An opinion on the microbial limit test with reference to harmonization.
By Daniel L. Prince, Ph. D., Christopher J. Waskewich, M.S., Kristah J. Kohan, B.S. and Danina G. Rinaldi, B.S. , Gibraltar Laboratories, Inc.
Contamination control for a non-sterile drug was first highlighted by Professor Kallings1 in Sweden in 1965. He detected strains ofSalmonella in an oral drug, Thyroidinum, USP, a dry thyroid powder from domestic animals. At this time there were no microbial specifications for non-sterile drugs or cosmetics. Soon thereafter, bacteriologists from the USP, FDA and the pharmaceutical industry fashioned a ground-breaking protocol: the microbial limit test. The USP XVIII2 microbial limit test was used internationally for 38 years until 2009, when it was harmonized by an international committee called the Pharmacopeial Discussion Group.
The harmonized test first appeared in 2009 as two chapters dealing with enumeration and specified organisms USP <61> and USP <62>, respectively3. USP <62> is substantially different from the previous non-harmonized test; these changes are the subject of this article. The procedures in preparing the sample for testing, method suitability and growth promotion are an improvement. We also believe that elimination of the selective media is of unproven value and potentially error-prone. A case-study is presented withSalmonellae and E. coli on the shift from classical microbiology techniques to polymerase chain reaction [PCR].
Three stages of the Microbial Limits Test
Stage I
Organisms – Specified bacteria are chosen on the basis of potential infectivity by non-parenteral routes, especially the oral, topical, rectal, vaginal, nasal and inhalation routes. Products that may be administered by these routes and their microbiological quality requirements are listed in USP <1111>4. This includes components and raw materials. The pre-harmonized methods consisted of Salmonella, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The danger that these organisms pose relates to five things:
1 their survivability in low water activity and/or nutrition-free product,
2 their susceptibility or multiplicity of infection,
3 host susceptibility,
4 portal of entry into the human body and
5 their indication of Good Manufacturing Process deficiencies during manufacturing5.
Culture and enrichment media – Directions include the name, incubation conditions, media and chemical composition of culture media recommended to propagate the organism to recovery (elution) from the product and for challenging the media for growth promotion and “method suitability.” Selective and differential media for subculturing from elution (enrichment) fluids are prepared and challenged to verify diagnostic capability.
Stage II
Isolation – Test samples in 1 or 10 gram amounts are inoculated into appropriate specialized enrichment media (see later) and incubated for a stated period of time and temperature. They are then streaked to selective/differential media and observed for growth (present/absent). The presence of pigment or other diagnostic colonial feature or reaction in the agar plate is noted and recorded. A notation of either negative or presumptive isolation is made.
Stage III
Identification – In the non-harmonized methods, presumptive subcultures from the above isolation plates are inoculated to more than one specified diagnostic test and observed for typical reactions, as shown in Table 2. Conclusions are rendered as to presence or absence of the designated organism based on their growth and reaction on the diagnostic media. In the harmonized test, only one medium is selected and specified organisms are narrowly defined in terms of reaction on a single medium. Presumptive isolations are required to be identified using methods not cited in USP <62>.
Table 1 shows how the two bench level stages are joined and how they differ in the pre- and harmonized MLT. Note the reduction in the number of isolations from 10 to four in the two methods. Both compendial and literature references cite the use of multiple cultures as shown in column A and the diminished sensitivity that occurs for detection of Salmonella with fewer cultures, as seen in column C6, 7.
In table 2, the pre-harmonized MLT included portions of the above 20 reactions for identifications of a monograph isolate. Our purpose in showing the information-rich review in section A and empty section B is to emphasize the value of a stand-alone document to one that requires searching for additional resource material.
Focus on Salmonella
The pre-harmonized methods in one form or another appeared in FDA BAM, AOAC and WHO references for over fifty years. In Table 1 we note that harmonization allows reduction of the original USP isolations from three to only one. For example, in the case of Salmonellae XLD was retained and Bismuth Sulfite and Brilliant Green agars were eliminated. Validation to demonstrate that one medium was as good as three was not performed by the three pharmacopeiae.
There are at least three reasons why more than one medium was recommended for many years for screening forSalmonella. First, hydrogen sulfide, a key metabolic determinant to separate Salmonella from Shigella, admittedly, is not always produced or visible in the culture media. Second, size, color and contour of the suspect colony is variable and very subjective. Third, atypical strains (as reported by the FDA and described in AOAC) will not always be recognized if only XLD is used. Therefore, according to AOAC the presence of an atypical strain of Salmonella sp. on XLD will be incorrectly identified in USP < 62> as absent. AOAC (Salmonella in Processed Foods) cautions only against XLD as follows, “. . . atypically a few XLD Salmonella cultures produce yellow colonies with or without black centers.” It also recommends without reservation the use of Hektoen Enteric Agar or Bismuth Sulfite Agar.
An inanimate surface or material can be permissive or hostile to a microorganism depending on its water activity (Aw). Most drugs are in this category. However, USP 36 still includes articles of plant or animal origin, as encountered by Kallings years ago (Table 3). All have a history of Salmonella contamination.
We stress that the pharmaceutical industry, like the food industry, must remain ever vigilant as to employing the most sensitive techniques for isolating and identifying this ancient, ubiquitous and dangerous organism.
One notes in Table 3 the occurrence of two of the organisms that often appear in the USP <1111> Staphylococcus aureus and Pseudomonas aeruginosa. What does this tell us? Of all the organisms mentioned and reviewed in this article, these species may be the most adept in crossing environmental boundaries, S. aureus from people and P. aeruginosa from water. Tracking down sources of contamination remains as challenging as ever.
Theory of the Selective Enrichment Test
The MLT relies on the use of selective agar (Table 1). The enrichment broth with the specified incubation conditions is finely crafted to promote viability of the designated organism and inhibition of the unwanted. There is sometimes a situation when the designated organisms are in pure culture and the only purpose of the enrichment phase is to bring it in to detectable levels. There are four inhibitory enrichment broths in use in the various MLT (Table 4). Note: Selenite cystine and tetrathionate were used exclusively in the U.S., and partially elsewhere, for many years. They are still used in USP <2022>9, addressing the microbiological limits of dietary supplements.
In Table 5, the commonly employed reagents used for inhibition and their mechanisms of action are shown. When the test material harbors a complex bioburden, selective microbial enrichment is a great aid in isolating the organism in question.
The Historical Most Favored Pre-Harmonized Tests
From review of the Japanese, British and European pre-harmonized methods, we have identified below the most common confirmation tests (Stage III) chosen over the years by the four nations. The committee chose none (Table 6).
For a comprehensive review of diagnostic microbiology, mechanisms of action, and various culture media, see Prince, Lechevalier and Laskin10. For future guidance the reader is referred to any early volume of the USP, JP, AOAC, BAM/FDA. For a current, more flexible, compendial view, one might read USP <2022>9. The reader is advised that related papers are available that address the subject of modifying the USP Microbial Limit Test, < 61>11.
The Chemistry of XLD Agar
XLD agar is recommended by USP for the detection of Salmonellae. XLD, like the other media which were eliminated in harmonization, is an example of a low-cost, proven approach that is available in developed and developing countries alike and does not depend on expensive software or technology.
XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram-positive microorganisms. Xylose is incorporated into the medium, since it is fermented by practically all enterics except for the Shigellae; this property enables the differentiation of Shigella species. Lysine is included to enable the Salmonellae group to be differentiated from the nonpathogens since without lysine, Salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. After the Salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme, lysine decarboxylase, with reversion to an alkaline pH which mimics the Shigellae reaction. To prevent similar reversion by lysine positive coliforms, lactose and saccharose (sucrose) were added to produce acid in excess. Differentiation of pathogenic12,13 Salmonellae is further achieved with an H2S indicator system, consisting of the enzyme thiosulfate reductase, sodium thiosulfate and ferric ammonium citrate for the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers.
Case Study: A Misleading Current USP <62> Evaluation of Salmonellae and E. coli
Regardless of theory, it is our experience that reliance on a single medium — like XLD agar — in the context of narrowly focusing in on the presence of a specified organism leads to error. In an investigation of starch, conducted by the authors, it was found that, from USP <62> when suspicious colonies were picked from XLD agar and subcultured onto MacConkeys agar, that E. coli was identified. Thus, if the step of sub-culturing from XLD agar to MacConkeys was not taken, the false conclusion reached would be that Salmonellae was present when in fact it was not. Furthermore, the false conclusion that E. coli was absent would be made, and thus the presence of E. coli would not be reported. The subjectivity that is troublesome in some phenotypic assays may be overcome by some newer genetic assays. In our laboratory, in certain cases, we supplement enrichment and classical cultivation ofSalmonellae with qPCR [see Figure 1].
Potentially Adding Objectionable Organisms
The four original USP indicator (specified in the newer terminology) organisms are important, representing three ubiquitous and highly pathogenic gram negative rods (typhoid fever, diarrhea and a germ that infects wounds and mucosal tissue) and the master Gram positive pathogen shed from the nostrils, skin, hair, throat, ears and eyes (Staphylococcus aureus). Now Bacillus cereus and Burkholderia cepacia complex are openly being discussed as possible new additional specified organisms. Equally as important as adding another organism is a means to quickly identify it in a rapid and certain way. Does the culture medium exist? Can it be picked out as mistakenly as E. coli on a MacConkey plate? This subject is thoughtfully explored by Sutton and Jimenez14. The term “objectionable organism” — as distinguished from “indicated organism” in the sense of our present discussion — is derived from the GMP regulations5 and has legal standing. They are very common and seen with greater frequency in 483 citations than the indicated organisms associated with a microbial limit test.
Summary
The Microbial Limit Test was published by the USP in 1970 derived in part on AOAC methodology. It is based on the premise that certain indicator pathogens can serve as a screen for the infectivity of the chemical or biological material described in a monograph or elsewhere. The method is a three-stage process of eluting potential bioburden in standardized manner in a process called enrichment (Stage I), transferring any growth to special selective subculture plates designed to presumptively suggest either the absence or possible presence of the pathogen (Stage II). Thereafter simple diagnostic procedures are supplied to the analyst to identify any growth observed in Stage II so as to rule out or confirm presence of the indicator organism (Stage III). This methodology existed in a non-harmonized manner for 38 years under the purview of the USP, JP, BP and EP with certain variation.
In 2009 the test method was harmonized with what the authors consider useful and non-useful changes. Stage I was improved by the addition of superior growth media and method suitability. An even greater and major change was to limit discovery to report absence — but not presence — of an objectionable organism. The presumptive stage for the examination of Salmonella was reduced (Stage II) from the standard of as many as three cultures (USP, JP, EP, BP, AOAC, ISO, BAM, WHO) to a single culture, a non-validated reduction, without showing equivalence. The test portion in certain cases was reduced from the 10 grams to 1 gram, a 90% reduction, again, without showing the two portions to be equivalent. The authors attempt to show the emerging value of genotypic testing (such as the PCR reaction) as an adjunct to standard biochemical phenotypic testing.
The harmonized test first appeared in 2009 as two chapters dealing with enumeration and specified organisms USP <61> and USP <62>, respectively3. USP <62> is substantially different from the previous non-harmonized test; these changes are the subject of this article. The procedures in preparing the sample for testing, method suitability and growth promotion are an improvement. We also believe that elimination of the selective media is of unproven value and potentially error-prone. A case-study is presented withSalmonellae and E. coli on the shift from classical microbiology techniques to polymerase chain reaction [PCR].
Three stages of the Microbial Limits Test
Stage I
Organisms – Specified bacteria are chosen on the basis of potential infectivity by non-parenteral routes, especially the oral, topical, rectal, vaginal, nasal and inhalation routes. Products that may be administered by these routes and their microbiological quality requirements are listed in USP <1111>4. This includes components and raw materials. The pre-harmonized methods consisted of Salmonella, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The danger that these organisms pose relates to five things:
1 their survivability in low water activity and/or nutrition-free product,
2 their susceptibility or multiplicity of infection,
3 host susceptibility,
4 portal of entry into the human body and
5 their indication of Good Manufacturing Process deficiencies during manufacturing5.
Culture and enrichment media – Directions include the name, incubation conditions, media and chemical composition of culture media recommended to propagate the organism to recovery (elution) from the product and for challenging the media for growth promotion and “method suitability.” Selective and differential media for subculturing from elution (enrichment) fluids are prepared and challenged to verify diagnostic capability.
Stage II
Isolation – Test samples in 1 or 10 gram amounts are inoculated into appropriate specialized enrichment media (see later) and incubated for a stated period of time and temperature. They are then streaked to selective/differential media and observed for growth (present/absent). The presence of pigment or other diagnostic colonial feature or reaction in the agar plate is noted and recorded. A notation of either negative or presumptive isolation is made.
Stage III
Identification – In the non-harmonized methods, presumptive subcultures from the above isolation plates are inoculated to more than one specified diagnostic test and observed for typical reactions, as shown in Table 2. Conclusions are rendered as to presence or absence of the designated organism based on their growth and reaction on the diagnostic media. In the harmonized test, only one medium is selected and specified organisms are narrowly defined in terms of reaction on a single medium. Presumptive isolations are required to be identified using methods not cited in USP <62>.
Table 1 shows how the two bench level stages are joined and how they differ in the pre- and harmonized MLT. Note the reduction in the number of isolations from 10 to four in the two methods. Both compendial and literature references cite the use of multiple cultures as shown in column A and the diminished sensitivity that occurs for detection of Salmonella with fewer cultures, as seen in column C6, 7.
In table 2, the pre-harmonized MLT included portions of the above 20 reactions for identifications of a monograph isolate. Our purpose in showing the information-rich review in section A and empty section B is to emphasize the value of a stand-alone document to one that requires searching for additional resource material.
Focus on Salmonella
The pre-harmonized methods in one form or another appeared in FDA BAM, AOAC and WHO references for over fifty years. In Table 1 we note that harmonization allows reduction of the original USP isolations from three to only one. For example, in the case of Salmonellae XLD was retained and Bismuth Sulfite and Brilliant Green agars were eliminated. Validation to demonstrate that one medium was as good as three was not performed by the three pharmacopeiae.
There are at least three reasons why more than one medium was recommended for many years for screening forSalmonella. First, hydrogen sulfide, a key metabolic determinant to separate Salmonella from Shigella, admittedly, is not always produced or visible in the culture media. Second, size, color and contour of the suspect colony is variable and very subjective. Third, atypical strains (as reported by the FDA and described in AOAC) will not always be recognized if only XLD is used. Therefore, according to AOAC the presence of an atypical strain of Salmonella sp. on XLD will be incorrectly identified in USP < 62> as absent. AOAC (Salmonella in Processed Foods) cautions only against XLD as follows, “. . . atypically a few XLD Salmonella cultures produce yellow colonies with or without black centers.” It also recommends without reservation the use of Hektoen Enteric Agar or Bismuth Sulfite Agar.
An inanimate surface or material can be permissive or hostile to a microorganism depending on its water activity (Aw). Most drugs are in this category. However, USP 36 still includes articles of plant or animal origin, as encountered by Kallings years ago (Table 3). All have a history of Salmonella contamination.
We stress that the pharmaceutical industry, like the food industry, must remain ever vigilant as to employing the most sensitive techniques for isolating and identifying this ancient, ubiquitous and dangerous organism.
One notes in Table 3 the occurrence of two of the organisms that often appear in the USP <1111> Staphylococcus aureus and Pseudomonas aeruginosa. What does this tell us? Of all the organisms mentioned and reviewed in this article, these species may be the most adept in crossing environmental boundaries, S. aureus from people and P. aeruginosa from water. Tracking down sources of contamination remains as challenging as ever.
Theory of the Selective Enrichment Test
The MLT relies on the use of selective agar (Table 1). The enrichment broth with the specified incubation conditions is finely crafted to promote viability of the designated organism and inhibition of the unwanted. There is sometimes a situation when the designated organisms are in pure culture and the only purpose of the enrichment phase is to bring it in to detectable levels. There are four inhibitory enrichment broths in use in the various MLT (Table 4). Note: Selenite cystine and tetrathionate were used exclusively in the U.S., and partially elsewhere, for many years. They are still used in USP <2022>9, addressing the microbiological limits of dietary supplements.
In Table 5, the commonly employed reagents used for inhibition and their mechanisms of action are shown. When the test material harbors a complex bioburden, selective microbial enrichment is a great aid in isolating the organism in question.
The Historical Most Favored Pre-Harmonized Tests
From review of the Japanese, British and European pre-harmonized methods, we have identified below the most common confirmation tests (Stage III) chosen over the years by the four nations. The committee chose none (Table 6).
For a comprehensive review of diagnostic microbiology, mechanisms of action, and various culture media, see Prince, Lechevalier and Laskin10. For future guidance the reader is referred to any early volume of the USP, JP, AOAC, BAM/FDA. For a current, more flexible, compendial view, one might read USP <2022>9. The reader is advised that related papers are available that address the subject of modifying the USP Microbial Limit Test, < 61>11.
The Chemistry of XLD Agar
XLD agar is recommended by USP for the detection of Salmonellae. XLD, like the other media which were eliminated in harmonization, is an example of a low-cost, proven approach that is available in developed and developing countries alike and does not depend on expensive software or technology.
XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram-positive microorganisms. Xylose is incorporated into the medium, since it is fermented by practically all enterics except for the Shigellae; this property enables the differentiation of Shigella species. Lysine is included to enable the Salmonellae group to be differentiated from the nonpathogens since without lysine, Salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. After the Salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme, lysine decarboxylase, with reversion to an alkaline pH which mimics the Shigellae reaction. To prevent similar reversion by lysine positive coliforms, lactose and saccharose (sucrose) were added to produce acid in excess. Differentiation of pathogenic12,13 Salmonellae is further achieved with an H2S indicator system, consisting of the enzyme thiosulfate reductase, sodium thiosulfate and ferric ammonium citrate for the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers.
Case Study: A Misleading Current USP <62> Evaluation of Salmonellae and E. coli
Regardless of theory, it is our experience that reliance on a single medium — like XLD agar — in the context of narrowly focusing in on the presence of a specified organism leads to error. In an investigation of starch, conducted by the authors, it was found that, from USP <62> when suspicious colonies were picked from XLD agar and subcultured onto MacConkeys agar, that E. coli was identified. Thus, if the step of sub-culturing from XLD agar to MacConkeys was not taken, the false conclusion reached would be that Salmonellae was present when in fact it was not. Furthermore, the false conclusion that E. coli was absent would be made, and thus the presence of E. coli would not be reported. The subjectivity that is troublesome in some phenotypic assays may be overcome by some newer genetic assays. In our laboratory, in certain cases, we supplement enrichment and classical cultivation ofSalmonellae with qPCR [see Figure 1].
Potentially Adding Objectionable Organisms
The four original USP indicator (specified in the newer terminology) organisms are important, representing three ubiquitous and highly pathogenic gram negative rods (typhoid fever, diarrhea and a germ that infects wounds and mucosal tissue) and the master Gram positive pathogen shed from the nostrils, skin, hair, throat, ears and eyes (Staphylococcus aureus). Now Bacillus cereus and Burkholderia cepacia complex are openly being discussed as possible new additional specified organisms. Equally as important as adding another organism is a means to quickly identify it in a rapid and certain way. Does the culture medium exist? Can it be picked out as mistakenly as E. coli on a MacConkey plate? This subject is thoughtfully explored by Sutton and Jimenez14. The term “objectionable organism” — as distinguished from “indicated organism” in the sense of our present discussion — is derived from the GMP regulations5 and has legal standing. They are very common and seen with greater frequency in 483 citations than the indicated organisms associated with a microbial limit test.
Summary
The Microbial Limit Test was published by the USP in 1970 derived in part on AOAC methodology. It is based on the premise that certain indicator pathogens can serve as a screen for the infectivity of the chemical or biological material described in a monograph or elsewhere. The method is a three-stage process of eluting potential bioburden in standardized manner in a process called enrichment (Stage I), transferring any growth to special selective subculture plates designed to presumptively suggest either the absence or possible presence of the pathogen (Stage II). Thereafter simple diagnostic procedures are supplied to the analyst to identify any growth observed in Stage II so as to rule out or confirm presence of the indicator organism (Stage III). This methodology existed in a non-harmonized manner for 38 years under the purview of the USP, JP, BP and EP with certain variation.
In 2009 the test method was harmonized with what the authors consider useful and non-useful changes. Stage I was improved by the addition of superior growth media and method suitability. An even greater and major change was to limit discovery to report absence — but not presence — of an objectionable organism. The presumptive stage for the examination of Salmonella was reduced (Stage II) from the standard of as many as three cultures (USP, JP, EP, BP, AOAC, ISO, BAM, WHO) to a single culture, a non-validated reduction, without showing equivalence. The test portion in certain cases was reduced from the 10 grams to 1 gram, a 90% reduction, again, without showing the two portions to be equivalent. The authors attempt to show the emerging value of genotypic testing (such as the PCR reaction) as an adjunct to standard biochemical phenotypic testing.
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