In a recent study, RAPD analysis has been used to examine the genetic relationship among strains of L. Great genetic variability among the isolates of L. Further, there was also great genetic variability within strains isolated from food processing environments even within the same company and in one plant the presence of some strains had persisted for years. The work did not support the presence of strains as defined by RAPD typing better suited for survival in specific food types or food processing environments, indicating that although differences may be found in the virulence of individual strains, all L.
The methods tested, number of laboratories engaged, isolates and the results and conclusions of the trial according to the authors are shown in Table 4. No single method was found to be optimal to type all isolates used in the exercise. One issue of major concern is the increase in the number of bacterial strains that are developing resistance to antibiotics, especially to several antibiotics simultaneously multi-resistance.
Rapid Detection and Characterization of Foodborne Pathogens by Molecular Techniques
Resistance to antimicrobials existed before the introduction of antibiotics for the treatment of human and animal infections. The increase in the number of resistant strains, as well as in the number of antibiotics to which they show resistance, is attributed to their being selected and their dissemination enhanced, primarily due to the misuse and abuse of these drugs for treatment and prophylactic purposes.
There is a genetic basis for resistance to antimicrobial substances. Susceptible strains may acquire the genes coding for the resistance mechanisms by conjugation between bacteria of the same or similar species , transformation uptake of DNA from unrelated species , or transduction the transfer of DNA via a bacteriophage European Commission, and references therein; McDonnel and Russell, Antibiotic abuse may unfortunately not be the only problem: the use of cleaning and disinfectant agents in hospitals, by the food industry, and at private households, seems to have had the undesirable side-effect of permitting survival and therefore selection of strains resistant to disinfecting agents.
Disinfectants based on quaternary ammonium compounds QAC, such as benzalkonium chloride BC , cetylpyridinium chloride, cetrimide, etc. Regrettably, there seem to be similarities between bacterial resistance to antibiotics and to biocides: the spread and maintenance of QAC-resistant genes in staphylococci isolated from clinical environments and the food industry may be due to the selective pressure caused by the use of cationic biocides Stickler and King, Gram-negative bacteria that have developed resistance to cationic biocides may also be resistant to some antibiotics Russell, , Outer membrane changes are believed to be responsible for the non-specific increase in resistance; but also increased tolerance to disinfectants can be due to the presence of energy-dependent efflux pumps in the cell membrane Gillespie, May and Skurray , ; Noguchi et al.
Among staphylococci, the genes encoding multidrug exporter proteins can be divided into two families based on DNA homologies and phenotypic properties Littlejohn et al. Known resistance genes to QACs described in clinical staphylococci are generally plasmid borne and widely distributed in the environment Lyon and Skurray, ; Mayer et al. Results of the WHO-sponsored international collaborative study to evaluate methods for subtyping of L. Each participant laboratory received a set of 80 L. Complete agreement between the six laboratories on 49 Reproducibility of serotyping L.
Serotyping of L. Problems may however, be encountered mainly with the flagellar antigenic factors. There is a need, therefore, for preparing antisera of good quality from which efficient antigenic factors can be obtained. Most of the epidemiologically related strains were correctly identified by the four groups of investigators. In general this study reconfirmed that PFGE is a very accurate and reproducible method for fine structure comparison and molecular typing of L.
Using three different mer primers the median reproducibility of the RAPD-results obtained by the six participants was Failure in reproducibility was mainly due to results obtained with one particular primer. The number of epidemiological groups found to be homogeneous varied from median However, for some groups an inhomogeneity was found by the majority of participants.
EcoRI was the least discriminatory. The profiles generated by Hha1 and CfoI were not fully stable for some closely related isolates. The size and the number of restriction bands generated by Hha1 in one laboratory and Cfo1 in another laboratory were directly comparable, indicating that REA may be used as a definitive typing method for L. The stability of the REA-types needs further elucidation in order to establish firm differentiation criteria for comparison of isolates. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types ETs varied between 14 and This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L.
Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility. Phage typing was performed using an international phage set in five laboratories and phage sets unique to two laboratories. Testing of cultures sent in duplicate showed similar levels of reproducibility to those previously reported. Analysis of results from groups of epidemiologically related cultures showed a high level of agreement in all laboratories.
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Patterns of phage susceptibility were relatively stable on retesting strains in the same laboratory after long periods of time. However, there was limited comparability between results obtained from testing the same cultures using the same phages in different laboratories. It is recommended that the phages in the international set be reviewed, and that better inter-laboratory reproducibility may be achieved by standardization of phage suspensions, propagation strains and methodology, together with the use of centrally propagated phages.
Restriction fragment length polymorphism RFLP analysis using ribotyping and Southern hybridization with two probes from L. The seventh laboratory used Ncil to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. The discriminating power of the combination of two probes used by lab 7 was 0. Ribotyping and the cloned probes used by lab 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains identified by other subtyping methods included in three apparently epidemiologically related groups.
Ribotyping did not discriminate between strains of serotypes 4b and 4b X in one epidemiologically related group of strains; one cloned probe used by lab 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants. Recently, Sidhu et al. The same group of researchers Anthonisen et al. Gene transfer both between different genera and between species within a genus may occur with the possible spread of resistance genes to pathogenic strains see Im et al.
Thus, clinical strains of QAC-resistant Staphylococcus aureus have been isolated in several countries from clinical environments Leelaporn et al.
One of the plasmids that confers QAC-resistance, called smr small multidrug resistance , has been found in enterococci and staphylococci Sasatsu et al. The commonly used household disinfectant pine oil has also been reported to induce the development of resistant strains. Moken, McMurray and Levy found that mutants of E. When pine oil was used to select resistant mutants of S. Consequently, it seems that the acquisition of genes conferring resistance to disinfectants may also help the bacteria to tolerate some antimicrobial substances.
The opposite, on the other hand, may not always be the case: as mentioned above, antibiotic-selected Mar mutants were resistant to pine oil Moken, McMurray and Levy, but Kucken, Feucht and Kaulfers found that multiple antibiotic-resistant Gram-negative bacteria are not necessarily more resistant to quaternary ammonium compounds benzalkonium chloride and cetyltrimethylammonium bromide. Similar results were obtained by Rutala et al. Plasmid, and also some chromosomal genes are considered to be involved in the development of resistance to disinfectants and antibiotics: the expression of the E.
The Opinion of the Scientific Steering Committee on Antimicrobial Resistance - 28 May European Commission, indicates very clearly the need to reduce the overall use of antimicrobials.
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This implies elimination of unnecessary and improper use, more precise diagnoses of infectious agents, and monitoring of antimicrobial resistance. In order to design sensible disinfection procedures, Langsrud and Sundheim suggested the convenience of alternating the use of QACs with chlorine, phenolics, and alkylaminoacetate, to avoid the build-up of resistant strains. In addition there is a need for more rapid and accurate microbiological diagnostic tools to identify the resistance genes for the different antimicrobials carried by the strain under examination. Known sequences are given in some of the listed publications cited above, but new sequences are continuously being published and added to the available databases.
Protozoa, platyhelminths and nematodes can also cause food-borne diseases. Of special relevance to fish products are trematodes and some nematodes, whose taxonomical classification is shown in Table 5. Trematodes, or flukes, infect the liver, lungs or blood of mammals. Paragonimiasis is caused by species of the genus Paragonimus.
Clonorchiasis, caused by Clonorchis Opisthorchis sinensis, the Chinese liver fluke, causes oriental biliary cirrhosis. Opisthorchiasis is caused by Opisthorchis felineus and O. Clonorchis and Opisthorchis are very similar both morphologically and in their life cycles Figure 4.
Methods for Rapid Detection of Foodborne Pathogens: An Overview
The infections are mostly contracted from the ingestion of raw or improperly cooked crabs or fish. Paragonimiasis is acquired by ingesting crustaceans infected with metacercariae which hatch and bore their way as young flukes through the walls of the duodenum and then move to the lungs, where they become enclosed in connective tissue cysts Figure 5. Golden brown eggs may appear in sputum 2 to 3 months later. Clonorchiasis is due to the ingestion of fish containing the metacercariae: the cyst wall dissolves in the intestine, the young flukes emerge and migrate through the body to the bile ducts of the liver Figure 4.
Both paragonimiasis and clonorchiasis are diagnosed by demonstrating the presence of eggs in sputum, stools or duodenal fluid. There are some ELISA tests available for the infection, not for the parasite but they give cross-reactions with infections caused by related species. Anisakiasis is caused by the ingestion of fish flesh infected with nematodes: either Anisakis simplex herring- or whaleworm or Pseudoterranova formerly Phocanema decipiens cod- or sealworm.
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