Listeriosis Outbreak in South Africa: A Comparative Analysis with Previously Reported Cases Worldwide

Listeria species are Gram-positive, rod-shaped, facultative anaerobic bacteria, which do not produce endospores. The genus, Listeria, currently comprises 17 characterised species of which only two (L. monocytogenes and L. ivanovii) are known to be pathogenic to humans. Food products and related processing environments are commonly contaminated with pathogenic species. Outbreaks and sporadic cases of human infections resulted in considerable economic loss. South Africa witnessed the world’s largest listeriosis outbreak, characterised by a progressive increase in cases of the disease from January 2017 to July 2018. Of the 1060 laboratory-confirmed cases of listeriosis reported by the National Institute of Communicable Diseases (NICD), 216 deaths were recorded. Epidemiological investigations indicated that ready-to-eat processed meat products from a food production facility contaminated with L. monocytogenes was responsible for the outbreak. Multilocus sequence typing (MLST) revealed that a large proportion (91%) of the isolates from patients were sequence type 6 (ST6). Recent studies revealed a recurrent occurrence of small outbreaks of listeriosis with more severe side-effects in humans. This review provides a comparative analysis of a recently reported and most severe outbreak of listeriosis in South Africa, with those previously encountered in other countries worldwide. The review focuses on the transmission of the pathogen, clinical symptoms of the disease and its pathogenicity. The review also focuses on the major outbreaks of listeriosis reported in different parts of the world, sources of contamination, morbidity, and mortality rates as well as cost implications. Based on data generated during the outbreak of the disease in South Africa, listeriosis was added to the South African list of mandatory notifiable medical conditions. Surveillance systems were strengthened in the South African food chain in order to assist in preventing and facilitating early detection of both sporadic cases and outbreaks of infections caused by these pathogens in humans.


Introduction
Listeria was reported for the first time in 1924 by Murray and colleagues due to the sudden deaths of six young rabbits and was named Bacterium monocytogenes [1]. Subsequently, in 1927, Pirie named while diarrhoea and arthralgia are common in adults. The incubation period ranges from 6 hours to 10 days and the symptoms span from 1-3 days, although they can last for up to one week. Hospitalisation caused by gastroenteritis due to listeriosis is usually prominent in children and the elderly. Blood cultures from gastroenteritis patients may yield Listeria [52].

Listeriosis in Pregnancy
Listeriosis infection in pregnant women presents a serious side-effect because of its invasive nature. A previous study revealed the mean incubation period of listeriosis in pregnancy as 27.5 days, with a range of 17-67 days. Major side-effects include mild flu-like symptoms, with fever, backache, and headache, and some might lead to neonatal sepsis. Listeria infection in pregnancy usually occurs at the early stages, and the side-effects are observed during the third trimester. The disease often results in poorer neonatal outcomes. Serious side-effects associated with infections in pregnant women are spontaneous abortion, stillbirth, or preterm birth [53].

Neonatal Infections Due to L. monocytogenes
Listeria infection in neonates occurs through an endogenous transplacental transmission, inhalation of infected amniotic fluid, or following colonisation from the maternal gastrointestinal or vaginal carriage. A study revealed the isolation of Listeria pathogens from the genital tract and blood cultures of majority of mothers. The premature offspring experienced the onset of the disease within 36 hours, which represented transplacental neonatal infection. Neonates exhibited sepsis (90%), respiratory distress or pneumonia (40%), meningitis (25%) and, sometimes, with inflammatory granulomata (granulomatosis infantiseptica). The appearance of rash occurs with maculopapular or papulovesicular lesions on the trunk or extremities. Between 5-30 days postnatal period, the late-onset of the disease progresses and patients present the expansion of non-specific symptoms, sepsis and meningitis [54,55].

Bacteraemia Due to L. monocytogenes
Listeria monocytogenes can disseminate into the blood, leading to bacteraemia, one of the clinical presentations in some Listeria-infected persons. Bacteraemia due to Listeria might lead to gastroenteritis and, in most cases, associated with pregnancy or neonatal infection. Bacteremia in adults might be a clinical effect of HIV infection, chronic renal disease, steroid use, underlying malignancy, chemotherapy or age >65 years [56,57].

Meningitis Due to L. monocytogenes
Acute meningitis and encephalitis are considered to be some of the most severe symptoms of listeriosis. Other symptoms include rhomboencephalitis, which is the involvement of the midbrain, pons and/or cerebellum linked to cranial nerve involvement or cerebellar signs (ataxia, tremor), or the development of hemiparesis. The incubation period of meningitis is estimated to be 0-21 days, with an average of 10 days. Studies revealed that cases of neuroinvasive listeriosis, neck stiffness was found in 75% of cases, focal neurological signs in 30%, seizures in 30% and coma in 7%. Focal neurological symptoms comprised single or multiple cranial nerve involvement (most commonly the 6th and 7th cranial nerves), hemiparesis, ataxia, and aphasia [58,59].

Pathogenicity Factors and Virulence Potential
The pathogenicity of L. monocytogenes is due to the expression of different genes, which are responsible for its ability to penetrate, proliferate and spread in cells [60]. These capabilities are attributed to their inlAB internalisation locus, Listeria pathogenicity island-1 (LIPI-1), and hpt intracellular growth locus, respectively ( Figure 1) [49]. InlAB internalisation locus encodes two surface proteins: InlA and InlB that bind their corresponding receptors on host cell surface through their Leucine-Rich Repeat (LRR) domains, i.e., InlA binds human E-cadherin, a calcium-dependent intercellular adhesion glycoprotein, while InlB binds hepatocyte growth factor receptor Met [61] and, together, mediate bacterial internalisation and invasion into host cells. LIPI-1 encodes a pore-forming toxin listeriolysin O (LLO) and two phospholipases C (PlcA and PlcB), which cooperate to lyse the phagocytic vacuole membrane of host cells [62]. The hpt intracellular growth locus encodes hexose-6-phosphate transporter and actin assembly inducing protein (ActA), which play an important role in intracellular bacterial growth, cell-to-cell spread and actin polymerisation [49].
The virulence of L. monocytogenes is mainly regulated by six genes (prfA, plcA, hly, mpl, actA and plcB) residing in PrfA-dependent virulent gene clusters and other virulence-related genes located outside this gene cluster [49]. Furthermore, L. monocytogenes carries a gene cluster of five genes termed stress survival islet 1 (SSI-1), contributing to the survival of cells under suboptimal conditions, including low pH and high salt concentrations especially within food environments [63].
The prfA virulent gene clusters of L. monocytogenes and L. ivanovii also contains a positive master-regulator that controls the expression of different virulence factors. In addition, these pathogenic strains possess the hlyA gene that encodes a 60-kDa sulfhydryl-activated pore-forming listeriolysin O (LLO) that is very essential for facilitating escape of invading bacterial cells from the phagosomes of host cells into the host cytosol. The LLO is, therefore, known to be the main virulence feature responsible for discharging bacterial cells from the primary and secondary intracellular vacuoles [64]. The species-specificity properties of the hlyA gene and the LLO makes them effective molecular identification targets for L. monocytogenes, especially in food samples [65], on-farm microbiological control initiatives [66], and for serological diagnosis of ovine listeriosis [67] as well as contamination in pregnant women [68]. On the other hand, the plcA and plcB code for phosphatidylinositol-specific phospholipase C and phosphatidylcholine phospholipase respectively that assist, can operate in concert to ensure lysis of host cell membranes [69]. Soni, Ghosh [64] state that virulent genes comprising members of the internalins protein family (inlA, inlB), encode cell wall proteins that are important for attachment and invasion of non-phagocytic host cells. These virulent genes can be expressed or repressed by a few environmental conditions, thus affecting the virulence of L. monocytogenes [70]. together, mediate bacterial internalisation and invasion into host cells. LIPI-1 encodes a pore-forming toxin listeriolysin O (LLO) and two phospholipases C (PlcA and PlcB), which cooperate to lyse the phagocytic vacuole membrane of host cells [62]. The hpt intracellular growth locus encodes hexose-6-phosphate transporter and actin assembly inducing protein (ActA), which play an important role in intracellular bacterial growth, cell-to-cell spread and actin polymerisation [49]. The virulence of L. monocytogenes is mainly regulated by six genes (prfA, plcA, hly, mpl, actA and plcB) residing in PrfA-dependent virulent gene clusters and other virulence-related genes located outside this gene cluster [49]. Furthermore, L. monocytogenes carries a gene cluster of five genes termed stress survival islet 1 (SSI-1), contributing to the survival of cells under suboptimal conditions, including low pH and high salt concentrations especially within food environments [63].
The prfA virulent gene clusters of L. monocytogenes and L. ivanovii also contains a positive masterregulator that controls the expression of different virulence factors. In addition, these pathogenic strains possess the hlyA gene that encodes a 60-kDa sulfhydryl-activated pore-forming listeriolysin O (LLO) that is very essential for facilitating escape of invading bacterial cells from the phagosomes of host cells into the host cytosol. The LLO is, therefore, known to be the main virulence feature responsible for discharging bacterial cells from the primary and secondary intracellular vacuoles [64]. The species-specificity properties of the hlyA gene and the LLO makes them effective molecular identification targets for L. monocytogenes, especially in food samples [65], on-farm microbiological control initiatives [66], and for serological diagnosis of ovine listeriosis [67] as well as contamination in pregnant women [68]. On the other hand, the plcA and plcB code for phosphatidylinositol-specific phospholipase C and phosphatidylcholine phospholipase respectively that assist, can operate in concert to ensure lysis of host cell membranes [69]. Soni, Ghosh [64] state that virulent genes comprising members of the internalins protein family (inlA, inlB), encode cell wall proteins that are important for attachment and invasion of non-phagocytic host cells. These virulent genes can be expressed or repressed by a few environmental conditions, thus affecting the virulence of L. monocytogenes [70].

Antibiotic Resistance in Listeria monocytogenes
The first L. monocytogenes strains resistant to antimicrobials were detected in 1988 [72]. Since then, an increasing number of strains resistant to one or more antibiotics, isolated from humans, animals, food [73], such as food processing environments [74] and dairy farms [75], were reported in recent years.
Although L. monocytogenes are considered vulnerable to a wide range of antibiotics, intrinsic antibiotic resistance to first generation Quinolones, Fosfomycin, Monobactams and broad-spectrum Cephalosporins were described [76]. Moreover, clinical use of antibiotics contributed to the selection of resistant strains, particularly for antibiotics commonly used to treat listeriosis. β-lactams (Penicillin and Ampicillin), with or without Gentamicin, are the main antibiotics considered for the treatment of listeriosis; Vancomycin and Trimethoprim / Sulfamethoxazole can be used as alternative therapy for Penicillin-allergic patients [77].
Veterinary use of antimicrobials in food-producing animals, commonly administered for disease therapy, prophylaxis and as growth promoters, could also contribute to the emergence of resistant strains [78]. Antibiotic resistance and particularly multi-resistance represent public health problems since they may cause failure of therapeutic treatment. Therefore, monitoring changes in antibiotic resistance of L. monocytogenes due to the continuing emergence of resistant strains, particularly by integration of phenotypic and genotypic techniques, is needed.

Outbreak of Listeriosis in the United States of America
In 1998, an outbreak occurred in 10 states linked to the consumption of hot dogs and possibly refrigerated, processed deli meat. Forty patients contaminated by a single strain, L.monocytogenes, were identified in early August 1998 from the States of Ohio (13 cases); New York (12); Tennessee, Massachusetts and West Virginia (three each); Michigan (two); Connecticut, Oregon, Vermont, and Georgia (one each). Out of the forty infected patients, information could only be accessed from 38 individuals comprising six newborns and 32 adults (median age: 69 years; range: 18-88 years) with 55% of patients female. Out of this number, deaths were recorded in one foetus and three elderly [79].
The same year, on 22 December, Bil Mar Food recalled the production lots EST P261 or EST 6911 of hot dogs and meat products that could be contaminated. This recall comprised the Ball Park, Bil Mar, Bryan Bunsize and Bryan 3-lb Club Pack, Grillmaster, Hygrade, Mr Turkey, Sara Lee Deli Meat and Sara Lee Home Roast brands [80].
Listeria monocytogenes was also isolated from 29 patients in 10 states between 17 May and 26 November 2000. Out of this number, majority of cases 26 (90%) occurred from 15 July to 26 November 2000, while fewer cases 3 (10%) were isolated in the earlier months. The 29 patients were distributed as follows: New York (15 cases); Georgia (3); Connecticut, Ohio and Michigan (2 each); California, Pennsylvania, Tennessee, Utah and Wisconsin (1 each). 21 patients were elderly (29-92 years) with a median age of 65 years while 8 were perinatal. Three cases of miscarriages or stillbirths and four deaths were reported [81]. Since then, outbreaks or sporadic cases of the infection have been reported almost every year. Table 1 provides a summary of outbreaks of listeriosis in the USA from 1976-2019.

Outbreaks of Listeriosis in Australia
Listeria monocytogenes has become an invasive pathogen in foods and processed food products. In 2003, Australia experienced an outbreak of the disease with the highest mortality (30%) [99].  Figure 2). The highest number of cases of Listeriosis was reported in 2014 and the most prevalent group affected were patients of 80 years and above (Figure 2). In 2018, the isolated L. monocytogenes strain was serotype 4b strain ST240 and affected 59% of females.

Outbreaks of Listeriosis in Nigeria
Nigeria is considered to be the "Giant of Africa" due to the large population and growing economy. Regardless of this, there have been limited reports documented and surveillance systems of outbreaks of Listeriosis in the country. However, researchers successfully isolated Listeria in humans, animals, the environment, and food samples in Nigeria (Table 5). Listeria was shown to affect pregnant women and infants while ruminants dominated reports of incidences in animals. Table 5. Different sources of Listeria in Nigeria, such as humans, fish, water, animals, soil, and the environment [112].

Source
Origin References

Outbreaks of Listeriosis in Asia
In the past few decades, Asia has experienced severe outbreaks of Listeriosis. Several reports in Asian countries such as Japan [106,107], Thailand (Bangkok) [108] and Taïwan [109,110] were documented for clinical cases of Listeriosis. Table 4 shows that L. monocytogenes targets immune-compromised individuals such as pregnant women and infants. This is displayed in reports of outbreaks in China in 1964 and 2010 [111].

Outbreaks of Listeriosis in Nigeria
Nigeria is considered to be the "Giant of Africa" due to the large population and growing economy. Regardless of this, there have been limited reports documented and surveillance systems of outbreaks of Listeriosis in the country. However, researchers successfully isolated Listeria in humans, animals, the environment, and food samples in Nigeria (Table 5). Listeria was shown to affect pregnant women and infants while ruminants dominated reports of incidences in animals. Table 5. Different sources of Listeria in Nigeria, such as humans, fish, water, animals, soil, and the environment [112].

Source
Origin References

Outbreaks of Listeriosis in South Africa
Currently, South Africa is experiencing severe outbreaks of Listeriosis. To date, this is the highest outbreak of Listeria in history affecting a single nation. A total of 820 cases of Listeriosis were reported by the National Institute of Communicable Diseases (NICD) from January 2017 to 23 January 2018. The highest number of cases originated from Gauteng Province (59%), followed by the Western Cape (13%) and Kwa Zulu-Natal (7%). Samples were collected from blood (71%) and Cerebral Spinal Fluid (CSF) with 23% in the public and private healthcare sectors. Approximately 66% were diagnosed from public and 34% from private healthcare sectors. The age distribution of cases ranged from birth to 93 years, with majority (96%) of cases neonates (≤28 days). The gender distribution was 55% females and 45% males [142]. Figure 3 shows the evolution of confirmed cases of Listeriosis from 1 January 2017 to 17 July 2018 in South Africa [23].
Fortunately, human Listeriosis is rare. However, there is a high case of fatality if the disease ensues. With the very long incubation period, which can range up to 60 days, attributing the illness to a specific food product becomes difficult [17]. In addition to several factors mentioned earlier, responsible for outbreaks of Listeriosis in the supply chain, resistance of microorganisms to antibiotics and disinfectants also play a role [143].
(13%) and Kwa Zulu-Natal (7%). Samples were collected from blood (71%) and Cerebral Spinal Fluid (CSF) with 23% in the public and private healthcare sectors. Approximately 66% were diagnosed from public and 34% from private healthcare sectors. The age distribution of cases ranged from birth to 93 years, with majority (96%) of cases neonates (≤28 days). The gender distribution was 55% females and 45% males [142]. Figure 3 shows the evolution of confirmed cases of Listeriosis from 1 January 2017 to 17 July 2018 in South Africa [23]. Fortunately, human Listeriosis is rare. However, there is a high case of fatality if the disease ensues. With the very long incubation period, which can range up to 60 days, attributing the illness to a specific food product becomes difficult [17]. In addition to several factors mentioned earlier,

Estimated cost of the outbreak of Listeriosis in South Africa
Food spoilage caused by microbial pathogens results in massive economic loss to both the company responsible and the country. In developing countries, such as South Africa, outbreaks of Listeriosis are poorly documented [23], including the major outbreak in 2017-2018, which caused a deadly epidemic. Approximately, 1060 cases were reported with 216 deaths [23]. Processed meat products (Polony), produced by Tiger Brands Limited (South Africa), was identified as the responsible source. The accountable factories were Tiger Brand (Polokwane, Limpopo and Germiston, Johannesburg) [144]. The economic loss was projected to be US $260 million (R 3, 79 Billion) in terms of lethality, while cost of hospitalisation associated with one-month recovery of affected patients by the disease was US $10.4 million (R1, 5 Billion). Furthermore, more than US $15 million (R2, 19 Billion) was attributed to loss due to productivity and export of food processing companies due to the outbreak [145].

Conclusions
It is confirmed in this review that South Africa recorded the highest outbreaks of Listeriosis with a high mortality rate. L. monocytogenes was successfully isolated from contaminated possessed meat (Polony). South Africa is considered the economic capital of Africa and developing exponentially. The country also has strong regulations on food safety management and laws aligned with international standards. Despite this, the recent outbreak of Listeriosis showed some limitations in food safety policies. Authorities in the Department of Health (South Africa) recently reviewed the list of notifiable diseases to include Listeriosis. Funding: The authors appreciate the financial assistance received from the North-West University.

Acknowledgments:
The authors acknowledge the financial assistance received from the Postgraduate Bursary Office of the North-West University.

Conflicts of Interest:
There are no conflicts of interests to be declared by the authors.