Staphylococcus aureus in broiler chickens and its virulence and resistance factors

Saad EAK, Garamoun*; Nahed AES, Naem* and Ahlam E, Yonis**

*Bacteriology Department, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute.

**Biotechnology Department, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute.

Agriculture Research Center (ARC), Giza, Egypt

Corresponding author: Saad Garamoun Email: saad.kotb@yahoo.com

Keywords: Staphylococcus aureus, enterotoxins, virulence factors, and food poisoning.

Abstract:

Staphylococcus aureus is the main cause of poultry Staphylococcosis, it is widely spread in the poultry farm environment, including the air, water, sewage, and dust, which usually contain it and they are significant nosocomial pathogens and members of the opportunistic bacteria. Additionally, Staphylococcal food poisoning is considered one of the most prevalent food-borne illnesses in the world. It is caused by enterotoxigenic strains of S. aureus in food. Broiler chickens may suffer from S. aureus infection and its enterotoxins resulting in a major public health hazard and economic losses due to decreased weight gain, mortality, and increased condemnation, so this review aims to summarize and address current knowledge about diseases caused by S. aureus in broiler chickens together with understanding the significant virulence and resistance factors which may help in control its occurrence in animal farms and to understand food poising in human.

Introduction:

Staphylococcus aureus is a Gram-positive, non-motile, non-spore-forming, catalase-positive, coccoid bacteria that appears in grape-like clusters in stained smears. It is considered an opportunistic commensal organism of animals and the most pathogenic species of the genus Staphylococcus (Quinn and Markey. 2003). It normally occurs on the skin and internal organs and is commonly associated with infection of skin, bones, joints, and nerves.  A key factor in the development of various avian illnesses, including omphalitis, arthritis, Staphylococcal septicemia, synovitis, and yolk sac infections (Smyth and McNamee. 2001).

Transmission occurs via skin wounds, minor surgical procedures (like beak, toe, or comb trimming), vaccine injection, and compromised intestinal mucosa can introduce Staphylococcus to local tissue or into the bloodstream. Infection can also occur in the hatchery as a result of contamination of an open navel. Once in the bloodstream, Staphylococcus can produce systemic disease or localized lesions in tissues. Staphylococcus aureus can invade the metaphyseal area of joints, leading to arthritis and osteomyelitis (Yuko Sato  and Mohamed El-Gazzar.  2022).

The isolation sites of  S. aureus were mostly from the proximal femur, proximal tibiotarsus, tendon sheaths, hock joints, pododermatitis lesions, heart, and liver   (Andreasen. 2020).

There is a significant death rate in broilers with swollen joints, gaseous exudates, damaged cartilage, and thickened synovial membranes with inflammatory cell infiltration (Gu et al., 2013).

Most Staphylococcus species are pathogenic and capable of producing toxins that result in health problems for both humans and animals, among them S. aureus produces the most toxins (Fetsch and  Johler. 2018).

Enterotoxin-producing S. aureus is the most common cause of food-borne human illness throughout the world (Do Carmo et al., 2004).

Prevalence of S. aureus in chickens:

Live birds are considered a significant reservoir for S. aureus strains from apparently healthy and diseased chickens (Mamza et al., 2010). Also (Suleiman et al., 2013) found coagulase-positive S. aureus in 54% of apparently healthy chicken samples. Ali et al., (2017) found that the isolation rate of  S. aureus from broilers was 90% of nasal and cloacal swabs. Benrabia et al., (2020 (reported that the prevalence of S. aureus from nasal swab samples in broilers was 48.4%. Moreover (Abd El-Tawab et al., 2017 and Amen et al., 2019) recovered S. aureus from 66% and 74.07% of the tested broiler chickens, respectively.

Virulence and Resistance Factors:

Staphylococcus aureus strains can grow between 15 and 45 °C in the presence of sodium chloride concentrations 10% (Behling et al., 2010). Staphylococci have a wide range of virulence factors (Wright and Nair. 2010).

Staphylococci produce a variety of toxins and exoenzymes that can harm host tissues and interfere with the immune system (Gordon and Lowy. 2008). Several virulence factors are produced: (1) Surface proteins that aid in tissue colonization. (2) Invasion-related bacterial spread in tissues (hyaluronidase, kinases and leukocidin). (3) Surface components (capsule, Protein A) that inhibit phagocytic engulfment. (4) Catalase synthesis and carotenoids are two biochemical traits that help phagocytes survive. (5) Immunological obscures (Protein A, coagulase, coagulation factor). (6) Membrane-damaging toxins (hemolysins, leukotoxin, leukocidin) that lyse eukaryotic cell membranes. (7) Exotoxins (SEA-G, TSST, and ET) that cause host tissue destruction or other illness signs. (8) Both inherited and acquired antimicrobial agent resistance (Toder. 2005).

Another classification according to Diep and Otto. (2008) who reported that S. aureus virulence genes can be divided into two groups: group coding for exotoxins (secreted) and group coding for cell-surface-associated (adhesion).

A superfamily of about 23 low-molecular-weight pyrogenic exotoxins with similar structural and functional characteristics is known as Staphylococcal enterotoxins (SEs). According to their capacity to cause emesis, Staphylococcal enterotoxins can be divided into two groups: classical SEs (A to E) and newly confirmed enterotoxigenic-like proteins. By activating T cells and causing them to produce inflammatory cytokines, Staphylococcal enterotoxins have strong super antigenic activity and compromise adaptive immunity (Fisher et al., 2018). According to Argudin et al., (2010), SEs and SEIs can be further categorized into classic (SEA to SEE) and new (SEG to SEIU2).

Important cell-surface proteins on S. aureus play a role in the bacterium's pathogenicity and ability to adhere to host cells. These proteins include clumping factors A and B (clfA and clfB genes) and an elastin-binding protein (Momtaz  et al., 2013). Clumping factor A (CFA) is a surface protein of S. aureus that binds to fibrinogen and acts as a virulence factor in certain infections by blocking phagocytosis and enhancing adherence to fibrin and fibrinogen (Higgins et al., 2006).

One of Staphylococcus' most potent pathogenic genes, is hemolysin (hlg gene). Gundogan et al., (2013) reported it as a crucial virulence factor that has a strong toxic effect on lymphocytes, macrophages, neutrophils, epithelial cells, fibroblasts, and other cell lineages, which is responsible for facilitating the formation of pores in red blood cells following the binding of the active proteins hlgA and hlgB.

The protein A gene, known as spa, is primarily employed to type S.aureus. Another illustration of a gene is the coagulase (coa) gene, which is a virulence gene of S. aureus considered significant because it forms an alliance with other genes that allows it to persist inside host cells and invade immune system cells in the host (Balaban and Rasooly. 2000). It is also a virulence marker of Staphylococcus which aids in the formation of fibrin around Staphylococcal abscesses, which promotes localized infection and guards against phagocytosis (Sawai et al., 1997).

In most cases, virulence genes are linked to Staphyloccocal infections. The initial stages of the infections are believed to include bacterial adherence. clf, fnb A, and a can according to Arciola et al., (2005), Staphylococcal adhesions A were the most significant.

Staphylococcus aureus strains that have the bla gene produce β-Lactamase enzyme which deactivates β-lactam antibiotics through cleavage with the β-lactam ring  (Kiliç and Çirak. 2006). Staphylococcus aureus strains have resistance to β-Lactam antimicrobials, including cephalosporins and carbapenems (penicillin, oxacillin, cloxacillin, methicillin, flucloxacillin, and dicloxacillin) antibiotics due to a plasmid-encoded penicillinase/β-lactamase (Watkins et al., 2019).

Delorme et al.,(2009) reported resistance of S. aureus isolates to other classes of antimicrobials, such as fluoroquinolones, aminoglycosides, tetracyclines, and macrolides especially those from broiler chicken origin, while 100% resistance to tetracycline, penicillin and erythromycin was reported by  Otalu et al., )2011)  of S. aureus isolates from live and slaughtered chickens.

Twenty different types of Staphylococcal enterotoxin were found including SEA through SEE, SEG through SER, and SEU. But only a small number of the Staphylococcal enterotoxin serotypes are regularly linked to outbreaks of food poisoning, since S. aureus does not produce enterotoxins in all strains, the enterotoxin genes are accessory genetic components. They are coded by plasmids, phages, and pathogenicity islands, among other mobile genetic components (Martin et al., 2004).

Mahmoud  et al.,  (2018)  found that S. aureus enterotoxins from chicken samples were (12.5%)  type A, (6.25%)  was type C, (6.25%) type A with B and in finally type B together with D was (6.25%) also they reported that 68.75% of the isolates without any enterotoxins secretion, while results obtained by Harvey et al., )1982) was different than enterotoxin type A was (2.74%), enterotoxin type D was (14.81%) and Staphylococcal enterotoxin C+D was (1.23%),  but Britta et al., )2016) found that enterotoxin type A was  (3.1%) and  (25%) for each of the enterotoxins types  G, I, M, N, O, and U.

Methicillin-resistant S. aureus (MRSA) is recognized as one of the most important kinds of resistant S. aureus since it is the cause of acquired infections linked to a high risk of bacterial death globally (Tiemersma et al., 2004).

Staphylococcus aureus Enterotoxins (SEs):

SEs are 20–30 kDa released toxins that disrupt intestinal activity and induce Staphylococcal food poisoning (SFP), which is characterized by nausea, vomiting, abdominal pain, and diarrhea without indications of toxic effects, such as fever or hypotension, based on antigenic heterogeneity, more than 20 SEs (SEA—SElV) have been determined (Hennekinne et al., 2012). Clinical indications of SFP have been connected to inflammatory mediators including leukotriene B4 and prostaglandin E2, both of which are produced in response to SEs, even though the receptors implicated in the emetic response to SEs have not yet been identified (Pezato et al., 2012).

Staphylococcal enterotoxin B (SEB) is associated with food poisoning, it has been studied for potential use as an inhaled biological weapon (Pinchuk et al., 2010).

S. aureus Toxic Shock Syndrome Toxin 1 (TSST-1):

Unlike SEs, TSST-1 (22-kD) induces the production of a significant amount of pro-inflammatory cytokines from the host T-cells and macrophages but does not cause emesis (Stach et al., 2014). Toxic shock syndrome (TSS) symptoms such as high temperature, rash, desquamation, hypotension, and hypovolemic shock are brought on by this cytokine outburst, and they can lead to multiorgan failure (McCormick et al., 2001).

S. aureus in humans:

S. aureus can produce as many as 25 different toxins causing severe food poisoning, toxins of S. aureus that present in human food after ingestion will be absorbed into the blood from the digestive tract, resulting in nausea, emesis, abdominal cramps, and diarrhea (Ortega et al., 2010). Symptoms varied according to individual susceptibility and the amount of enterotoxin ingested (Do carmo et al., 2004). Sometimes, severe symptoms occur which require hospitalization and can end in death in some cases (Martin et al., 2004). The concentration of S. aureus ranges from 10⁶ to 10⁸CFU/g in ingested food samples needed to induce food poisoning, and for sensitive persons even 10⁵ CFU/g can produce enough amount SEs (around 1µ /g) to cause symptoms of food poisoning (Alarcon et al., 2006). According to Nagarajappa et al., (2012), SEs are highly thermostable; heat treatment, such as regular cooking, cannot completely inactivate them. They therefore resist thermal treatment and result in food poisoning. Outbreaks when the offending food had previously undergone heat treatment were brought on by enterotoxin in food (Asao et al., 2003). Some of the characteristics of SEs that lead to food poisoning include super antigenic activity, stimulation of T-cell proliferation, enhancement of endotoxic shock, suppression of immunoglobulin synthesis, and pyrogenicity (Le loir et al., 2003).

Recent reports of S. aureus food-borne illness outbreaks have been mentioned by  (Le et al., 2021). The presence of thermostable enterotoxins of S. aureus in contaminated chicken meat products may cause Staphylococcal food poisoning in humans (Balaban and Rasooly. 2000). According to Larsen et al., (2000), infections such as gastroenteritis, heat shock-like syndrome, skin infections, lung infections, urinary tract infections, and immune-mediated disorders might occur due to enterotoxins of S. aureus. Also (Fisher et al., 2018) reported that  Staphylococcal food poisoning (SFP) is associated with toxic shock syndrome (TSS), sepsis-related illnesses, and pneumonia.

Prevention and control:

According to Wideman et al., (2015) and Andreasen. (2020), therapeutic antimicrobial administrations can lower the prevalence of Staphylococcal infections, but (McNamee and Smyth. 2000) found that antimicrobial therapy is not appropriate to be a long-term solution to the issue due to the large number of Staphylococci strains that exhibit resistance to a variety of antimicrobial agents. In addition to complicating matters sick and lame birds frequently experience depression, exhibit a lack of appetite, and have trouble accessing food and water. They are less likely to receive the prescribed medication. However, bumblefoot (pododermatitis) caused by Staphylococcus infection when treated with antibiotics frequently leads to clinical improvement in the diseased birds, particularly when coupled with bettering the rearing environment. Birds with bumble foot have been successfully treated with levofloxacin (Youssef et al., 2019) and minocycline (Satterfield and O'Rourke. 1981).

Vaccination against S. aureus has been developed over many years, with some encouraging outcomes, for both humans and livestock. But to date, no such vaccination is available (Chang et al., 2008; Proctor. 2012, Miller et al., 2020). Despite continued work throughout the previous years till now no licensed vaccines against Staphylococcosis in poultry (Kaul et al.,  2001 and El-Maghraby et al.,  2020). The effectiveness of the S. aureus vaccine against one disease may be lost against another since Staphylococci cause a variety of illness forms. Also, the development of vaccines is considered a challenge due to the genetic diversity of S. aureus isolates and the presence of virulence factors.

Antibiotic alternatives such as probiotics prevent Staphylococcal infections by reducing the amount of Staphylococci that enter the bloodstream through the intestinal epithelium (Wideman et al., 2015).

Conclusions:

Management of Staphylococcal infections is challenging. Staphylococci are widespread in the environment of chicken farms causing different diseases such as omphalitis, arthritis, septicemia, and synovitis. Also, Staphylococcal food poisoning (SFP) is a common illness that results from the presence of different types of enterotoxins  (for example: type A, B, C, D, and E)  which are widely affected by several factors including misdiagnosis, underreported mild outbreaks, incorrect sample collection, and improper laboratory investigations leading to social and economic importance. The presence of a wide range of virulence and resistance factors together with antibiotic resistance was reported for S. aureus strains. Access to genome-based technologies like using PCR to identify these factors has great importance to control the infection and minimize its economic effect.