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Surgical Resources
Position Statement on the Use of Alcohol as a Sterilant / Disinfectant for Rodent Survival Surgery
(Prepared by Michael J. Huerkamp, DVM, Diplomate ACLAM)
Background
The NRC Guide for the Care and Use of Laboratory Animals discourages the use of alcohol as a liquid chemical sterilant/disinfectant for the preparation of surgical instruments with the statement that “alcohol is neither a sterilant nor a high level disinfectant (1).” This statement seemingly is the basis for the rejection of alcohol as a skin and surgical instrument disinfectant in rat and mouse survival surgery by regulatory and accreditation bodies.
According to the guidelines of the Association for Practitioners in Infection Control (APIC), ethyl and isopropyl alcohol are readily bactericidal against vegetative forms of bacteria and tubercle bacilli as well as being fungicidal and virucidal (2). The APIC states that alcohol works effectively in disinfecting hard, metal surfaces, with the caveat that it is not appropriate for surgery in the presence of (a) protein-rich contaminants or (b) bacterial spores (2). The relevance of these admonitions as applied to rodents are questioned given (a) the historical performance of alcohol in rodent survival surgery and (b) the low risk, providing that basic principles of aseptic technique are followed, of encountering either proteinaceous contaminants or bacterial spores in the rodent surgical theater.
With respect to the need to penetrate protein-rich materials on metallic surgical instruments, proper physical cleaning of instruments to remove gross contaminants and proteinaceous materials (i.e., blood) prior to exposure to alcohol ostensibly reduces the risk of impaired disinfection to a low level. Thus, proper care of surgical instruments prior to contact with alcohol (i.e., cleaning with soap and water) as well as physical removal of macroscopic contaminants from instruments between sequential surgeries should be sufficient to remove proteinaceous contaminants and permit alcohol to work effectively.
Infection by spore-forming bacteria in theory may be caused by organisms common to the gastrointestinal tract or environment of rodents or acquired either via fomite or direct contact with humans or other animals. Various clostridial species and Bacillus anthracis are the most important pathogenic spore-forming bacteria in mammals (3). However, the risk of infection related to bacterial spores in rats and mice is quite low given (a) their resistance to clostridial infection or intoxication, (b) the inability of their indigenous enteric spore-forming flora to cause disease, and, by virtue of high quality production practices and containment in animal research facilities, (c) the low risk of environmental exposure to pathogenic spore-forming bacteria that cause disease in other species. These premises are supported by definitive sources of information on natural rodent pathogens that suggest that spontaneous or iatrogenic clostridial infections in rodents are rare (4-6).
Bergey’s Manual of Systemic Bacteriology provides evidence that rats and mice are resistant to colonization, infection or intoxication by many clostridia. The listing and description of 88 species of clostridia showed that 20 species were avirulent, 2 species were rarely pathogenic for humans and of unknown pathogenicity for rodents, and 52 additional species were specifically not pathogenic or toxigenic for mice. Fifteen species of clostridia have the potential to cause disease in mice or rats: Clostridium botulinum, Clostridium chauvoei, Clostridium carnis, Clostridium difficile, Clostridium hemolyticum, Clostridium histolyticum, Clostridium limosum, Clostridium novyi, <em>Clostridium piliforme</em>, Clostridium perfringens, Clostridium septicum, Clostridium sordelli, Clostridium spiroforme, Clostridium tetani, and, a relatively new transfer to the genus, <em>Clostridium piliforme</em>. However, based upon a comprehensive literature search done by the DAR veterinary staff, surgical infections with these or any other spore-forming bacteria have not been documented to occur naturally in immunocompetent, microbiologically-defined rodents used in research. Searches were done on Medline on March 9, 1998, December 31, 1999 and January 7, 2000 covering the period of 1966 through 1999 and using the following key words: mice, rats, Clostridium, Clostridium botulinum, Clostridium chauvoei, Clostridium carnis, Clostridium difficile, Clostridium hemolyticum, Clostridium histolyticum, Clostridium limosum, Clostridium novyi, <em>Clostridium piliforme</em>, , Clostridium perfringens, Clostridium septicum, Clostridium sordelli, Clostridium spiroforme, Clostridium tetani, bacterial spore, infection, clostridial infection, wound infection, surgical wound infection, bacteremia, septicemia, gas gangrene, and postoperative complication.
With the exception of Clostridium perfringens and <em>Clostridium piliforme</em>, the aforementioned pathogenic spore-forming organisms are not known to naturally colonize laboratory rodents and are absent from well-managed colonies of laboratory mice and rats. While mice have been shown to be experimentally susceptible to wound infections with Clostridium chauvoei and Cl septicum, infection must be facilitated by chemically-induced tissue trauma (3,7). When given orally to mice, Clostridium chauvoei survives only transiently in the gut (7). By the same token, Clostridium tetani and Cl botulinum are pathogenic for in rats and mice only when given by intramuscular injection (8,9). Only short-lived GI colonization follows oral inoculation with either organism (10-15). Logically, mice and rats are at little risk of ever harboring these pathogens or shedding them into the environment where they could subsequently be seeded into surgical wounds. Other pathogens such as Clostridium piliforme and Clostridium difficile cause infection by routes and means not requiring a surgical insult (6) and rats and mice, unless rendered germfree, are resistant to infection by Clostridium difficile (3,16,17). Other than characterization of the effects of toxins, the literature is vague and unremarkable with respect to the pathogenicity of Clostridium carnis, Clostridium hemolyticum, Clostridium histolyticum, Clostridium limosum, Clostridium novyi, Clostridium sordelli, and Clostridium spiroforme for rats and mice.
Clostridium perfringens is a pathogen of many species, more widespread in nature than any other pathogenic microorganism, and commonly a component of the indigenous intestinal flora of mice and rats (3,18). However, in the history of the use of rodents in research there is only one case report of natural infection in a rat or mouse (19). This anecdotal report involved mortality in suckling mice, not associated with surgery, but Clostridium perfringens type D enterotoxemia. However, attempts to establish a clear pathogenic role of Cl perfringens in this case were inconclusive. Experiments have shown that Clostridium perfringens (untyped) infection could not be established in adult mice when pure cultures are given orally, intravenously or intraperitoneally (20). Gas gangrene occurred only when large numbers of type A or C organisms were given to adult mice by intramuscular injection (21,22) or when bacteria (type unknown) were seeded into fractures in rats (23). Most revealingly, a series of experiments published over 20 years ago established that the indigenous spore-forming microflora of rats were not important in wound or intra-abdominal infection. These studies involved the subcutaneous or intraperitoneal inoculation of rats with rat cecal contents (which included untyped endogenous Clostridium perfringens), but not resulting in clostridial infection (18,24,25). In one study using an established polymicrobial cocktail, including untyped Clostridium perfringens, that was inoculated into the abdomen of rats in the presence of a foreign body (barium sulfate), Cl perfringens was found subsequently in only 4% of resultant abscesses (26). The establishment of Clostridium perfringens types A and C infections in the peritoneal cavity has only been accomplished reliably in association with concomitant surgical trauma in germfree rats (27,28). This suggests that gross fecal contamination during a laparotomy will not lead to infection involving spore-forming bacteria.
The spore-forming organisms that colonize rats and mice in high number are generally non-pathogenic commensals that are highly associated with the intestinal epithelium (29) and include well over 100 strains of non-pathogenic clostridia (30) such as Clostridium baratii (3), Cl cocleatum (3), Cl sartagoformum (18), Cl paraputrificum (18), and Cl tyrobutyricum (18). Of these bacteria, only Clostridium piliforme has a pathogenic potential and that is restricted to a role in hepatic carcinogenesis of gnotobiotic mice (31). In general, enteric clostridia are considered to be a minor and unimportant part of the indigenous microflora (32). Given these considerations, the inoculation of spores into surgical sites is not likely to involve those of pathogens or in sufficient number to cause disease unless surgical technique is grossly deficient and/or host immunity is impaired.
Finally, alcohol is commonly recommended and used internationally as a skin and surgical instrument decontaminant for the production of genetically mutant mice (33). The production of mice using transgene insertion, targeted gene mutagenesis, homologous recombination and other techniques are expensive and time-consuming processes with little margin for inefficiency or failure. Scientists developing such mice would not imperil production or its efficiency by using surgical techniques that did not have a proven record of success. Alcohol, when used on clean instruments and with a suitable contact time, has such a record. It is interesting to note that with over a century of rodent research in the United States and with countless numbers of surgeries done on rodents, there has not been one report of a surgically-related infection with a spore-forming organism. Anecdotally, at my institution alcohol is permitted in rodent survival surgical procedures in compliance with uniform IACUC surgical standards (adopted in 1990) and has not been associated with any postoperative infections in rodents.
Unarguably there are more effective or expeditious methods of sterilization or high level disinfection of surgical instruments, but alcohol, because of convenience, cost, and a lack of drawbacks specifically in rats and mice, has an appropriate role in rodent survival surgery.
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(last updated 02/18/2000)
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