Biofilm formation, multidrug-resistance and clinical infections of Staphylococcus haemolyticus: A brief review

Coagulase-negative staphylococci (CoNS) have been associated with a range of human health issues such as medical device-related infection, localized skin infection, or direct infection, are recognized as comprising the main part of human normal microbiota and associated with severe and intensive infections, causing infections in humans, especially immunocompromised patients and neonates. S. haemolyticus is, after Staphylococcus epidermidis , the second most frequently isolated CoNS from clinical cases, notably from blood infections, including sepsis. The most important factor might be the ability to acquire multiresistance against available antimicrobial agents, even glycopeptides. It is widespread in hospitals and among medical staff, resulting in being an emerging microbe causing nosocomial infections. This review discuss aspects of S. haemolyticus bloodstream infections associated, virulence factors, and the ability of biofilm formation on medical devices surfaces. The great adaptability and the ability to survive in the hospital environment, especially on medical devices, S. haemolyticus becomes a crucial factor in nosocomial infections caused by multiresistant strains. carbapenems and monobactams. Methicillin resistance in staphylococci is because of the expression of a modified penicillin-binding protein, PBP2a, which is encoded by mec A or mec C genes. Research showed that most


Introduction
Staphylococcus haemolyticus is one of the most frequently coagulase negative staphylococci (CoNS) isolated from healthcare-associated infections, mainly those related to implanted medical devices. CoNS are considered as inhabitants of the human and animal microbiota, have been increasing reported as relevant nosocomial pathogens, especially due to microbial biodiversity and virulence mechanisms, involving resistance to antimicrobial agents (Heilmann et al, 2019;Al-Tamimi et al, 2020;Wolden et al, 2020). It is an increasing cause of nosocomial infections associated with indwelling medical devices, particularly affecting immunocompromised patients and neonates (Pain et al, 2019).
In recent years, CoNS has been reported as pathogenic causes of infections in human and veterinary medicine, especially in immunocompromised patients, critically ill, long-term hospitalized and in those harboring invasive medical devices such as catheters (Pereira-Ribeiro, et al 2019;Eltwisy et al, 2020). Clinicians and microbiologists are frequently confronted with determining whether retrieved CoNS are contaminants that intrude during sampling or sample processing, are regular skin or mucous membrane commensals, or are clinically relevant, because many CoNS species form part of the skin and mucous membrane microbiota, the line between innocuousness and pathogenicity may be indistinct and comes down to virulence strategies employed by the various species as well as host defense mechanisms. Although, today the CoNS are recognized as causative agents of clinically significant infections (Becker et al, 2014;Sued-Karam & Pereira-Ribeiro, 2022).
The advancement of diagnostic techniques has increased to understanding of the molecular mechanisms of pathogenicity. The identification is important to distinguish between coagulase-positive staphylococci and CoNS as a basic principle, a practice carried out in most laboratories by the slide and tube coagulase tests. Identification down to species level is important particularly for species suspected to cause infections and informs accurate clinical decision making by clinicians.
Therefore, the identification of CoNS species is important in detecting exact of the causes of infection in environment nosocomial and fast identification methods are required for effective diagnoses and prompt treatment (Bouchami et al, 2011;Asante et al, 2020). Speciation with biochemical methods is gradually being replaced by molecular methods such as polymerase chain reaction (PCR) and spectrometric methods, such as matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Molecular methods such as PCR affords higher accuracy, typeability, and reproducibility and are therefore recommended in CoNS speciation. MALDI-TOF MS as an identification technique also has a useful application in toxin detection and has been shown to exhibit high accuracy and reproducibility when used to identify CoNS. Furthermore, MALDI-TOF has been reported as unable to detect rare species in close to 50% of cases (Bouchami et al, 2011;Becker et al, 2014;Pereira-Ribeiro, et al, 2019).
In several cases where rare species are suspected, sequencing of phylogenetically important target genes such as the 16S rRNA gene can prove useful. Certainly, amplification and sequencing of target housekeeping genes such as elongation factor Tu (tuf) gene, RNA polymerase B (rpoB), and 16S rDNA gene have been shown to assist in discriminating between Staphylococcus species and subspecies with varying levels of discriminatory power. The multiple approach to pathogen identification improves the accuracy of identification up to species level, such as Staphylococcus haemolyticus that has high resistance to antimicrobials, thus helping the clinician in a more effective treatment (Lamers, et al, 2012, Asante et al, 2020.
This review aims to briefly discuss aspects of S. haemolyticus bloodstream infections associated, their clinical relevance and virulence factors, as well as the ability of biofilm formation on medical devices surfaces.

Methodology
Articles were consulted to identify studies relevant of the literature review in the period of march to July of 2022, utilizing articles from the last 10 years for its writing, although articles most relevant in the last 5 years. Articles in Portuguese and English were included in this study. Databases were search from Scielo and Pubmed, Search terms included: "coagulasenegative Staphylococcus", "Staphylococcus haemolyticus", Staphylococcus biofilm and resistance", "Staphylococcus haemolyticus biofilm-formation" and "Staphylococcus haemolyticus multiresistance". The Table 1 shows the number of articles included and excluded for this study and the term used to search for articles. Research, Society and Development, v. 11, n. 11, e228111133605, 2022 (CC BY 4.

Staphylococcus haemolyticus
Staphylococcus haemolyticus is one of the CoNS that inhabit the skin as a commensal. Studies of the skin metagenome have propped the long-known assertion that Staphylococcus species prefer areas of higher humidity such as the plantar foot region, axillae, and umbilicus (Asante et al, 2020). It is increasingly implicated in opportunistic infections in S. haemolyticus has also been seen in skin and soft tissue infections especially associated with hidradenitis suppurativa lesions (HSL). The HSL is a chronic, recurrent, and debilitating skin disease results from an inflammatory disorder of the follicular epithelium, associated or not with a bacterial infection (Lee et al, 2017). The hypothesis of the association of S.
haemolyticus and HSL bacterial infection may be attributed to the higher colonization of S. haemolyticus in the axillary and inguinal regions with a higher concentration of hair follicles and associated apocrine glands (Hessam, 2016). Most infections caused by this group are characterized by subacute and chronic courses of infection with subtle clinical symptoms. However, more aggressive courses and sudden lethal outcomes in the case of inadequately managed patients with chronic foreign body related infections (FBRIs) have been reported and are associated with significantly longer hospital and intensive care unit stays, higher mortality rates and higher hospital costs. Delayed diagnosis of CoNS infections contributes to increased mortality.
Immunosuppression or an immature immune system represent further risk factors for foreign body-independent Staphylococci infections, including, preterm newborns are predisposed to invasive infections such as sepsis, endocarditis, meningitis and FBRIs, leading to prolonged hospital stay, morbidity and mortality (Horasan, et al, 2011;Pereira et al, 2014;Heilmann et al, 2019).
S. haemolyticus has a feature the ability to form biofilms, which play an essential role in the establishment of infections. The produced exopolysaccharides can inhibit the growth of other bacteria and decrease their ability to form biofilms. This species has gained an increased clinical significance due to its genome plasticity, which allowed a great adaptation and development of resistance to different antibiotics, including methicillin and its ability to survive in the hospital environment. The remarkable S. haemolyticus ability of to acquire antibiotic resistance, especially to oxacillin, limits the available therapeutic options for catheter-related infections caused by S. haemolyticus methicillin-resistant isolates and may predispose to sepsis and increase patient's morbidity and mortality (Pereira et al, 2014;Ahmed et al, 2019;Pereira-Ribeiro et al, 2019;Eltwisy et al, 2020).
Further than virulence factor acquisition, staphylococci have evolved additional mechanisms to sustain within the host and even to modulate the course of an infection. Similar to S. aureus, S. haemolyticus are able to switch from a more aggressive style of infection to an intracellularly adapted lifestyle, which causes a diminished inflammatory response and facilitates chronic and relapsing infections (Tuchscherr et al 2010).

Staphylococcus haemolyticus multidrug-resistant
The increasing number of S. haemolyticus in hospital-acquired infections (HAIs) is closely related to their antimicrobial resistance and the ability to survive in a hospital environment. Among all the CoNS studied, this specie is known to be resistant to most antibiotics, including cephalosporins, penicillins, macrolides, tetracyclines, quinolones, and aminoglycosides (Manoharan et al, 2020).
It is well known that the mechanism of methicillin resistance determines the resistance to all β-lactam antibiotics: penicillin, cephalosporins, carbapenems and monobactams. Methicillin resistance in staphylococci is because of the expression of a modified penicillin-binding protein, PBP2a, which is encoded by mecA or mecC genes. Research showed that most isolates are cefoxitin resistant and also some strains are negative for the mecA gene, wherein resistance might have occurred due to other mechanisms such as mecC or hyperproduction of penicillinase, although methicillin-resistant S. haemolyticus isolates to be negative for the mecA gene. Analysis of mecA gene sequences in GenBank reference strains of S. aureus, S.
Horizontal gene transfer is a major source for acquisition of antibiotic resistance genes in staphylococci, facilitated by phage transduction and plasmid conjugation. Plasmids can be reservoirs and vectors for antibiotic resistance and virulence, even in the absence of selective pressure. Horizontal transfer of plasmids can occur not only between staphylococcal strains of the same species but also between species, with S. haemolyticus believed to act as reservoirs for antibiotic resistance genes (Mores et al, 2021).
The growing of infections for MDR bacteria strains, associated with the lack of new antimicrobial development is a global concern. It is stipulated that by 2050 antimicrobial resistance will be responsible for a global economic impact of $ 100 The common antibiotics used for the treatment of the infections caused by CoNS are penicillin, oxacillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and vancomycin. Linezolid is an oxazolidinone group of antibiotics with activity against Gram-positive bacteria. It is used for the treatment of serious infections caused by Gram-positive bacteria resistant to other antibiotics, Linezolid is approved as an alternative drug to be given for catheter-related bloodstream infections. In earlier studies, linezolid-resistant staphylococci have been reported increasingly all over the world (Grupta et al, 2020). Several potential resistance genes were found in S. haemolyticus genome, such as norA, norB, van, cfr, lmrB (efflux pumps), blaZ (β-lactamase), fusB (fusidic acid resistance EF-G-binding protein), ermA (rRNA adenine N-6methyltransferase) and AAC(6')-Ie-APH(2'')-Ia (bifunctional aminoglycoside N-acetyltransferase and aminoglycoside phosphotransferase) and most of the identified antibiotic resistance genes were associated with transposon elements, suggesting that the genes can be spread to other strains or species (Kim;Jang, 2017).
The glycopeptides resistance was reported since 1990 and has been considered an emergent problem. The teicoplanin resistance has become more common reported than vancomycin resistance in CoNS strains. This resistance is attributed to teicoplanin resistant operon (tcaRAB) formed by tcaR, tcaA and tcaB genes; the deletion or mutation of the genomic region of tcaRAB locus could promote to increase the level of teicoplanin resistance. Some S. haemolyticus strains present the capacity to inactive tcaA gene increasing the teicoplanin resistance (Veach et al, 1990;Bakthavatchalam et al, 2017).
S. haemolyticus infections are often difficult to treat because of MDR and different pathogenicity. The resistance to oxacillin and aminoglycosides in this specie have been proposed as surrogate markers for invasiveness, while the absence of these traits indicates a commensal flora. Vancomycin has been considered the antibiotic of first choice in treating severe infections caused by methicillin-resistant CoNS. However, the increased use of vancomycin has resulted in the development of vancomycin heteroresistance in this specie, due to multiple and complex molecular mechanisms, phenotype instability, variable vancomycin selective pressure and the lack of specific genetic markers for reliable detection. Vancomycin heteroresistant strains causing bloodstream infections is a growing and unrecognized clinical concern in intensive care patients and its clinical impact are well studied in S. aureus and has also been reported in S. haemolyticus, frequently associated with catheter-related bloodstream infections (Szabo et al, 2009;Bathavatchalam et al, 2021).

Adherence and/or biofilm formation in medical devices
Staphylococcus haemolyticus is one of the most frequently CoNS isolated from healthcare-associated infections, mainly those related to implanted medical devices, whose the colonization and infections by resistant strains are associated to contamination during the placement of medical devices in hospital environments and breaking of aseptic barriers, causing morbidity and mortality in patients. The ability to adhere and colonize implanted biomaterials in addition to biofilm formation Biofilm-associated infections are persistent and difficult to treat. The biofilms represent a perfect structure to protect the microorganisms to the host immune response, the action of antibiotics, and to contributing to environmental agents. The steps of biofilm formation start with the attachment of bacteria to the biotic or abiotic surface, following to the proliferation and expression of intercellular adhesion traits leading to the formation of multilayered cell clusters; maturation of the biofilm and dissolution and dissemination with the blood stream leading to infection in other regions on the body, where after initial attachment of staphylococci, they multiply and grow into multilayered cell clusters requiring intercellular adhesion.
Intercellular adhesion is mediated by biopolymers, such as polysaccharides and proteins (Figure 1) (Heilmann et al, 2019;Pereira-Ribeiro et al, 2022). Biofilm formation also plays a role in the transmission of infection by S. haemolyticus, that is the second most frequently isolated CoNS that colonize different parts of human bodies, causing serious infections in patients with implanted devices associated with nosocomial infections and related to biofilm formation and high levels of antimicrobial resistance. In most chronic infections, bacteria exist in biofilm communities rather than in a planktonic form to adapt in the direction of unfavorable conditions. A broad spectrum of microorganisms, including most pathogens, will form biofilm by attaching to biotic or abiotic sites to prolong their survival. Biofilms may be formed on abiotic surfaces of medical devices or on biotic surfaces, such as host factor coated foreign material or host tissue. Within the biofilm, bacteria are protected against the patients' immune system and antibiotic therapy, thus explaining why biofilm-associated infections are extremely difficult to eradicate (Costerton et al, 2005, Pereira et al, 2014Feßler et al, 2017).
S. haemolyticus has a potential to adhere and form biofilms on tissues, implants and catheters. A biofilm can be produced by a single species or by multiple species, including fungi, bacteria and virus, embedded in a secreted extracellular matrix. This complex biofilm matrix acts as a protective layer allowing bacteria to be more resistant to antibiotics and disinfectants (Chandra et al, 2001;Pais-Correia et al, 2010;Yong et al, 2019).
The contamination for CoNS in medical devices is a reality, especially in patients with the catheter use. Studies report this contamination as an emergent problem in hospital environments, due to a multi-drug resistance (MDR) profile of clinical samples isolated and low drug alternatives to antimicrobial therapy, however infectious diseases involving biofilms are difficult to treat (Rogers et al, 2009;Asaad et al, 2016;Argemi, et al 2019). Some of the mechanisms involved in the resistance of bacteria in biofilms to antimicrobials differ from the mechanisms responsible for the resistance of planktonic bacteria to the same antimicrobials. Biofilm resistance to antimicrobials is multi factorial and there are several resistance mechanisms that act together to provide increased biofilm resistance. Biofilm growth of staphylococci also increased horizontal transfer of plasmid-carried antibiotic resistance and heritable antibiotic resistance through spontaneous mutation (Savage et al, 2013;Yong et al, 2019).
As part of the early biofilm maturation process, the bacteria also start to produce extracellular polymeric substance (EPS), which eventually forms the biofilm matrix. EPS mainly consists of polysaccharides, proteins, teichoic acids, and eDNA.
In polysaccharide-dependent biofilms, the main EPS component is polysaccharide intercellular adhesin (PIA). The production of PIA is dependent of the intracellular adhesion operon (icaADBC). The ica locus, constituted by icaR (regulador/repressor) and icaADBC (biosynthetic) genes, is essential to biofilm formation and is induced by anaerobiosis through the staphylococcal respiratory response regulator (SrrAB) but they can also be regulated by other environmental factors, such as glucose, ethanol, osmolarity, temperature and some antimicrobials, such as tetracycline. These genes are responsible for polysaccharide intercellular adhesin (PIA) expression, also known as poly-Nacetylglucosamine (PNAG), essential for cell-cell adhesion.
Staphylococcus haemolyticus is commonly identified as one of the CoNS that takes virulence genes, such as, icaADBC, aap, atl, fbp, bap, fnb, sesB are involved in biofilm formation. Data showed a correlation with the presence of icaA, aap, atl and fbp genes colonizing abiotic surfaces that the process of biofilm formation is complex and may not be related to the icaA gene (Pereira-Ribeiro, 2019;Yong et al, 2019).
The adhesion on biotic surfaces is associated to express of a variety of surface proteins, being fibronectin-binding protein (FnBP) one of the most important. The capacity to express this protein is associated with the presence of fnbA and fnbB genes.
The presence of this protein is crucial to adhesion on biotic medical devices, tissues, invasion, and internalization on the host cell (Eltwisy et al, 2020).
A study demonstrated the correlation of the concomitant presence of resistance gene and high number of virulence genes associated to biofilm formation and adhesion surface protein in different clones of S. haemolyticus isolated from catheterrelated bloodstream infections in neonates and adults, suggesting the correlation of the biofilm production and adhesion profile with the antimicrobial resistance (Pereira-Ribeiro, 2019).

Conclusion
S. haemolyticus has a great ability to survive in the hospital environment, especially on medical devices, becomes a crucial factor in nosocomial infections caused by multiresistant staphylococci. This pathogen can exchange resistance genes and enhance the biofilm forming ability for spreading of infections. The important function of S. haemolyticus as reservoirs for antibiotic resistance and virulence traits is still an underestimated issue that requires more scientific attention. This may also have practical consequences for hospital hygiene by including resistant and virulent CoNS isolates into future infection control and surveillance measures. Further studies are, however, required to understand the genetic constitution of Staphylococcus haemolyticus in contributing to biofilm formation. More research is needed to clarify the mechanisms of resistance and biofilm formation of this specie, as well as the recognition of health professionals, especially in the correct hand washing, handling of medical devices and inappropriate use of antimicrobials.