Cronobacter Support
02-19-2009, 07:30 PM
Subtyping of E. sakazakii
To identify sources of infection, subtyping has been conducted on both environmental and clinical isolates. Phenotypic typing is based on secondary characteristics of bacteria including biochemical reactions, antibiograms, serotyping, and bacteriophage typing. However, the power of these phenotypic methods to differentiate E. sakazakii isolates has been poor. For example, fewer differential patterns have been discerned through antibiograms than have been found with molecular typing methods such as ribotyping. Variability in antibiotic resistance that occurs among different colonies (clones) selected from each isolate upon re-testing also severely hampers the usefulness of antibiograms.
The most common molecular methods used for epidemiologic subtyping of E. sakazakii include chromosomal DNA restriction analysis, plasmid typing, ribotyping, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) typing. A study by Health Canada investigators characterizing E. sakazakii isolates from dried infant formulas and clinical isolates revealed that biotyping should be used in outbreak situations as a screening tool, whereas RAPD or PFGE should be used to more definitively establish relatedness among isolates. Distinguishable differences in isolates of E. sakazakii have been reported with these latter genomic subtyping methods, even in situations when no differences were observed by ribotyping. Overall, a high discriminatory index of 1.000 was reported in PFGE analysis of 18 E. sakazakii isolates that indicated a high level of genomic diversity in these strains. The disadvantage in using the electrophoretic typing techniques, however, is the inability to compare results between laboratories because such methods have not been standardized.
Antibiotic Susceptibility of E. sakazakii
Reports from 1960-1999 of antibiotic susceptibility of E. sakazakii indicate the organism is typically susceptible to ampicillin, tetracycline, chloramphenicol, gentamicin, and the third-generation cephalosporins. However, a study of E. sakazakii isolates from patients at the University of Massachusetts Medical School in 1995–1996 revealed these bacteria were uniformly resistant to ampicillin, cefazolin, and extended-spectrum penicillins, and were not uniformly susceptible to the third-generation cephalosporins or the quinolones. The occurrence of antibiotic resistance observed in the UMass isolates is consistent with the recent trend of increased acquisition of antibiotic resistance among all Enterobacter species. Although E. sakazakii isolates are typically susceptible to aminoglycosides, such antibiotics are not recommended for primary treatment because of poor penetration into the central nervous system.
Pathogenicity of E. sakazakii
To date, specific mechanisms of pathogenicity or associated virulence factors of E. sakazakii have not been identified. Hence, it is unknown whether the predilection of E. sakazakii for neonates reflects intrinsic virulence of the organism or the pathogen’s opportunity to be an early colonizer. Given that there is an immunologic similarity between enterotoxins of E. coli and other coliforms, there is a strong possibility that such toxins, mediated by plasmid(s) transmissible to other organisms resident in the human intestine, are also virulence factors of E. sakazakii. In a review article, Nazarowec-White and Farber reported that E. sakazakii was pathogenic in suckling mice when inoculated orally and intraperitoneally at levels of 105 and 103 CFU, respectively. They reported the production of an enterotoxin-like compound by E. sakazakii, but provided no supporting data.
Ecology of E. sakazakii
Powdered milk substitute infant formulas are the principal sources from which E. sakazakii has been isolated. E. sakazakii was isolated from 20 of 141 dried infant formulas from 35 countries in a 1988 survey. A Canadian study of dried infant formulas determined that E. sakazakii was present in contaminated samples at cell numbers less than 1 cfu/100g. Detection of such low levels of contamination requires the use of enrichment culture methods. Lack of effective enrichment procedures may account for the inability of investigators prior to 1990 to detect the organism in environmental samples, including surface water, soil, mud, rotting wood, grain, bird dung, rodents, domestic animals, cattle and raw cow’s milk. Since 1990, E. sakazakii has been isolated from ultra-hightemperature (UHT) milk in cartons, ground meat, fermented bread (khamir) and thermal mineral water springs.
In formula powders, the source of contamination is not known, but post-process contamination from the factory environment seems likely because these products receive thermal treatments prior to and during drying. Dupont Qualicon reports on their internet site (qualicon.com/entsak.html) that E. sakazakii contamination was detected in three factories used to produce dried infant formulas. Using the RiboPrinter® system to discriminate 30 E. sakazakii isolates from one factory, eight RiboGroup pattern sets were identified. Personnel movement and unhygienic practices were identified by Dupont Qualicon personnel as mechanisms for cross-contamination within the processing facilities. A study sponsored by Orkin Pest Control, Atlanta, GA, reveaed the importance of proper sanitation practices in foodservice to prevent E. sakazakii contamination, as household flies collected at the backdoor areas and rear dumpsters of four restaurants in Gainesville, FL, were found to carry E. sakazakii. Similarly, E. sakazakii has been isolated from the gut flora of fruit flies, with no differences in distribution between males and females. Surprisingly, the organism was only isolated from a culture of fruit flies propagated in 1998 and not from a colony of flies over 30 years old. Since flies have been demonstrated to spread bacteria to equipment surfaces, it is important that they be prevented from entering food processing facilities.
To identify sources of infection, subtyping has been conducted on both environmental and clinical isolates. Phenotypic typing is based on secondary characteristics of bacteria including biochemical reactions, antibiograms, serotyping, and bacteriophage typing. However, the power of these phenotypic methods to differentiate E. sakazakii isolates has been poor. For example, fewer differential patterns have been discerned through antibiograms than have been found with molecular typing methods such as ribotyping. Variability in antibiotic resistance that occurs among different colonies (clones) selected from each isolate upon re-testing also severely hampers the usefulness of antibiograms.
The most common molecular methods used for epidemiologic subtyping of E. sakazakii include chromosomal DNA restriction analysis, plasmid typing, ribotyping, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) typing. A study by Health Canada investigators characterizing E. sakazakii isolates from dried infant formulas and clinical isolates revealed that biotyping should be used in outbreak situations as a screening tool, whereas RAPD or PFGE should be used to more definitively establish relatedness among isolates. Distinguishable differences in isolates of E. sakazakii have been reported with these latter genomic subtyping methods, even in situations when no differences were observed by ribotyping. Overall, a high discriminatory index of 1.000 was reported in PFGE analysis of 18 E. sakazakii isolates that indicated a high level of genomic diversity in these strains. The disadvantage in using the electrophoretic typing techniques, however, is the inability to compare results between laboratories because such methods have not been standardized.
Antibiotic Susceptibility of E. sakazakii
Reports from 1960-1999 of antibiotic susceptibility of E. sakazakii indicate the organism is typically susceptible to ampicillin, tetracycline, chloramphenicol, gentamicin, and the third-generation cephalosporins. However, a study of E. sakazakii isolates from patients at the University of Massachusetts Medical School in 1995–1996 revealed these bacteria were uniformly resistant to ampicillin, cefazolin, and extended-spectrum penicillins, and were not uniformly susceptible to the third-generation cephalosporins or the quinolones. The occurrence of antibiotic resistance observed in the UMass isolates is consistent with the recent trend of increased acquisition of antibiotic resistance among all Enterobacter species. Although E. sakazakii isolates are typically susceptible to aminoglycosides, such antibiotics are not recommended for primary treatment because of poor penetration into the central nervous system.
Pathogenicity of E. sakazakii
To date, specific mechanisms of pathogenicity or associated virulence factors of E. sakazakii have not been identified. Hence, it is unknown whether the predilection of E. sakazakii for neonates reflects intrinsic virulence of the organism or the pathogen’s opportunity to be an early colonizer. Given that there is an immunologic similarity between enterotoxins of E. coli and other coliforms, there is a strong possibility that such toxins, mediated by plasmid(s) transmissible to other organisms resident in the human intestine, are also virulence factors of E. sakazakii. In a review article, Nazarowec-White and Farber reported that E. sakazakii was pathogenic in suckling mice when inoculated orally and intraperitoneally at levels of 105 and 103 CFU, respectively. They reported the production of an enterotoxin-like compound by E. sakazakii, but provided no supporting data.
Ecology of E. sakazakii
Powdered milk substitute infant formulas are the principal sources from which E. sakazakii has been isolated. E. sakazakii was isolated from 20 of 141 dried infant formulas from 35 countries in a 1988 survey. A Canadian study of dried infant formulas determined that E. sakazakii was present in contaminated samples at cell numbers less than 1 cfu/100g. Detection of such low levels of contamination requires the use of enrichment culture methods. Lack of effective enrichment procedures may account for the inability of investigators prior to 1990 to detect the organism in environmental samples, including surface water, soil, mud, rotting wood, grain, bird dung, rodents, domestic animals, cattle and raw cow’s milk. Since 1990, E. sakazakii has been isolated from ultra-hightemperature (UHT) milk in cartons, ground meat, fermented bread (khamir) and thermal mineral water springs.
In formula powders, the source of contamination is not known, but post-process contamination from the factory environment seems likely because these products receive thermal treatments prior to and during drying. Dupont Qualicon reports on their internet site (qualicon.com/entsak.html) that E. sakazakii contamination was detected in three factories used to produce dried infant formulas. Using the RiboPrinter® system to discriminate 30 E. sakazakii isolates from one factory, eight RiboGroup pattern sets were identified. Personnel movement and unhygienic practices were identified by Dupont Qualicon personnel as mechanisms for cross-contamination within the processing facilities. A study sponsored by Orkin Pest Control, Atlanta, GA, reveaed the importance of proper sanitation practices in foodservice to prevent E. sakazakii contamination, as household flies collected at the backdoor areas and rear dumpsters of four restaurants in Gainesville, FL, were found to carry E. sakazakii. Similarly, E. sakazakii has been isolated from the gut flora of fruit flies, with no differences in distribution between males and females. Surprisingly, the organism was only isolated from a culture of fruit flies propagated in 1998 and not from a colony of flies over 30 years old. Since flies have been demonstrated to spread bacteria to equipment surfaces, it is important that they be prevented from entering food processing facilities.