Introduction

Klebsiella pneumoniaeis a gram-negative bacteria, considered an opportunistic pathogen because it can grow in a wide range of hosts and environments. Over in the past years, there has been an increase in infections caused by resistant K. pneumoniae strains, leading to serious consequences, especially in hospital areas, such as longer hospitalization time, the absence of therapeutic options, and also an increase in morbidity and mortality numbers [1]. Normally, the infections caused by K.pneumoniae are asymptomatic and can reach the gastrointestinal tract, the skin, the nose, and the throat of healthy individuals [2]. The transmission occurs mainly through hand contact. Nevertheless, when there is a homeostatic imbalance, this microorganism can cause a variety of infections depending on the site, generating urinary and respiratory infections but also more severe complications in the blood as well as hepatic abscess, meningitis, and sepsis [3]. The infection can be transmitted mostly through direct or indirect contact between patients, health professionals, contaminated objects, visitors, or travelers [4]. In hospitals, for example, the high number of people circulating in an enclosed area leads to possible bacterial resistance. In addition, the absence of hand sanitization from professionals and visitors contributes to the dissemination of these resistant strains in different places, such as public transport, supermarkets, beaches, and parks [5]. In this sense, there are several factors involved in the dissemination of K. pneumoniae in a hospital environment. Considering that this bacteria is an opportunistic microorganism, and it possesses a great ability to produce biofilms, and it has been the subject of extensive studies in the development of nosocomial infections [6]. Among these, catheters use, time of stay in these environments, long hospitalization time in the Intensive Care Unit (ICU), prolonged antimicrobial therapy, reutilized instruments in surgeries and implants, and/or devices can all provide open access to K. pneumoniae infections [7,8]. Susceptible individuals such as diabetics, transplanted patients, and people with compromised hepatic function and dialysis patients are among the group at risk considering their immunodeficiency, which results in a defect in the immune system that can no longer work properly against the infection. This immunodeficiency is a side effect of the therapies as a consequence of their treatment, considering that in these conditions, the immune system does not work properly [4,9]. Biofilms are classified as a microbial community that sits in a thin layer of either biotic or abiotic surfaces, established through the extracellular polymeric substances that originate from the biofilm itself. K. pneumoniae biofilm formation has been linked to an important pathogenic feature of this bacteria, particularly in the use of medical catheters. Biofilm matrix provides the necessary conditions to mitigate drug delivery regarding its dense polysaccharide and protein layer in which the antibiotic can't diffuse properly, resulting in significant exposure to bacteria and, therefore, the establishment of chronic infections in the urinary tract [4, 10, 11]. Alternative methods are being studied to minimize the consequences caused by biofilm formation. Among these methods is the research on the activity of antibiofilm formation of essential oils (EOs) derived from plants. EOs are a viable therapy option for contaminated patients who have been infected with resistant strains. There has been a tremendous surge in research on aromatic and medicinal plants in recente years [12-22]. However, to obtain an adequate and commercial formula, there are a few factors to consider, such as geographical morphology, climate, and cultural conditions [13]. The primary purpose of this study is to conduct a systematic review of the usage of essential oils in the administration of K. pneumoniae biofilms using scientific databases.

Materials and Methods

The current study was undertaken using a bibliographic search of PUBMED, MEDLINE, Scientific Electronic Library Online (SCIELO), Academic Google, and other relevant websites, from 2012 to 2022. "Biofilm," "K. pneumoniae and biofilm," "biofilm and medical catheters," "biofilm and essential oils," and "K. pneumoniae and essential oils" were the top search terms because they addressed the study's major goal. The initial search was undertaken by reading the titles and abstracts and only full and published papers were chosen to participate in this review follows the careful reading.

Results and Discussion

The keyword "biofilm" was the most comprehensive among all others regarding the present study in our search. From the period selected, there were 100,951 published papers, whilst for the term "Klebsiella pneumoniae and biofilm," 10,979 studies were found. 7.387 publications were found for the term biofilm and medical catheters, and for the term "biofilm and essential oils," there were 42 studies. As for the term "Klebsiellapneumoniae and essential oils," only 20 publications were found. These results are presented in Table1 and suggest that the majority of the studies are not focusing on the main goal of this review.

Search term	Number of published papers between 2012 and 2022
Biofilm	100,951
K. pneumoniae and biofilm	10,979
biofilm and medical catheters	7,387
biofilm and essential oils	42
K. pneumoniae and essential oils	20

Table 1: Number of published papers in the last 10 years according to the search term

This review included the main studies discovered through the selected databases. The relevance of biofilm formation was stated in the work of Bergamo G, et al., in which the author observed a significant increase in the number of publications since 1983 [23]. These data showed that biofilms had been a relevant subject over the years for the scientific community and the need for further studies to identify the causes and possible solutions for this matter.

Use of essential oils in K. pneumoniae strains

Total number of studies found correlating the use of EOs in K. pneumoniae strains was 20. As of today, K. pneumoniae is the most nosocomial pathogen that carries the carbapenemase gene (KPC) that gives resistance to carbapenem, a classic beta-lactam antibiotic that is used for the treatment of bacterial infections. As a result, the search for novel antimicrobials is necessary. Natural products, such as EOs, are a promising source due to their complex composition. These products are demonstrated to be effective against resistant strains, as shown in Figure 1, but the mechanisms underlying their actions are not fully understood.

Figure 1: EO action sites and mechanisms in a bacterial cell

Eugenol promotes disruption of the cytoplasmic membrane, increasing its nonspecific permeability, causing ion leakage and excessive loss of other cellular components, including intracellular proteins, leading to cell death. Geraniol, linalool act on the wall, changing permeability and modifying membrane proteins and periplasmic enzymes, as well as altering the membrane, ion transport, and energy production. Palmarosa oil penetrates the lipophilic part of the cell membrane generating na imbalance in the membrane potential and losing its selective permeability. The compounds methyl carvacrol, menthol, citronellol, and thymol also cause a widening of the cell membrane that leads to the passive diffusion of ions between the phospholipids. They also act on the cell wall of bacterial cells, causing rupture and extravasation of the extracelular contents. Carvacrol induces leakage and loss of ATP from bacterial cells. Cinnamaldehyde inhibits ATPase enzymes and disrupts the outer cell membrane.

A better understanding of the molecular mechanisms of EOs when used as an antibacterial agent against KPC-K. pneumoniae could lead to improved clinical efficacy. According to Yang SK, et al., the proteomic perspective was investigated toward KPC-K. pneumoniae cells when comparing the untreated cells with cells treated with cinnamon essential oil (Cinnamomumverum J. Presl) (CBO) [16]. The authors reported that cells exposed to CBO express a higher level of oxidative stress tht eventually tears the bacterial membrane, possibly by interacting with the lipid bilayer.

Interestingly, different repair membrane pathways were affected by oxidative stress and hence contributed to cell viability loss. Iseppi R, et al. investigated the antibacterial activity of Cinnamomum cassia L [19]. (CCeo) isolated and in combination with polymyxin B (a comercial antibiotic) against carbapenemase-producing Klebsiella pneumoniae and Serratiamarcenscens. The association between CCeo and polymyxin B showed a reduced number of viable cells in a 4-hour treatment. When associated with polymyxin B, a synergistic effect was displayed, and the bacterial growth was rapidly inhibited, demonstrating that the CCeo is a promising candidate in the development of an alternative method for carbapenem-producing strains. Cinnamomum verum essential oil has been used as an alternative treatment for different diseases. Wijesinghe GK et al. showed the antibacterial and antibiofilm properties of essential oil leaves from Cinnamomum verum against, K.pneumoniae, P. aeruginosa, and S.aureus [24]. All tested strains displayed sensitivity towards cinnamon oil steamer. Cinnamaldehyde are one of the major compounds in cinnamon EO, and its concentration ranges between 60 and 80% when obtained from the bark of the Cinnamomum genus [25]. Different studies demonstrate the antimicrobial activity of cinnamaldehyde against different bacteria, including K. pneumoniae, L. monocytogenes, P. aeruginosa, E. coli, E.faecalis, S. aureus, S. epidermis,V. parahemolyticus and Salmonella spp. [25].

Dhara L, et al. tested the cinnamaldehyde and eugenol antibacterial activity against Enterobacteriaceae ESBL quinolone-resistant (QR) strains along with their in vivo toxicity in a murine pharmacological model [26]. In this study, the authors observed the high efficiency of cinnamaldehyde and eugenol as promising antibacterials with no considerable toxicological and behavioral effects. Anandhi P, et al. add that wound infection is a great site for pathogenic microorganisms [27]. Cinnamon and clove oil are effective antibacterial agents due to their ability to reduce virulence and pathogenicity in a wound healing invivo model against K. pneumoniae, P. aeruginosa, and E.coli.

The ethanolic extract (CEE, 0,01-1 mg/plate), essential oil (CNO, 0.125-1 mg/plate) from cinnamon bark, and cinnamaldehyde (CLD, 0,125-1 mg/plate) were eficiente against different pathogens, such as K.pneumoniae, E. coli, S. aureus, Proteus vulgaris, and S.typhimurium (S. typhimurium) in an agar diffusion protocol. CEE and CLD showed promising antimutagenic and antimicrobial properties, respectively. Therefore, their properties should be explored in depth in an in vivo condition.

Aromatic herbs such as oregano (Origanumvulgare), sage (Salvia officinalis), and thyme (Thymusvulgaris) is used for ornamental, culinary, and phytotherapy purposes all over the world. The antibacterial activity of these plants has attracted the scientific community's interest as a potential alternative to conventional antibiotics in addressing microorganism resistance. There has been a tremendous increase in the growth of several aromatic and therapeutic plants in recent years. Fournomiti M, et al. showed that K. pneumoniae displayed a higher sensitivity towards thyme oil, followed by oregano, whilst sage oil did not present any antibacterial activity [13].

Souza ERL, et al. tested the hybrid Lavandula essential oil against K. pneumoniae strains, in which the authors observed a moderate antibacterial activity along with a bacteriostatic property [28]. These results were in agreement with Kozics K, et al., which confirmed the strong antibacterial activity of oregano (Origanum vulgare), sage (Salviaofficinalis), thyme (Thymus vulgaris), arbovitae (Thujaplicata), Cassia (Cinnamomum cassia), lemongrass (Cymbopogonflexuosus) against P. aeruginosa, P. vulgaris,C. koseri and K. pneumonia [15]. The enzyme extended-spectrum beta-lactamase (ESBL) is synthesized by the Enterobacteriaceae family, which includes K. pneumoniae and E. coli. Penicillin, cephalosporin, cephamycin, and carbapenem are among the beta-lactam antibiotics that the ESBL can hydrolyze. The bacteria that produce ESBLs spread predominantly from ranches to slaughterhouses and human meals derived from animals.

Ginting EV, et al. discovered that Syzygiumaromaticum (clove) and Cinnamomum verum (cinnamon) have effective antibacterial action against resistant K. pneumoniae and E.coli ESBL-strains [21]. Iseppi R, et al. analyzed the antibacterial effects of four different EOs, Melaleuca alternifolia, Eucalyptus globulus, Menthapiperita, and Thymus vulgari as an alternate method to prevent the progress and dissemination of K. pneumoniae and E.coli ESBL-strains, Metallo beta-lactamase (MBL)-producing P. aeruginosa and KPC-producing K. pneumoniae.M. alternifolia and T. vulgaris EOs presented the most effective antibacterial activity in all tested strains, with M.alternifolia as the best candidate even at low concentrations [18].

The antibacterial activity of oregano (Origanumsyriacum L.), thyme (Thymus syriacus Boiss.), cinnamon (Cinnamomum zeylanicum L), and clove (Syzygium aromaticum) EOs were the most effective among 28 different EOs in a study performed by Al-Mariri A, et al. against four different Gram negative strains, K. pneumoniae, E. coli O157:H7, Yersinia enterocolitic O9 (Y.enterocolitica O9) and Proteus spp [29].

The authors compared their efficiency with that of cephalosporin and ciprofloxacin, two well-established antibiotics that were effective. The discussed studies demonstrate promising antibacterial activity against K. pneumoniae, especially for its resistance to conventional treatments. As a result, it is necessary to discover new alternative methods to control the spread of antimicrobial-resistant strains.

Elements of biofilm formation in a hospital environment

One of the causes for researching bacterial biofilm formation in medical devices, as evidenced by the papers chosen for this study, is that they are responsible for a high infection rate, accounting for roughly 80% of human illnesses.

Bacterial resistance has been a major factor in the high rate of hospital infections worldwide [30, 31]. There has been an increase in the mortality rate, demonstrating the seriousness of biofilm formation and the need for a clean hospital environment, contributing to the reduction of health costs and also patient quality of life.

This the investigation is essential considering that the microorganism's ability to develop a biofilm is directly proportional to its aptitude to cause na infection. Research conducted by Ferreira ACB, et al. showed the risk of infection in catheters in hemodialysis patients in Brazil [30]. Several other studies also demonstrated a high number of infections in patients with catheters and prosthetic devices [31-37].

Biofilm formation is a prokaryotic adaptation used by several microorganisms, called resistant microorganisms, that have developed a growth strategy to overcome antibiotics and vaccines. These evolutionary steps allow the survival of organisms in hostile environments and the colonization of new sites through dispersion mechanisms [23]. As a result, these microorganisms acquire strong intercellular communication and can rapidly respond to subtle changes in their surroundings through a signaling mechanism called quórumsensing [38].

Among the several microorganisms that can produce biofilm are K. pneumoniae, P. aeruginosa, E.coli, S. aureus, S. epidermidis, and C. albicans. K. pneumonia is a gram-negative bacteria and an important opportunistic pathogen that can cause several health problems, including tract infections, bacteremia, pneumonia, and hepatic abscesses. There is an alarming situation regarding multi-resistant (MDR) and hypervirulent (hvKP) K. pneumoniae strains, whose mechanisms are not yet fully understood. Therefore, it is necessary to elucidate the pathogenic and resistance pathways of this microorganism [39].

Bergamo G, et al. discussed that microorganism resistance is related to poor penetration from antimicrobial agents to the polysaccharide membrane in which the bacteria develop the biofilm [23]. Assays that can evaluate both the action and properties of antimicrobials against biofilms are of great value the minimizing infection rates. Several other factors can influence biofilm formation, such as altered bacterial growth and gene expression [41-43].

The well-ordained tridimensional biofilm structure is a result of different molecular associations compared to Other non-sessile microorganisms. Among the molecules expressed on the biofilm surface that allow substrate adhesion and, consequently, an elaborated structure are fibrinogen-binding proteins, fibronectin, capsular polysaccharide-adhesion factors, autolysin, and teichoic acid. This explains the antibiotic selectivity towards bacterial strains and the lack of capacity to eliminate biofilm formation from resistant microorganisms. In addition, virulence factors and nutrient optimization contribute to higher antibiotic resistance [41].

During biofilm formation, the microorganisms can be found in a progressive accumulation phase of virulence factors, increasing the risk of infection in patients. As a result, research on biofilm formation in a medical environment must be kept up to date for academic and scientific communities [38, 40].

Biofilm formation in hospital catheters

Biofilm formation in hospital catheters is a serious worldwide health problem because it is responsible for triggering several infectious diseases [44]. Figure 2 shows different catheters and probes used in hospitals daily.

Figure 2: Catheters and probes that are used daily in hospitals

Contamination of urinary catheters, for example, can result in urinary infections [44]. Likewise, a central venous catheter can also present an infection. This demonstrates the subject's relevance to ensuring a high-quality service from hospitals and providing a better quality of life for patients [44,45,46].

Viega PIM highlights that catheter-associated urinary infections are common in patients with long urinary catheterization periods [47]. The factors related to biofilm formation are crucial for choosing the best intervention strategy to minimize the risk of infection. Furthermore, contamination of a central venous cateter is very concerning because microorganisms can circulate throughout the entire organism, exacerbating an already severe infection [44-46].

Central venous catheters are widely used in hospitals because they provide conveniente access to a patient's vascular system. As a result, it allows for the administration of drugs and other vital components, particularly in hospital circumstances [48].

On the contrary, urinary infections are responsible for high morbidity and mortality rates [40]. One of the main causes of infections in hemodialysis patients is material manipulation by health professionals [30,49]. The use of urinary catheters is essential in patients where access to the arteriovenous fistulae is not possible and, or access to the blood needs to be fast [49].

The use of urinary catheters is associated with a high infection rate as a result of biofilm formation (Figure 3), restricting the benefits that they could provide for patients [49,50]. On catheter surfaces, the most reported microorganisms are S. aureus, S.epidermidis, P. aeruginosa, K. pneumoniae, and C.albicans [49,51].