ISOLATION AND IDENTIFICATION OF KLEBSIELLA ON SEA FOOD (catfish, prawns, Titus fish, shrimps, periwinkles)

INTRODUCTION

Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety of infectious diseases in humans, including septicaemia, liver abscesses, diarrhea, and pneumonia (Bi and Xu, 2013; Cao et al., 2014; Guo et al., 2017). It is a well-known hospital-acquired pathogen and associated with increased patient morbidity and mortality (Brisse et al., 2009; Cabral et al., 2012). In addition to the clinical environment, K. pneumoniae is frequently found in foods including raw vegetables, powdered infant formula, meat, fish, and street foods, and has been considered as an important food-borne pathogen (Haryani et al., 2007; Sun et al., 2010; Puspanadan et al., 2012; Overdevest et al., 2014; Kim et al., 2015; Davis and Price, 2016). In powdered infant formula, K. pneumoniae is included in the hazard identification category “B” according to the FAO and WHO guidelines on microorganisms (FAO-WHO, 2004). In recent years, an increasing number of food-borne outbreaks caused by K. pneumoniae have been reported in different countries (Calbo et al., 2011; Tambekar et al., 2011; Zhou et al., 2011; Bi and Xu, 2013; Yu and Zhou, 2013).

K. pneumoniae can express a variety of virulence factors including capsules, endotoxins, siderophores, iron-scavenging systems, and adhesins, which have been shown to play important roles in its pathogenesis. Capsule is an important virulence factor, which is involved in at least two pathogenic mechanisms: (1) protection of the bacteria from phagocytosis, and (2) direct inhibition of the host immune response (Kang et al., 2015). Some capsular (K) types, particularly K1, K2, K54, K57, K20, and K5, are often associated with community-acquired invasive pyogenic liver abscess syndrome, septicemia, and pneumonia (Fang et al., 2007; Siri et al., 2011; He, 2012). K1, K2, K20, K54, and K57 are highly virulent in experimental infections in mice and are often associated with severe infections in humans and animals (Turton et al., 2008; Yu et al., 2008; Cheng et al., 2015; Wei et al., 2016). K2 and K5 are frequent causes of metritis in mares and are associated with community-acquired pneumonia (Brisse et al., 2009). K3 is generally associated with rhinoscleroma (He, 2012). Capsule typing is currently the most widely used technique for typing K. pneumoniae isolates and exhibits good reproducibility in differentiating clinical isolates (Siu et al., 2011). Several PCRs targeting the wzy genes have been developed for capsule typing of K. pneumoniae (Turton, 2010; Cheng et al., 2015). Other virulence factors such as the rmpA gene (regulator of mucoid phenotype A); allS gene (encoding the activator of the allantoin regulon, associated with allantoin metabolism); endotoxin-related genes wabG, uge, and wcaG; iron acquisition system-related genes iucB, iroNB, ybtA, and kfuBC; adhesin gene fimH (type I fimbriae); and ureA gene (α-subunit of the urease, invasin related) are also believed to be involved in virulence processes (Brisse et al., 2009; Turton, 2010; El et al., 2013; Calhau et al., 2014). The detection of such virulence factors is important in understanding the pathogenic characteristics of K. pneumoniae isolates and enhancing our knowledge of the health risks posed by this pathogen.

The emergence of antimicrobial resistance in Klebsiella spp. isolates is of great concern worldwide in human medicine (Hu et al., 2013). Multidrug-resistant K. pneumoniae strains have been isolated from different samples (Nawaz et al., 2012; Falomir et al., 2013; Guo et al., 2016; Yaici et al., 2017). Dietary intake is one of the primary routes for the introduction of antibotic-resistant bacteria and their genes into the human digestive tract. Consumption of specific food categories might influence gut antibiotic resistance gene diversity (Milanović et al., 2017). Moreover, such bacteria may transfer antibiotic resistance determinants to other pathogenic bacteria (Machado et al., 2008). Therefore, surveillance and monitoring of drug-resistant bacteria in foods is important for implementing targeted control strategies and selecting effective drugs for treatment.

Molecular typing is a useful tool for determining the genetic relationships of food-borne bacteria and identifying probable sources of infections. This is particularly important in endemic and epidemic nosocomial outbreaks of K. pneumoniae infections to improve the management of such outbreaks. A variety of methods have been used for K. pneumoniae typing, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD), and (GTG)₅ oligonucleotide PCR (Haryani et al., 2007; Ryberg et al., 2011; Barus et al., 2013; Sachse et al., 2014). The ERIC, RAPD, and (GTG)₅-PCR assays are relatively simple and cost-effective methods that have been successfully used for genotyping K. pneumoniae isolates from various sources (Ryberg et al., 2011; Barus et al., 2013).

In recent years, the number of food-borne illness outbreaks caused by K. pneumoniae has increased. However, until now, limited information has been available on the characteristics of K. pneumoniae isolated from foods. The purpose of the present study was to determine the biotypes, serotypes, virulence genes, and antimicrobial resistance patterns of food isolates and to further analyse their genetic diversity using ERIC-PCR and (GTG)₅-PCR molecular typing.

ISOLATION AND CHARACTERISTICS OF SALMONELLA SPECIES FROM FROZEN FOODS IN OWERRI

ABSTRACT

A study was conducted to determine the presence of Salmonella species in Frozen Foods in Owerri in August, 2000. A total of 30 Food samples were collected from the different market in ten different locations in Owerri and examined for the presence of Salmonella. After the culturing and biochemical tests, three different Salmonella species were isolated and they include: Salmonella typhi, Salmonella typhimurium and Salmonella paratyphi A. out of the 30 Foodsamples examined, Salmonella were found in 20 (66.67%). Out of the total samples examined, Salmonella typhi were isolated with the prevalence rate of 12 (40%) from 30 samples, Salmonella paratyphi A were isolated with the prevalence rate of 5 (16.67%), and Salmonella typhimurium were isolated with the prevalence rate of 3 (10%).

CHAPTER ONE/INTRODUCTION

Illnesses from food are one of the most important economic and health problems among industrial and non-industrial countries. In recent years, Salmonella has been one of the most common causes of food born disease Salmonellosis is an infectious disease which often occurs through contaminated food, especially food products with an animal origin such as meat, chicken, egg, animal foods and sometimes vegetables in the food chain. In developing countries, estimation of Salmonelosis is difficult because there has not been sufficient surveillance. Therefore, globally, many studies have been performed reporting that the prevalence and kind of salmonella serotypes are different based on geographical regions. Salmonella entericaserovarsEnteritidis and Typhimurium were reported to be the two most frequent serotypes of Salmonella isolated in Iran and other countries. During the two past decades, the emergence of antibiotic-resistant Salmonella has become a serious problem worldwide. Wide usage of antibiotics in the diet of domestic animals has made drug resistant bacteria which could betransfered to human beings. Also, in recent years, problem of resistant strains to multiple drugs (MDR) is increasing and most studies in Iran and other countries have shown high resistance of Salmonella strains to several antibioticsIt belongs to the family Emtero-bacteriaceae and it is a facultative anaerobic gram-negative bacillus that parasitizes the intestine of a large number of vertebrate species. It also infects man leading to enteric fever, gastroenteritis and septicemia, collectively known as Salmonellosis infection.

Of all the enterobacteriaceae, the genus salmonella is the most complex with more than 2,000 closely related bacteria that causes illness by reproducing in the digestive tract. (Aora, 2004). Each salmonella subgroup or serotype share common antigen and has its own name and the antigenic complexity of the bacteria have been described by Kauffman-White scheme. According to this scheme, the terminology that identifies the particular protein coats, or serovars, is not well settled, and what previously were thought to be various species of the genus Salmonella are now thought to be serovars of only two species by many researchers, Salmonella enterica and Samonellabongori.

However, these designations are not always accepted in the scientific literature and so common serovars that have been named in the past are still used (for example, S. typhi, S. typhimurium, S. enteritidis, S. cholerasuis, S. saintpaul ). The Serovars are identified by the Kauffman-White classification that uses two major types of antigens (Somatic O and flagella H) to distinguish the over 2, 500 types of samonella bacteria. Initially each Salmonella species was named according to clinical consideration, e.g. Salmonella typhi-murium (mouse typhoid fever), S. cholerae-suis  (hog cholera). After it was recognized that host specificity did not exist for many species, new strains (or serovar, short for serological variants) received species name according to the location at which the new strain was first isolated.

SerovarTyphimurium has considerable diversity and may be very old. The majority of the isolates belong to a single clonal complex. Isolates are divided into phage types, but some phage types do not have a single origin as determined using mutational changes. Phage type DT104 is heterogeneous and represented in multiple sequence types, with its multidrug-resistant variant being the most successful and causing epidemics in many parts of the world. Serovartyphi is relatively young compared to Typhimurium, and probably originated approximately 30,000-50,000 year ago. The common salmonella serotypes that are important in Animals and mammals include;

*     Salmonella enterica subspecies arizonae – is found in cold-blooded animals, poultry, mammals.

*     Serovars of Salmonella  enterica subspecies enterica.

*     Salmonella choleraesuis (Bacillus paratyphoid B and C). This is an intestinal commensalists in pigs; it becomes pathogenic if resistance is weak; humans can be infected by ingesting sick animals; the bacteria cause septicemic salmonellosis in swine.

*     Salmonella enteritidis – It is found in the intestines of cattle, rodents, ducks and humans; it causes calf paratyphoid fever and acute gastroenteritis in humans.

*     Salmonella paratyphi B, in central Europe usually human pathogen, causes paratyphoid fever.

*     Salmonella typhi, occurs in temperate and subtropical zones. It is the human pathogen of typhoid fever; 3-5% of persons falling ill remain permanent carriers of the pathogen.

*     Salmonella typhimurium, causes a wide range of infections in birds to severe systemic paratyphoid diseases. It causes salmonella enteritis (food poisoning) in humans.

*     Salmonella dublin, one of the pathogens causing cattle salmonellosis.

*     Salmonella typhisuis, one of the pathogen causing hog Salmonellosis.

*     Salmonella brandenburg, causes abortion and enteritis in shepherd cattle.

*     Salmonella anatum  and  Salmonella montevideo.

Animals consume Salmonella from the soil or contaminated processed food e.g. feeds. The bacteria are then shed alive in the infected animals’ faeces.

Humans on the other hand consume Salmonella through contact withUnclean food, particularly in institutional kitchens and restaurants

Excretions from either sick or infected but apparently clinically healthy people and animals (especially endangered are caregivers and animals)

Polluted surface Food and standing Food (such as in shower hoses or unused Food dispensers)

Unhygienically thawed fowl (the melt Food contains many bacteria).

An association with reptiles (Pet tortoises and snakes) primarily aquatic turtles is well described.

Salmonella bacteria can survive several weeks in a dry environment and several months in Food; thus, they are frequently found in polluted Food, contamination from the excrement of carrier animals being particularly important. Aquatic vertebrates, notably birds and reptiles, are important vectors of salmonella.  Poultry, cattle, and sheep are frequently agents of contamination. Salmonella can be found in food, particularly meats and raw eggs.

Salmonella infections other than typhoid fever are termed enteric fever, salmonella food poisoning and gastroenteritis. These diseases are usually less severe than typhoid fever and are scribed to one of the many serotypes of Salmonella enteritidis. Most outbreaks of epidemics and food poisoning in humans and animals are caused by salmonella enteritidis  and  Salmonella typhimurium,  others are Salmonella Dublin, Salmonella Thompson. Non-typhodialsalmonellosis are more prevalent than typhoid fever and currently holding steady at 40,000 to 50,000 case a year. All strains are zoonotic in origin though human may be some carriers under certain circumstances by contrast. The type of salmonella that affect humans usually cause disease which can be recognized by diarrhea, vomiting.

Diarrohoeal disease seems to be one of the major health hazards both for human and animals. Many salmonella grow in the gut of their host and once in the gut, salmonella wave-long filament called flagella moves about until they touch a suitable cell. The bacteria stick to the cell and are “swallowed” smaller “hairs” called fimbriae are important in stimulating immunity in the host.

Screening of Bacteria Producing Chitosan

abstract