The efficacy of disinfectants on abattoirs’ Candida albicans isolates in Niger Delta region

Test materials and reagents

A selective medium Sabouroud Dextrose Agar (SDA) was used for the cultivation of Candida albicans. The following equipments were used-Microscope, Microscopy slides, cover slips, cotton wool, sterile swab sticks and blood serum, safranin, lugos iodine, gram differentials, crystal violet.

Source of sample

Five different abattoirs (slaughter floor) in an open environment were sampled using sterile swab sticks, within Port Harcourt metropolis, Rivers State Nigeria. The abattoirs include Agip, Woji, Trans Amadi, Rumuokoro and Rumuodara. Samples were collected with sterile cotton swabs moistened with 1ml of 0.1% NaCl peptone solution, from a surface area of preferable 20cm, marked with sterile template and swabbed 10 times from top to bottom¹⁰. Then for wet areas dry sterile cotton swabs were used. The swabs were held in sterile forceps and the sampled surfaces were randomly collected every other week for a period of one and a half year starting from January 2005 to June 2006. The samples were taken to the laboratory immediately and cultured.

Media

Growth media used for enumeration and characterization were selective according to the cultural requirements of fungi. Sarboroud Dextrose Agar (SDA) was used for Candida albicans cultivation and the same agar medium for stock culture. The medium was steam sterilised at 121°C for 15 minutes.

Isolation of the fungi

The spread plate method was used, serial dilutions of the swab sample was made by adding 1.0ml of phosphate buffer into the swab container and mixed thoroughly. From this, serial dilution was carried by adding 9ml of sterile distilled water to give 10^-1, 10^-2, 10^-3, 10^-4 and so on. 0.1ml was plated in triplicate onto sterile selective medium mentioned above. The sets of plates were then incubated at 37°C. Resulting pure colonies were transferred onto SDA agar for subsequent characterization and identification.

Characterisation and identification

Pure cultures of fungi (yeast) Candida albicans isolated were characterized and identified on the basis of their cultural, morphological and biochemical properties and based on their macroscopic appearance on culture medium, and type of asexual spores produced and germ tube test, also identified by reference to Illustrated General of Imperfect Fungi¹¹ and Fungi in Agricultural soil¹². The test yeast used is Candida albicans.

Preparation of yeast suspension

Organisms used in this experiments were, Candida albicans. Candida albicans were grown in Sarboroud Broth overnight. Cultures were centrifuged at 512g (sigma model 3k-1) for 10mm and resulting cell pellets resuspended in 0.1% peptone.

Preparation of test disinfectants

Disinfectants parachlorometaxylenol (PCMX) (dettol), savlon, purit and Sodium hypochlorite (jik) active were diluted in sterile distilled water prior to use to 10%, 20%, 40% and 70% concentrations these products were obtained from Reckit Benckiser Nigeria Limited Lagos, Nigeria, Johnson & Johnson, Lagos; CAP Plc (Chemical and Allied Products), Lagos respectively and used without further purification. The pH of the mixtures was controlled by the addition of HCL or NaOH as appropriate.

Suspension test (Traditional Plate Count Method)

Approximately 0.1ml of a yeast suspension approximately 1 × 10⁹ bacteria (Cfu/ml) was added to the test disinfectants (10ml), mixed thoroughly and left at room temperature for a specified contact time starting from 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min. This experiment occupied 21 test tubes each, for the experiment was carried out in triplicate. Following contact an aliquot (1ml) was transferred to universal quenching agent, UQA (9ml of a solution containing 1g peptone, 5g. Tween-80, 1g sodium thiosulphate and 0.7g lecithin 1^-1 of deionised water pH7), for up to 60 min to inactivate the disinfectants. The quenched solution were serially diluted in sterile distilled water and survivors enumerated on SDA Agar (Oxoid) using 0.1ml spread plates. The colonies on the plates were counted after incubation at 37°C for 48hrs. Control test did not contain the disinfectants, but only the serially diluted suspension was plated and counted.

Composition of disinfectants used

Dettol’s composition include Chlorxylnol B.P.C. 4.8% w/v, Oleum Pini Aromaticum 9.0% v/w Denatured spirits 11.3% w/v, sapo vegetails 5.8% w/v, Saccharum Ustumqs, aqua ad 100vol. Purit contains Chlorhexidine Gluconate B.P. 0.3% w/v and Centrimide B.P. 3.0% w/v. Savlon constitutes n-Propyl alcohol 2.84% m/v, preservative Chlorhexidine gluconate 0.3g and Centrimide 3.0g all in 100ml. Hypochloride contains sodium hypochlorite as an active ingredient.

Phenol co-efficient test

Rideal-Walker Phenol co-efficient experimental test method for the evaluation of test disinfectants as jointly reported by S. Rideal and J.T.A. Walker in 1903¹³ was the first real attempt to evaluate the effectiveness of disinfectants on a qualitative basis. The standard procedure for this test was published by the British Standard Institution.

The microbial culture for phenol co-efficient testing

The test organism was Salmonella typhi NCTC 786 obtainable from the National Collection of typed cultures in London. It was supplied in the freeze-dried form and reactivated by weekly sub-culture on a Rideal-Walker agar slope, incubated at 37°C for 24hrs and then stored at room temperature until the time for another sub-culture. Only cultures that were 22 to 26 hours old and only those from the 3^rd to the 14^th subculture were used for the test.

Disinfectant dilutions

The standard phenol solution contained 1g of phenol in 95, 100, 105, 110 and 115ml of solutions made with sterile distilled water. For the test disinfectants dettol, purit, savlon and jik, four dilutions were made, varied in arithmetic series, changing by the unit of 50; these are 1:200, 1:250, 1:300 and 1:350.

Procedures

A volume of 5ml of each of the dilutions of test disinfectants and standard phenol was placed in separate sterile test tubes. A 24 hour broth culture of the test organism was also prepared. The dilutions and the culture were placed in a water bath maintained at 17–18°C. When the contents of the tubes and the culture had attained the operational temperature, 0.2ml of the culture was transferred to each of the dilution and shook gently to begin the action of the germicide on the cells. At 2½ minute interval following the incubation of the tubes, a standard loopful of the reaction mixture was transferred to 5ml of sterile nutrient broth (the recovery medium) in a tube. In this way, each reaction mixture was sub-cultured into four separate test tubes of the recovery medium at intervals of 2½, 5, 7½ and 10 minutes respectively. The tubes of recovery medium were incubated at 37°C for 48 hours after which the presence or absence of growth in each tube was recorded.

Discussion

Laboratory testing of disinfectants was carried out by assessing the sensitivity of various Candida albicans strains to biocidal agents using microbial suspension test. Similar observations have been made on bacterial suspensions such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis, Proteus mirabilis and Escherichia coli on disinfectants¹⁴. Disinfectants used for the test are chlorine based and these included dettol, savlon, purit and jik containing sodium hypochlorite.

From the results of this investigation, it was discovered that Candida albicans species were readily isolated from raw meat and from the equipments used in abattoirs. Similar observation has been made by Haniel (1989)¹⁵ from his investigation and he stated that undetected Salmonella species in instrument or food products such as raw meat could pose a health risk, because they are not heat treated and raw animal tissues may harbor many potentially pathogenic organisms, including bacteria and yeasts e.g. (Salmonella, Escherichia coli, Candida species) and parasites. For these reasons, the Food and Dietary Association, FDA has promulgated a regulation specifying that meat scraps or other similar animal products are adulterated when they are found upon examination to be contaminated with Candida and Salmonella microorganisms. According to the investigation of Kirk (2005)¹⁶ from Minnesota Department of Agriculture, title 21 code of Federal Regulations (CFR) Part 500.35 stated that not only are the animals consuming the product at risk of infection by organism contained in the raw tissues, but the people handling the product are also at risk. He then stated that adequate heat treatment is the most effective and efficient means of mitigating this risk. Because he reported some processes currently used, such as freezing or freeze drying, do not achieve the same degree of effectiveness as heat treatment. Candida species were readily isolated from the tables where meat carcasses are displayed. Similar observation was made by dEnfert and Hube (2007)² where he stated that these fungi and bacteria are potentially fatal to human especially when ingested and when the immune system is compromised. Ingestion of these yeasts in meat products could be very dangerous or when it has a contact with urinogenital tracts, during defecating or urinating, from contaminated hands and fingers. These fungi cause vomiting and diarrhea, many ailments such as urinary tract infection, even the long term effects in the tracts could result to blockage of fallopian tubes in women and sterility in men. This could be severe in immunocompromised infected people, while healthy people can recover from these infections. Symptoms of candidiasis usually occur within two to ten days of ingesting the yeast (Cotter and Kavanagh, 2000).

Candida albicans strains were isolated in number virtually from all the studied samples which are cutting tables. Similar observation was made by Gardner (1993)¹⁷, and dEnfert and Hube, (2007)². The authors worked on the importance of Quality Assurance and Food Safety in Modern Food Production System, they stated that in countries that have implemented a consistent mandatory meat inspection, this harvest food safety procedure and the more and more stringent rules for post-harvest safety measures improving the hygiene standards during slaughter, meat process and distribution have led to a remarkable decline of meat related food-borne disease. And in respect to meat management, it is advised that any animal that arrives at the abattoir should be slaughtered immediately, but animals which have not been slaughtered within 12 hours of the arrival should be fed, and subsequently be given moderate amounts of food at appropriate intervals. Also suitable drinking water should be available to the animals on their arrival and at all times to animals in lairages unless they are to be slaughtered immediately. There has been criticism of the methods of preparation hutching, and killing within some slaughter houses and in particular of the speed with which the slaughter is sometimes conducted^18–21 (also see PETA video taken inside AgriProcessors Inc. in Iowa in 2004. Warning: graphic images). There has also been criticism of the methods of transport of the animals, who are driven for hundreds of miles to slaughter houses in conditions that often result in crush injuries and coma en route, a good condition for microbial contamination and infection^22,23. Vibrio cholera the causative agent of cholera, is sensitive to the low pH found in the human stomach, a high infectious dose of 10⁸ bacteria is required for the onset of severe cholera; however the infectious dose can drop to 10⁴ bacteria in individuals who produce less stomach acid including young children, the elderly and those who take antacids.

From the overall report of this study, dettol at 10% is an appropriate disinfectant or sanitizer that can be used to sanitize the abattoirs cutting tables before any meat products are displayed for sale and should be rinsed off with sterile and clean water after 10 minutes of application. The need for good quality water becomes more and more pressing in the face of the increasing pollution of our water bodies by human activities such as those related to abattoirs services, agriculture and industries. Good water is essential for washing the slaughtered animals, displaying tables, equipments use such as knives, apron, slaughter floor, besides animals themselves need portable water for drinking before slaughter while kept in lairage (see: "Molecule of the Month: Dettol" and "'Dettol Man’ cleans himself to death"). Revankar and Lester (2007)²⁴ stated that overuse of dettol could provide a selective environment in which existing resistant bacteria can grow with little competition. In Nigeria, there is a need for the receiving streams around these abattoirs to be treated because people living in these environments defecate into the water bodies, this act should be discouraged since these are flowing rivers and could serve as sources of drinking and cooking to neighboring villages nearby. This could be the cause of high Vibrio isolates in these abattoirs.

This law of meat inspection makes food safer and is the principle of food hygiene being the most important means of protecting consumer against food-borne health risks, in order to reduce food infection. Nevertheless, the receiving streams around these abattoirs should be treated because people living in abattoirs environment defecate inside these water bodies, this act should be discouraged since, they are flowing rivers and could serve as sources of drinking water to neighboring villages where their flow goes. This may be one of the reasons why greater numbers of Candida species were isolated from these study abattoirs.

The federal government and local government should be blamed for these vices, because the abattoirs’ chairman, the butchers, the sellers and the inhabitants of these areas have the propensity to live in such a stench environment due to lack of both intuitive and acquired knowledge in boosting the standard of hygiene in abattoirs. Therefore, the government should enforce a legislative law with respect to the standard of hygiene that should be practiced among the abattoirs users, and a circular should be passed round in such a way that everybody should be a participant and there should not be a room for exculpation by any member whatsoever. And anyone that violates this rule should be brought to face the music.

It is advised, due to the overall report of this study, Candida albicans is more sensitive to savlon than to other test disinfectants in Port Harcourt abattoirs, hence savlon should be recommended as a potent sanitizer on the abattoirs’ displaying tables and in Nigeria as a whole. Savlon was recommended for destroying Candida albicans contaminants within 10 to 20 minutes of exposure to this disinfectant. Phenol co-efficient experimental calculation showed that phenol was less potent than dettol having phenol co-efficient of 7, on Salmonella sp. 2 for Dettol on Pseudomonas sp. but having 1 on Pseudomonas sp. purit, and savlon were more potent than phenol having phenol co-efficient of 1.7, 2 on Salmonella sp. and Pseudomonas sp. respectively, but equal in phenol co-efficient with phenol on Staphylococcus sp. of 1. According to the investigation of De bruin (1976)²⁵ who stated that phenol is generally protoplasmic poison and was the first antiseptic to be employed by Joseph Lister (1912–1927) so it does not make it more potent than the newly produced antiseptics or disinfectants due to the development science and technology. Though, Keswick et al. (1985)²⁶ stated that chemical agents used as germicides destroy microbial pathogens using a variety of biochemical mechanisms, including cell membrane.

The results of this study are in agreement with that of Le Chevalier et al. (1998)²⁷ which stated that pathogens resistance to chemical disinfectants is typically an inherent characteristic and not one that is acquired or initiated due to exposure to disinfectants. He also stated that mechanisms of resistance of microorganisms have evolved with specialized structures aiding in their survival. Certain Candida albicans strains produce a gelatinous material known as proteins to form a biofilm. Biofilm help organisms stick to environmental surfaces and physically protect them from exposure to harmful disinfectants or other detrimental environmental conditions²⁸. This slimy substance can be 100 or more times the mass of the bacterial cells. While the bacteria that initiate biofilm are often not harmful, pathogenic bacteria may also stick to the biofilm and share the protective nature of the environment Hardie et al. (2009)²⁹. Candida species was very sensitive to most of the tested disinfectants in this study. These sensitivity and resistant attributes exhibited by these microbial isolates may be due to some of the innate characteristics stated above. The high incidence of water and food borne sanitation diseases is one of the major problems confronting our country, Nigeria, due to unhygienic abattoir environments.

In conclusion, the result of this research has shown that for our meat products to be free from Candida contaminants, savlon is an effective sanitizer on abattoirs cutting tables and should be applied at 10% concentration for 10 minutes and then rinsed off with sterile water before meat products are displayed for sale. This automatically reduces the yeast load on these objects.

The following recommendations are made as follows:

The federal government should set up a group of Environmental Health workers which will comprise of Environmental Microbiologists, Veterinary Doctors, Medical Doctors and Medical Laboratory Scientists that would play a significant role in the promotion of the health of the people. Adequate sanitation and safe water are fundamental prerequisites for Nigeria abattoirs as a whole for better health, otherwise other programs may not succeed and there can be lasting improvement of public health. These should be vigilantly implemented by these personnel listed above including sanitary engineers and inspectors, and should be directly involved in this exercise.

It was discovered from this study that every abattoir has a receiving stream and this stream or river could serve as a source of drinking water to some rural dwellers around the vicinity, hence the butchers and other inhabitants in abattoirs defecate into this same river. The government should abrogate these vices by providing good toilet facilities for them, for water quality control, proper excreta disposal and Food sanitary inspection should be practiced (Food Law internet Project (FLIP) 2000. Food safety Administration. Authorized information provided to the WHO. University of Reading UK, Michigan State University, U.S.A)³⁰.