MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 25
ENUMERATION OF SALMONELLA AND SHIGELLA

25.1 Introduction

Salmonella is a group of bacteria that can cause diarrhoeal illness in people. Salmonellosis is a bacterial disease commonly manifested by an acute enterocolitis, with sudden onset of headache, abdominal pain, diarrhoea, nausea and sometimes vomiting. Deaths are uncommon, except in the very young, in the very old, the debilitated and immunosuppressed. However, morbidity and associated costs of salmonellosis may be high. Salmonellosis is classified as a food borne disease, because contaminated food, mainly of animal origin, is the predominant mode of transmission. Epidemiologically, Salmonella gastroenteritidis may occur in small outbreaks in the general population. However, large outbreaks in hospitals, institutions for children, restaurants are not uncommon and usually arise from food contaminated at its source, or, less often, during handling by an ill person or a carrier, but person-to-person spread can occur. They cause illnesses in humans and many animals, such as typhoid fever and enteritis. Salmonella (e.g. Salmonella enterica subsp. enterica serovar enteritidis) can cause diarrhoea. According to the World Health Organization over 16 million people worldwide are infected with typhoid fever each year, with 500,000 to 600,000 of these cases proving to be fatal.

A large outbreak of Shigella sonnei gastroenteritis occurred in Murcia Region (Southeast Spain) in the winter of 1995–1996. More than 200 people were affected. Epidemiological investigations implicated a regionally manufactured fresh pasteurised milk cheese as the vehicle of infection. The dispersed sale of the cheese resulted in a regional dissemination of the organism and people were affected in eight townships. Research suggested that an infected food handler at the cheese factory might have been the source of contamination and that the processing method might have allowed cross-contamination to occur. This study emphasises the importance of increasing the control of strict hygiene during the processing of fresh cheese, since legislation does not forbid direct contact by hand that could result in contamination of cheese even when the milk pasteurisation process was correctly performed. The higher susceptibility in young children of contracting Shigellosis and typhoid fever in addition to the high prevalence of Salmonella and Shigella—found to grow rapidly in liquid infant formula—has focused the attention of the scientific community to study the survival capabilities of these organisms in foods. In addition, the wide distribution of this commodity throughout the world creates the risk of a bioterrorism attack directed against the infant population. In this study, we investigated the survival of S. Typhi and S. dysenteriae over a 12-w period in dehydrated infant formula under ambient air or nitrogen atmospheric conditions.

25.2 Transmission and Source of Infection of Salmonella

Salmonella infections are zoonotic; they can be transmitted by humans to animals and vice versa. Infection via food is also possible. Salmonella is usually transmitted to humans by eating foods contaminated with animal faeces. Contaminated foods usually look and smell normal. Contaminated foods are often of animal origin, such as beef, poultry, milk, or eggs, but any food, including vegetables, may become contaminated. Thorough cooking kills Salmonella. Food may also become contaminated by the hands of an infected food handler who did not wash hands with soap after using the bathroom. Salmonella may also be found in the feaces of some pets, especially those with diarrhoea, and people can become infected if they do not wash their hands after contact with pets or pet feaces.

25.3 Isolation and Enumeration Principles of Salmonella and Shigella

The examination of various products for the isolation of Salmonella often requires the use of methods, different from those used in clinical and public health laboratories. Many methods used for examining these foods are essentially similar in principle and employ the step of pre-enrichment, selective enrichment, differential and selective plating for isolation and identification of selected isolates.

25.3.1 Pre-enrichment

Twenty-five (25) grams or ml of sample is added to 225 ml of buffered peptone water and incubated at 37°C for 24 hours.

25.3.2 Selective enrichment

Transfer one ml portion from pre-enrichment step to each 10 ml of selenite eosine broth and tetrathionate broth and incubated at 37°C for overnight. Then the contents of both tubes were mixed and a loopful was streaked on to the xylose lysine deoxycolate agar (XLDA), and bismuth sulphite agar (BSA) plate and Hektoen enteric agar (HEA). These plates were incubated at 37°C for 24 hours. The incubation may be continued up to 72 hours before report as nil.

25.3.2.1 Typical salmonella and Shigella colonies on different selective media

a. Xylose lysine deoxycolate (XLDA) agar

XLDA was developed to improve the recovery of enteric pathogens, especially Salmonella and Shigella species. Lactose, Sucrose, and Xylose are the fermentable carbohydrates present and phenol red is used as the pH indicator. Bacteria that ferment none of these sugars, e.g., Shigella, appear as red, translucent colonies. Yellow colonies indicate a rapid fermentation of lactose and acid pH, as demonstrated by Escherichia coli. Since Salmonella ferment xylose as readily as coliforms, a second differential mechanism, lysine decarboxylase, is utilized. Those organisms that ferment xylose as well as decarboxylate lysine exhaust the xylose rapidly and the lysine reaction causes a pH reversal to the alkaline reaction similar to Shigella. Lactose and sucrose are added in excess to prohibit this same reversion by lysine-positive coliforms. Sodium thiosulfate and ferric ammonium citrate are indicators of H₂S production only when alkaline conditions exist; Salmonella will, therefore, form red colonies with black center in 24 hours. Sodium deoxycolate is added to inhibit gram-positive growth and to retard the growth of many strains of coliforms. Many cultures of Salmonella may produce pink colonies with or without glossy black centres or may appear as almost completely black colonies (Fig. 25.1).

Fig. 25.1 Pink and black color colony of Salmonella on XLD agar

b. Bismuth sulphite agar (BSA)

Brown, grey, or black colonies; sometimes they have a metallic sheen. Surrounding medium is usually brown at first, but may turn black in time with increased incubation, producing the so-called halo effect. Bismuth Sulphite Agar is a modification of the original Wilson and Blair selective medium for the isolation and preliminary identification of Salmonella typhi and other Salmonellae from pathological material, sewage, water supplies, food and other products suspected of containing these pathogens. In this medium freshly precipitated bismuth sulphite acts together with brilliant green as a selective agent by suppressing the growth of coliforms, whilst permitting the growth of Salmonellae. Sulphur compounds provide a substrate for hydrogen sulphide production, whilst the metallic salts in the medium stain the colony and surrounding medium black or brown in the presence of hydrogen sulphide.

Fig. 25.2 Black color colony of Salmonella on BSA

c. Hektoen Enteric Agar (HEA)

HEA is used for isolating and differentiating enteric pathogens such as Salmonella, Shigella and other Gram-negative Enterobacteriaceae. It is used particularly in foods where multi-steps are followed to isolate the pathogens of gastroenteritis. The nutrients for growth are provided by the meat, peptone and yeast extract. The increased content of the peptone and the three fermentable carbohydrates (lactose, sucrose, salicin) as sources of carbon and energy reduce the inhibitory action of the bile salts on Salmonella and Shigella spp. Bromothymol blue and acid fuchsin are pH indicators. Sodium thiosulphate provides sulphur and ferric ammonium citrate is the indicator for H2S production. H2S positive colonies are blue-green to blue colonies with or without black center (Fig. 25.3).

Fig. 25.3 Blue-green to bluish colony of salmonella on HEA

d. RAMBACH Agar (chromogenic medium) for Salmonella

RAMBACH Agar is a differential diagnostic culture medium for identifying non-typhi Salmonella in foodstuffs and clinical samples. The nutritive substrates in the RAMBACH Agar enable Enterobacteriaceae to multiply readily. Sodium deoxycolate inhibits the accompanying Gram-positive flora. RAMBACH Agar enables species of Salmonella to be differentiated unambiguously from other bacteria by means of a new procedure, for which a patent application has been submitted. This is made possible by adding propylene glycol to the culture medium. Salmonellae form acid with propylene glycol, so that, in combination with a pH indicator, the colonies have a characteristic red color (Fig. 25.4). In order to differentiate coliforms from Salmonellae, the medium contains a chromogen indicating the presence of ß-galactosidase, a characteristic for coliforms. Coliform microorganisms grow as blue-green or blue-violet colonies. Other Enterobacteriaceae and Gram-negative bacteria, such as Proteus, Pseudomonas, Shigella, S. typhi and S. parathyphi grow as colorless to yellow colonies.

Fig. 25.4 Red color colony of Salmonella on RAMBACH agar

25.3.3 Biochemical tests for detection of Salmonella and Shigella

Lysine-decarboxylase broth, phenol red dulcitol broth, TSI Agar slant, tryptone broth, potassium cyanide (KCN) broth, malonate broth, indole production test, phenol red sucrose broth or purple sucrose broth, MR-VP broth, etc.

Indole and H₂S production

It is done to determine the ability of organisms to degrade the amino acid tryptophan to indole. Tryptophan is an essential amino acid that can undergo oxidation by some bacteria possessing the enzyme tryptophanase to indole. Presence of indole is detected by adding Kovac’s reagent composed of p-dimethyl amino benzaldehyde, butanol & hydrochloric acid. Indole is extracted from the medium into the reagent layer by the acidified butanol and forms a complex with p-di methyl amino benzaldehyde to give cherry red color (Fig. 25.5). Some bacteria are capable of degrading cystine & certain other sulphur containing compounds with the formation of H2S. Heavy metals are usually incorporated to detect the H2S production. Addition of 3 – 4 drops of Kovac’s Indole reagent on the surface after incubation indicated production of Indole from tryptophan amino acid.

Fig. 25.5 Indole test

Citrate Utilization

A slant of Simmons Citrate Agar was used & streaking was done from inoculum and incubated at 37°C for 24 hour, color change from green to blue shows citrate utilization positive. This test determines the ability of organism to use citrate as a source of carbon in the absence of glucose & lactose this ability depends upon citrate permeases that facilitates its transport into the cell. Citrate is the major intermediate of Kreb’s cycle & produced by condensation of active acetyle CoA with oxaloacetic acid. During its conversion to pyurvic acid the medium becomes alkaline as CO₂ generated combines with sodium and water to form Na₂CO₃ whose presence is detected by the bromothymol blue indicator which undergoes color changes from green to deep blue.

A slant of Simmons Citrate Agar was used and streaking was done from inoculums and incubated at 37°C for 24 hours. Color change from green to blue shows citrate utilization positive.

Urease Test

It depends upon the ability of micro organism to degrade urea in the presence Urease enzyme produced by bacteria and is indicated by phenol red indicator which undergoes color change from peach to pink. If on streaking a Urease agar slant and on incubating it at 37°C for 24 hrs color changes to pink the bacteria is Urease positive (Fig. 25.6).

Fig. 25.6 Urease test

Methyl red reduction (MR) Test)

This test enables the microbiologist to determine the pathway being used to ferment glucose, and in the process helps to determine the species of bacteria that is most likely present. MR/ VP is actually two tests: The methyl red (MR) test determines whether or not large quantities of acid have been produced from mixed acid fermentation of glucose. End products of this pathway include lactic, acetic, formic and succinic acids. The Voges-Proskauer (VP) test determines whether a specific neutral metabolic intermediate, acetoin, has been produced instead of acid from glucose. Acetoin is the last intermediate in the butanediol pathway. The pH indicator methyl red detects the acidic end product by formation of red color (Fig. 25.7). A tube of MR-VP broth is inoculated with suspected colony incubated at 37°C for 24 hours. After incubation pour 2-3 drops of methyl red.

Fig. 25.7 Methyl red reduction test

Triple sugar iron (TSI) Test

Triple sugar iron agar is composed of three sugars viz. glucose, sucrose and lactose. Ferrous sulphate and phenol act as indicator, which is employed for detection of fermentation of sugars, indicated by the change in color due to production of organic acid. The appearance of bubbles in the butt or pushing of the entire slant indicates production of gas from the fermentation of sugar by an organism. H2S production by an organism is indicated by reduction of ferrous sulphate of medium to ferric sulphate, which is manifested as black precipitate (Fig. 25.8). TSI slant stabbed and streaked and incubated at 37°C for 24 hours. After incubation sugar fermenting bacteria has fermented the sugars and may produce H2S.

Fig. 25.8 Triple sugar iron test

Reactions observed on TSI slants

Biochemical reactions of Salmonella and Shigella