobial resistance, particularly in healthcare settings [8,10,11,[14][15][16][17]. These products, which contain live microorganisms beneficial to the cleaned environment, may restore microbial balance and reduce selective pressure driving drug-resistant bacteria. They may also have a more favorable environmental profile due to their use of natural, biodegradable ingredients.
Our review of the efficacy of probiotics in reducing the presence of ESKAPEE pathogens identified numerous studies that analyzed the bactericidal properties of probiotic strains against E. coli and S. aureus, which are well-known causes of gastroenteritis [168] particularly relevant to the use of probiotics in the gastrointestinal tract. The probiotic isolates examined in these studies were primarily Lactobacilli, aligning with the focus on using probiotics in the gut. In addition, most studies simulated conditions present in the gastrointestinal tract. P. aeruginosa, a microorganism known to cause food spoilage [169], was also frequently examined for its susceptibility to probiotics. However, there were very few studies that investigated the effects of probiotics against the majority of other ESKAPEE pathogens.
It is important to note that the greater number of studies demonstrating the bactericidal effects of Lactobacillales does not necessarily indicate superior performance in suppressing ESKAPEE pathogens compared to Bacillus species. Rather, evaluating probiotics for use on hospital surfaces requires a more nuanced analysis that considers factors such as survivability, biofilm formation, and bactericidal activity at room temperature and under nutrient-poor conditions, which may instead make Bacillus species more attractive candidates. It is also crucial to recognize that significant strain-level variations within the same species can impact the desired properties [170,171].
Despite the generally recognized safety of probiotics and the fact that most were derived from benign edible sources some of which are even consumed in large quantities, the use of these microorganisms in hospitals warrants further investigation to ensure their safety, particularly in dysbiotic, immunocompromised, leukopenic or even agranulocytic patients [172][173][174][175], as these are patient groups that could particularly benefit from such innovative solutions [176][177][178][179][180][181][182][183][184]. It is desirable for these microorganisms, which may be transferred from the environment to the patient, to retain their protective effects in the patient as well.
Conducting more targeted studies on hospital or similar surfaces and the specific pathogens to be displaced provide valuable insights and aid in the development of new and promising approaches for preventing or eradicating bacterial biofilms in medical settings [185][186][187]. Comparison of different probiotic-based cleansing formulations should also be considered, as the efficacy or inefficacy of one product may not necessarily apply to other products with different strains.

Materials and Methods
A systematic search was conducted using the PubMed and Web of Science databases. For the assessment of search results, results were imported into CITAVI Database. Two reviewers independently screened the titles and abstracts of the identified studies for eligibility. Studies were eligible for inclusion if they examined the effects of Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Limosilactobacillus, Latilactobacillus or Bacillus spp. or their products/supernatants on one or more ESKAPEE pathogens. ESKAPEE pathogens were defined as Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli. Full-text articles were then obtained for all potentially eligible studies and assessed for inclusion based on the predefined inclusion and exclusion criteria. Any discrepancies between the reviewers were resolved through discussion and consensus.
Overall, a total of 143 studies were included in the review (see Figure 1). The studies were conducted in a variety of settings and involved a range of bacterial strains and ESKAPEE pathogens. The experiments included both in vitro and in vivo studies, and a variety of methods were used to assess the effects of the probiotic bacteria on the growth, colonization, and survival of the pathogens.

Conclusions
The evidence collected from the multitude of scientific studies shows that many Lactobacillaceae and Bacillus species are able to suppress the proliferation of ESKAPEE pathogens. However, the experimental conditions under which these studies were conducted do not allow conclusive assessments of the efficacy of the species in protecting hospital surfaces, which could contribute to the development of new promising approaches to prevent or eradicate bacterial biofilms in medical settings. More targeted studies on hospital surfaces and pathogens to be displaced are needed to understand the potential of each species and strain in more detail, along with their respective safety profiles. Once a safe and effective formulation is identified, conducting large-scale studies seems imperative, given the potential they carry in tackling several issues infection control and medical practice in general are currently facing. Data were extracted from the included studies using a standardized data extraction form. The following data were extracted: examined bacteria, ESKAPEE pathogens they were tested against, environment tested in, type of experiment, and, if applicable, additional relevant information. The extracted data were synthesized and analyzed using a narrative synthesis approach. The results were organized by bacteria and pathogens tested and summarized in a tabular format.
Our review aimed to provide a comprehensive overview of the available evidence on the effects of Lactobacilli and Bacilli or their products against ESKAPEE pathogens. By synthesizing and analysing the data from the included studies, we aimed to evaluate the potential of these probiotic bacteria as means to combat infections caused by these pathogens.

Conclusions
The evidence collected from the multitude of scientific studies shows that many Lactobacillaceae and Bacillus species are able to suppress the proliferation of ESKAPEE pathogens. However, the experimental conditions under which these studies were conducted do not allow conclusive assessments of the efficacy of the species in protecting hospital surfaces, which could contribute to the development of new promising approaches to prevent or eradicate bacterial biofilms in medical settings. More targeted studies on hospital surfaces and pathogens to be displaced are needed to understand the potential of each species and strain in more detail, along with their respective safety profiles. Once a safe and effective formulation is identified, conducting large-scale studies seems imperative, given the potential they carry in tackling several issues infection control and medical practice in general are currently facing.