Food infection- Brucellosis

 The genus Brucella is named after Sir David Bruce who in 1887 recognized it as the causative organism of undulant fever (brucellosis, Malta fever, Mediterranean fever).

Four species that are human pathogens is associated with a particular animal host,

• B. abortus (cattle)
•  B. melitensis (sheep and goats),
• B. suis (pigs),
• B. canis (dogs)

 Brucellosis is principally contracted from close association with infected animals and is an occupational disease of farmers, herdsmen, veterinarians and slaughterhouse workers. It can also be contracted by consumption of milk or milk products from an infected animal, although the risk is lower.

The illness has been effectively eliminated from the United States, Scandinavia, the UK and other countries by campaigns to eradicate the organism in the national dairy herds through a programme of testing, immunization of young calves and compulsory slaughter of infected cattle.

Brucellosis has sometimes been associated with the consumption of inadequately cooked meat from an infected animal but raw milk or cream are the principal food vehicles. Brucella is readily killed by normal milk pasteurization conditions so there is no risk from pasteurized milk or products made from it. Cheeses made from unpasteurized milk can sometimes pose a problem  since the organism can survive the cheese making processes and subsequent storage in the product.
 

Brucella are Gram-negative, catalase-positive, oxidase-positive, short oval rods, which are non-motile and usually occur singly, in pairs, or, rarely, in short chains. It grows optimally around 37^oC and is killed by heating at 63^oC for 7–10 min. When shed in the milk of an infected animal it can survive for many days if the acidity remains low.

Brucellosis is a protracted (long lasting) and debilitating (causing weakness) illness characterized by an incubation period of from one to six weeks. Symptoms are a chronic, relapsing fever with accompanying lassitude (physical or mental weariness), sweats, headache, constipation, anorexia, pains in the limbs and back, and weight loss. After the temperature has returned to normal for a few days, another bout of fever may ensue and such episodes recur a number of times over several months. Treatment is commonly with a mixture of tetracycline and streptomycin.

It is a facultative parasite and can live intracellularly or in extracellular body fluids. During the febrile stage, caused by circulating endotoxin, the organism may be isolated from the bloodstream but in the majority of laboratory-confirmed cases diagnosis is based on serological tests rather than cultural techniques.

Brucella are quite fastidious organisms and do not grow in conventional laboratory media. Liver infusions or calf serum are normally added. The organism grows slowly and cultures are normally incubated for three weeks before they are considered negative. In view of this, testing foods for the organism is not practically feasible or useful. 

Cattle are tested for the presence of antibodies to the organism in the ‘Ring Test’. Stained antigen is mixed with the test milk, if antibodies to Brucella are present (indicative of infection) then they will cause the antigen to clump and rise with the milkfat on standing to form an intense blue-violet ring at the top of the milk.

Food infection-Amoebiasis & Hepatitis

 Amoebic dysentery 

•        Amoebic dysentery can be very widespread wherever there is poor hygiene

•        Causative organism: Entamoeba histolytica

•        a protozoan that causes intestinal amoebiasis as well as extraintestinal manifestations.

•        Although 90 percent of E. histolytica infections are asymptomatic, nearly 50 million people become symptomatic, with about 100,000 deaths yearly.

•        usually transmitted by the faecal–oral route.

•        usually through contaminated food or water sources.

•        The organism is an aerotolerant anaerobe which survives in the environment in an encysted form.

•        affects males and females of all ages equally.

•        Factors that are associated with increased risk for complicated infection and mortality are associated with the following: pregnancy, corticosteroid treatment, malignancy, malnutrition, and alcoholism. 

•        There are two forms E. histolytica can take: the cyst form, which can survive in the environment for a prolonged period, and the trophozoite stage, which is the active and invasive form.

•        After the ingestion of the cyst form, trophozoites can be formed, which can invade and penetrate intestinal mucosa destroying epithelial cells and inflammatory cells.

•        The pathological range includes mucosal inflammation, thickening, ulcers, and necrosis, leading to perforation.

•        Indeed, a person with amoebic dysentery may pass up to fifty million cysts per day.

•        most infections remain symptomless

•         illness may start with the passing of mucous and bloody stools, due to ulceration of the colon, a few weeks after infection and progress to severe diarrhoea, abdominal pain, fever and vomiting.

•        Entamoeba histolytica infection is endemic in many poor communities in all parts of the world

 

Hepatitis A

•        Hepatitis A is an inflammation of the liver

•         caused by the hepatitis A virus (HAV). 

•         non enveloped RNA virus

•         spread through food or water that is contaminated with the faeces of an infected person.

•        Outbreaks are usually due to contamination by an infected food handler.  

 

•        The incubation period varies between two and six weeks.

•        During this period the virus multiplies in the cells of the gut epithelium before it is carried by the blood to the liver.

•        In the later part of the incubation period the virus is shed in the faeces.

•        Early symptoms are anorexia, fever, malaise, nausea and vomiting, followed after a few days by symptoms of liver damage such as the passage of dark urine and jaundice.

•        Transmission by oral fecal route

•        Infection is by ingestion

•        It occurs sporadically or as outbreaks

•        Caused by contaminated food and water or milk

•        Shellfish is also responsible

•        Fruits such as strawberries and raspberries, salad vegetables such as lettuce are common food vehicles

•        Domestic or institutional spread is common children.

•        Overcrowding and poor sanitation favor its spread.

 

Hepatitis E

•        Hepatitis E is a liver infection caused by the hepatitis E virus

•        Non enveloped RNA virus

•        The source of infection is fecal contamination of drinking water and the environment.

•        Incubation period –: 2-9weeks

•        Mainly occur to middle aged adults (15-40 years)

•        Infection is self-limiting and resolves within 2–6 weeks.

•        Occasionally a serious disease, known as fulminant hepatitis (acute liver failure) develops, can cause death.

•        Poor sanitation, ingestion of undercooked meat or meat products derived from infected animals (e.g. pork liver) and rarely, transfusion of infected blood products; vertical transmission from a pregnant woman to her baby.

•        Initial phase of mild fever, reduced appetite (anorexia), nausea and vomiting, lasting for a few days; some may also have abdominal pain, itching (without skin lesions), skin rash, or joint pain.

•        Jaundice, with dark urine and pale stools-    a slightly enlarged, tender liver (hepatomegaly).

•        High fatality in pregnant women-   Pregnant women with hepatitis E, particularly those in the second or third trimester, are at increased risk of acute liver failure, fetal loss and mortality.

 Control

•        Safe public water supplies and proper disposal of human faeces.

•        Maintain hygienic practices;

•        Avoiding consumption of water and ice of unknown purity

•        A recombinant subunit vaccine registered in China- not yet been approved in other countries.

Escherichia coli

 Family Enterobacteriacieae

 

• Genus is named after Escherich (1885), who was the first to describe it
• Escherichia coli is part of human enteric flora.
• Unlike other coliforms, E. coli is a parasite living only in the human or animal intestine.
• Excreted in feces, it remains viable in the environment only for a few days.
• Detection of thermotolerant E. coli in drinking water indicates fecal contamination of the water.

Morphology

• E. coli is a gram-negative, straight rod arranged singly or in pairs, nonspore forming organism.
• Motile - peritrichous flagella.
• Capsules and fimbriae are found in some virulent strains. 
  

Cultural Characters

• Aerobe, facultative anaerobe
• Optimum temperature 37^oC (grow at 10-40^oC)
• Good growth on nutrient agar (ordinary media)-large colonies, thick, moist, smooth (S forms), opaque/partially translucent colonies when fresh
• R forms produce irregular dry colonies-autoagglutinable
• S-R variation due to repeated subculture; loss of surface antigens and virulence
• Pathogenic strains -have polysaccharide capsules & are hemolytic on blood agar
• MacConkey’s medium- bright pink colonies-lactose fermenting
• Growth inhibited on selective media for Salmonellae/Shigella (DCA/SS agar)
• Growth uniform & turbid in broth
  
  
  

 Antigenic Structure

 Serotyping/Antigenic typing of E. coli is based on three antigens: somatic O antigen, flagellar H antigen and Capsular K antigen. In addition, it also has fimbrial or F antigens.

• O antigen -somatic antigen- O antigens are associated with the virulence of the organism
• Around 170 types of heat-stable, lipopolysaccharide O antigens have been recognised..
• H antigen (flagellar antigen)
• H antigens are more specific since cross-reaction between other members is not common. 75 H antigens have been recognised so far.
• K antigen (capsular antigens)
• About 100 K antigens have been recognised till date. This is an acidic polysaccharide antigen located in the envelope or microcapsule-enclose O antigens-contribute to virulence by inhibiting phagocytosis

 Virulence Factors

 Two types of virulence factors have been recognised in E. coli:

1. Surface antigens

O antigen (somatic lipopolysaccharide)

• Has endotoxic activity.
• protects the organism from phagocytosis and the bactericidal effects of complements.
• The envelope or K antigens also offer protection against phagocytosis.
• KI envelope antigen -Strains causing neonatal meningitis and septicaemia carry this antigen

F antigens

• Fimbriae are important virulent factors- important in initial attachment and colonisation.
• Plasmid coded - found only in small numbers-important in virulence
• heat-labile and get detached when the organism is heated to 100°C
• P fimbriae, seen in uropathogenic strains,
• Fimbriae seen as surface Colonisation factor antigens (CFA) in enterotoxigenic E. coli, causing human diarrhea.

2. Toxins

E. coli produces two kinds of exotoxins: hemolysins and enterotoxins.

Hemolysins-not relevant in pathogenesis

CNF1 (cytotoxic necrotising factor-1) and siderophores are virulence factors in uropathogenic E. coli and are important components of biofilm production and adhesion.

Enterotoxins

Enterotoxins are important in the pathogenesis of diarrhea. Three distinct types of E. coli enterotoxins have been identified:

1 Heat-labile toxin (LT),

3 Heat-stable toxin(ST)

3 Verotoxin (VT), also known as Shiga-like toxin (SLT).

 

1 Heat-labile toxin (LT)

 Heat-labile toxin (LT), of E. coli was discovered in 1956  by De and colleagues in isolates from adult diarrhea cases in KoIkata, E . coli LT resembles the Cholera toxin in its structure, antigenic properties and mode of action. It is a complex of polypeptide and subunits-each unit Consisting of one subunit A A for active) and five subunits B (B for binding)

The Al fragment activates adenylyl cyclase in the enterocyte to form cyclic adenosine 5' monophosphate (¢AMP), leading to the increased outflow of water and electrolytes into the gut lumen, with consequent watery diarrhea.

2) Heat-stable toxin (ST) of E. coli was first identified in 1970 and comprises low-molecular-weight polypeptides which are poorly antigenic.

3) E. coli verocytotoxin or verotoxin is so named because of its cytotoxic effect on Vero cells (cell line derived from African green monkey kidney cells). It is also known as Shiga-like toxin (SLT) because of its similarity to Shigella dysenteriae type 1 toxin, in physical, antigenic and biological properties. It acts by inhibiting protein synthesis. 

Clinical Infections

Four main types of clinical syndromes are caused by E. coli:

1. Urinary tract infection

2. Diarrhea

3. Septicaemia, neonatal sepsis and neonatal meningitis

4. Pyogenic infections                 

(I) Urinary tract infection                                

E. coli and other coliforms account for the large majority of naturally acquired urinary tract infections (UTI).

The E. coli serotypes commonly responsible for community acquired UTI are O groups 1, 2, 4, 6, 7. One serotype isolated from infected urine, at a time. Recurrence may be due to different serotypes

About 5-7 per cent of pregnant women have been reported to have asymptomatic bacteriuria, which, if undetected and untreated, may lead to symptomatic infection later in pregnancy, pyelonephritis and hypertension, leading to prematurity and perinatal death of the fetus.

The P-pili-positive E. coli are generally uropathogenic.

Laboratory diagnosis

1. Specimen

• Freshly voided urine -following urogenital toilet, a clean-catch midstream sample of urine is collected. Process immediately-to avoid contamination- refrigerate if delayed
•  Suprapubic aspiration in obstructive uropathy, when voiding is not possible, a suprapubic aspiration may be done.
  
• Bacteriological diagnosis of UTl is done by demonstrating significant bacteriuria-100,000 bacteria or more per ml in urine, in case of active infection, in quantitative cultures (developed by Kass)
  

For quantitative culture, serial tenfold dilutions of urine are tested by pour plate or surface culture methods

Semiquantitative techniques include- transferring a standard loopful urine on a noninhibitory medium such as blood agar and another loopful on an indicator medium-MacConkey agar

Antibiotic sensitivity test of the isolate is mandatory-resistance is often to multiple drugs and is transferable easily

 2. Screening test for UTI

UTI is common so, several screening techniques have been introduced for the rapid presumptive diagnosis of significant bacteriuria:

 i) Griess nitrite test

 The presence of nitrite, detectable by a simple colorimetric test, indicates the presence of nitrate-reducing bacteria in urine; normal urine does not contain nitrites.

ii) Catalase test

The presence of catalase as evidenced by frothing on addition of hydrogen peroxide indicates bacteriuria (positive obtained in hematuria, also)

ii) Triphenyl tetrazolium chloride (TTC) test

This is a dye reduction test -pink-red precipitate in the reagent, caused by respiratory activity of growing bacteria causing UTI.

iv) Microscopic demonstration

The bacteria can be demonstrated on Gram-stained films of uncentrifuged urine.

v) Glucose Test paper

Based on the utilisation of minute amounts of glucose present in normal urine, by bacteria causing infection.

v) Dip slide culture methods

 Agar-coated slides are immersed in urine or exposed to the stream of urine during voiding. They are incubated and the growth estimated by colony counting or by colour change of indicators in the medium.

Screening methods is not as reliable /sensitive as culture

 3. Localisation of the site of UTI- Antibody coated bacteria test

 This is based on the assumption that bacteria coated with specific antibodies are present in the urine only when the kidneys (upper UTI) are infected and not when the infection is confined to the bladder (lower UTI). Antibody-coated bacteria are detected by immunofluorescence using fluorescent-tagged antihuman globulin/ staphylococcal coagglutination

 None of the screening methods is as sensitive or reliable as a culture.

 4. Cultural characteristics

It grows aerobically and is a facultative anaerobe. The temperature range required for its growth is 10-40°C (optimum 37°C). In ordinary media, it grows well. Many pathogenic isolates have polysaccharide capsules. Some may occur in the mucoid form.

On blood agar, many strains, especially those associated with infection, are hemolytic.

On MacConkey medium, colonies are bright pink due to lactose fermentation.

On EMB- small, dark centred/nucleated colonies with or without metallic sheen.

 5. Biochemical reactions

Glucose, lactose, mannitol, maltose and several other carbohydrates are fermented with the production of acid and gas. Typical strains do not ferment sucrose.

IMViC test Four biochemical tests widely employed in the classification of Enterobacteriaceae are (i) indole, (i) methyl red (MR), (iii) Voges-Proskauer (VP) and (iv) citrate utilisation, generally referred to by the mnemonic IMViC

Antimicrobial susceptibility test E. coli and other common urinary pathogens develop drug resistance frequently. Isolates are often resistant to multiple antibiotics, which are transferable.

 Treatment

Treatment of uncomplicated UTI caused by antibiotic susceptible E. coli is with cotrimoxazole or ciprofloxacin. However, treatment would depend on the susceptibility pattern of the clinical isolate.

                     

(II) Diarrhea

1885-Ehlrich first isolated E.coli from feces of infants with enteritis- since then suspected to be causative of diarrhea-but no method to differentiate diarrheagenic strain from commensal E. coli.

In 1945, Bray established a type of E. coli specifically with childhood diarrhea- Many enteropathogenic E. coli subsequently identified.

 

There are six diarrheagenic E. coli which are as follows-

• Enteropathogenic E. coli (EPEC)
• Enterotoxigenic E. coli (ETEC)
• Enteroinvasive E. coli (EIEC)
• Enterohemorrhagic E. coli (EHEC)
• Enteroaggregative E. coli (EAEC)
• Diffusely adherent E. coli (DAEC)

1. Enteropathogenic E. coli (EPEC)

These have been associated mainly with diarrhea in infants and children -usually occurring as institutional outbreaks. They occasionally cause sporadic diarrhea in children and less often, in adults.

EPEC are identified by their O antigens.

The pathogenesis of EPEC diarrhea is not fully understood.

EPE are non-invasive and do not produce toxins.

EPEC adherence factor (EAF), plasmid encoded protein- for adherence, in infantile enteritis. Show localised adherence.

 2. Enterotoxigenic E. coli (ETEC)

Two epidemiological types:

 a) Endemic in developing countries of the tropics - seen in all age groups. Its severity varies from mild watery diarrhea to a fatal disease indistinguishable from cholera.

 b) Traveller's diarrhea seen in persons from non-endemic areas visiting endemic areas. Serotypes- 06, 08, 015, 025, etc.

It adheres to intestinal mucosa by fimbriae called colonisation factor antigens, (CFA I, II, II, and IV).

ETEC produce enterotoxins which may be either LT or ST or both.

 3. Enteroinvasive E. coli (EIEC)

They have the capacity to invade interstitial epithelial cells as seen in shigellosis and termed Enteroinvasive E. coli. This ability to penetrate cells is determined by a large plasmid- detection of which can also be a diagnostic test.

EIEC strains belong to serogroups 028ac, 0112ac, 0124, 0136, 0143 etc.

Molecular serotyping of clinical isolates is presently used to diagnose EIEC infection. HeLa or HEp-2 cell invasion in culture can also be used as a diagnostic test.

 4. Enterohemorrhagic E. coli (EHEC)

These strains produce two potent toxins, verocytotoxin (VT) or Shiga-like toxin (SLT). They can give rise to fatal haemorrhagic colitis and haemorrhagic uremic syndrome (HUS), particularly in young children and the elderly. The primary target is the vascular endothelial cells. Appearance of a characteristic renal lesion -capillary microangiopathy (a disease of the capillaries, in which the capillary walls become so thick and weak that they bleed, leak protein, and slow the flow of blood)- pathogenesis of HUS.

E.coli serotype O157:H7 or rarely, O26:H1 are associated with EHEC diarrhea

Laboratory diagnosis of VTEC diarrhea can be made by detecting VT in faeces directly or in culture isolates.

Real-time PCR with specific DNA probes for the VT₁, and VT₂, genes can be done.

VT can also be detected by its cytotoxic effects on Vero or HeLa cells.

5. Enteroaggregative E. coli (EAEC)

 Associated with persistent diarrhea, these appear aggregated in a 'stacked brick' formation on HEp-2 cells or glass.

6. Diffusely adherent E. coli (DAEC)

These strains are defined by a pattern of diffuse adherence (DA), in which the bacteria uniformly cover the entire cell surface. DEAC are less well established as pathogens and may cause diarrhea in children above 12 months of age.

  

(III) Pyogenic infections

E. coli are the most common cause of

• intra-abdominal infections such as peritonitis and abscesses.
• Pyogenic infections of perianal region
• Neonatal meningitis.

 

(IV) Septicemia

• Bloodstream invasion by E. coli may lead to fatal conditions like septic shock and systemic inflammatory response syndrome (SIRS).
• Uro-sepsis -emerging complication of urinary tract infection among immunocompromised individuals, especially diabetics and the elderly. This is detected by isolating the same organism from the urine and blood of the patient and noting the similarity in the antibiotic susceptibility pattern. They generally respond to broad-spectrum antibiotics.

 

Treatment

 E. coli commonly show multiple drug resistance, so antibiotic sensitivity testing of strains is important in treatment. Multidrug-resistant (MDR) E. coli including isolates producing extended-spectrum beta-lactamase (ESBL) have been associated with hospital-acquired UTI, catheter-associated UTI (CAUT) and urosepsis.