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Epidemiology of antibiotic resistance in Gram-negative bacteria
Journal of Hospital Infection (1988) 11 (Supplement
A), 13&l 34
Epidemiology of antibiotic resistance Gram-negative bacteria Rosamund Department
in
J. Williams
of Medical
Microbiology
The London Hospital Medical College, Turner Street, London El 2AD Introduction The epidemiology of an infectious disease may be usefully evaluated in terms of the determinants, occurrence, distribution and control of the disease in a defined population. This review is intended to examine antibiotic resistance in Gram-negative bacteria in these terms, drawing examples from the opportunist Gram-negative rods which cause infections in hospitalized patients. The determinants of antibiotic resistance are genes which code for proteins which directly or indirectly result in the resistance of the cell to specific antibiotics. The gene products most clearly understood are the enzymes, such as beta-lactamases and aminoglycoside-modifying enzymes, which destroy or modify the antibiotic and thus prevent it from reaching its target site in the cell. Other mechanisms that the cell can employ to render it resistant include the synthesis of an altered target site, e.g. a change in a single ribosomal protein results in failure of streptomycin to bind to the ribosome, or synthesis of an altered cell wall or cell membrane structure, e.g., changes in porin proteins in the outer membrane of Escherichia coli cell walls result in reduced susceptibility to beta-lactams. The occurrence of antibiotic resistance in opportunist Gram-negative rods has been estimated for various antibiotics and bacterial species in many different localities. Table I shows the results of several surveys of gentamicin resistance in Pseudomonas aeruginosa carried out in the last 10 years. From these data one may conclude that gentamicin resistance is much more common among P. aeruginosa isolates in Japan and Greece than it is in isolates from Switzerland, UK or USA. However large surveys such as these are only capable of giving a rather general view of the situation. For example, in the UK multi-centre survey (Williams et al., 1984a) four of the 24 centres had no gentamicin-resistant isolates while in one centre the rate 01954701/88/02A130+05
SO,.OO/O
0
130
1988 The Hospital
Infection
Society
Resistance
in Gram-negative
bacteria
131
was 10%. Thus although national trends are interesting it is important to discern local differences in resistante rates especially because these may be indicators of antibiotic abuse *outbreaks of infection with resistant strains. Table Date of survey
1977-79 1981 1982 1983 1976 197677 1982-83 *Multi-centre
I. Aminoglyco#de
Location
-Worldwide* Switzerland J”aEt* Ohio, !JSA Chfe;$o, USA
resistance
in Pseudomonas
aeruginosa
Number of isolates examined
% isolates resistant to gentamicin
Authors
657 2235 1866 153 870 693 145
29 5.9 5.5 37 11.6 7.5 58
Price et al. (1981) Pitton (1981) Williams et al. (19840) Furusawa et al. (1986) Kauffman et al. (1978) Weinstein et al. (1980) Giamarellou et al. (1983)
surveys.
The large numbers of isolates collected in many surveys make analysis of the mechanisms of resistance an enormous task and survey data often fail to reveal whether the mechanism of resistance was the same in all isolates or in different localities. To gain a fuller understanding of the epidemiology of antibiotic resistance it is necessary to consider the occurrence and distribution of the determinants of resistance. This is complicated by the fact that the resistance genes may be located on plasmids or on the bacterial chromosome, or on transposons which are capable of integrating into plasmids and into the chromosome. The position of the genes affects their stability and transmissibility to other bacterial and human hosts. The frequency of occurrence and transmissibility of plasmids varies between different bacterial species. This was clearly exemplified by a survey of ampicillin resistance in hospital Gram-negative rods (Whitaker, Hajipieris & Williams, 1983). These workers showed that 48% of E. coli isolates were ampicillin resistant and in 90% of resistant isolates, the resistance was plasmid-encoded. In contrast all isolates of P. aeruginosa were resistant to ampicillin but in only two isolates could plasmid-mediated beta-lactamases be detected. Plasmid-mediated resistance may spread by three main routes. First, the resistant strain can spread from person-to-person carrying its plasmid with it. This route was exemplified by an outbreak described by Falkiner and his colleagues (1982) in which a multi-resistant P. aeruginosa spread to infect several patients in a leukaemia ward. The isolates from the patients and some from the environment were shown to be the same by several epidemiological typing methods, and all carried the same plasmid which encoded resistance to carbenicillin and aminoglycosides. The plasmid itself was non-transmissible and thus could only spread through the population as the bacterial host spread.
132
R. J. Williams
Second, the resistance genes can spread by means of plasmid transfer to another bacterial host of the same or different species. This may occur within a single human host or in environmental reservoirs. It is an important route of spread of resistance in hospitals because it allows rapid dissemination of resistance genes and they may be acquired by bacterial species that are particularly well-adapted to a pathogenic role. The dissemination of a single resistance plasmid in this manner was described by O’Brien and his colleagues (1980) when a plasmid carrying a gentamicin-modifying enzyme was identified in several different species of Enterobacteria. Third, resistance genes on transposons may move between plasmids or from a plasmid to the chromosome. For example, a move from a non-transmissible to a transmissible plasmid may result in greater spread of the resistance determinant. A move to the chromosome may result in greater stability and conservation of the resistance gene. For example, the genetic determinant of PSE4, the most common plasmid-mediated beta-lactamase in UK isolates of P. ueruginosa (Williams et al., 19846) has been shown to be on a chromosomally-integrating transposon (Sinclair & Holloway, 1982) and this may account for its frequency in this species. Despite the importance of resistance plasmids and the concern over the spread of ‘infectious resistance’ among hospital Gram-negative rods, it is important to remember that plasmid-mediated resistance is rare in some species of opportunists, such as P. aeruginosa. In a survey of beta-lactam resistance in P. ueruginosu (Williams et al., 1984b) less than one quarter of the resistant isolates produced plasmid-mediated beta-lactamases. By far the most common mechanism of resistance did not involve beta-lactamase and did not appear to be plasmid-mediated. This mechanism has been termed ‘intrinsic resistance’ and probably involves a decrease in the permeability of the outer membrane of the cell wall (Williams et al., 19846). Resistance determinants which are incorporated in the chromosome provide the cell with a stable mechanism of resistance that is less likely to be lost when antibiotic selection pressure is removed, and the epidemiology of the resistance reflects the epidemiology of the bacterial species itself. In recent years there has been concern over the emergence of resistance to and the failure of treatment with some of the newer beta-lactams. The bacterial species involved in treatment failures (P. ueruginosu, Enterobucter spp., Proteus vulgaris, Providenciu stuurtii, Morganella morguni and Serrutiu murcescens) are all capable of producing inducible, chromosomallymediated beta-lactamases. Production of the enzymes is normally under repressor control but in the presence of a beta-lactam inducer, repression is lifted and enzyme synthesis proceeds at a high level (Jaurin et al., 1981). Although induction may occur during therapy, the resistant isolates which emerged after therapy were no longer inducible but were permanently switched on to high level production. Such organisms are known as stablyderepressed mutants, and they are at a considerable advantage if their
Resistance in Gram-negative
bacteria
133
enzymes can hydrolyse the beta-lactams used in therapy. It appears that many of the newer beta-lactams, althnugh almost totally stable to hydrolysis by plasmid-mediated beta-lactmases, nevertheless have a high affinity for and are susceptible to hydrolysis by chromosomal enzymes (Livermore & Yang, 1987). It appears that stably-derepressed mutants arise only in bacterial species which have inducible chromosomal beta-lactamases. More than 90% of clinical isolates of P. aeruginosa, Proteus vulgaris, Providencia stuartii and Morganella morgani have been shown to produce inducible beta-lactamases, and among 48 Enterobacter cloacae isolates three were stably derepressed, 38 were inducible and 20 of these also produced plasmid-mediated beta-lactamases (Yane, personal communication). These species, even in their ‘susceptible’ form, present a challenge to antimicrobial chemotherapy but the broad cross-resistance exhibited by stably-derepressed mutants leaves our antibiotic armamentarium seriously depleted. There is no evidence to suggest that stably-derepressed mutants have lost their virulence or their ability to spread and thus they have the potential to cause outbreaks of infection in the same manner as their less resistant counterparts. rods is influenced by the location of the genetic determinants of resistance within the cell and by the epidemiology of the host bacterium. It is important to recognize that opportunist Gram-negative rods do not all behave in the same way in terms of sites both of the body and of the environment which they prefer to colonize. They also differ in the frequency with which they contain resistance plasmids and their ability to receive and disseminate plasmids. Plasmid-mediated resistance provides a mechanism by which strains can acquire multiple resistance and can disseminate it rapidly to other species in the patient and in the environment. Chromosomally-mediated resistance is a more stable property of the cell that may be considered as the ‘irreducible minimum’ which will be difficult or impossible to eradicate. It is in the face of chromosomal mechanisms such as stable derepression of beta-lactamase production that alternative therapeutic strategies may have to be considered.
References I. B. R., Rennie, R. P. & Duncan, N. H. (1981). A long-term study of Duncan, gentamicin-resistant Pseudomonas aeruginosa in a general hospital. Journal of Antimicrobial Chemotherapy 7, 147-l 55. Falkiner, F. R., Jacoby, G. A., Keane, C. T. & McCann, S. R. (1982). Amikacin, gentamicin and tobramycin resistant Pseudomonas aeruginosa in a leukaemic ward. Epidemiology and genetic studies. Journal of Hospital Infection 3, 253-261. Furusawa, T., Uete, T., Kawada, T., Kuwahara, M. & Okuma, A. (1986). Resistance to cefsulodin and gentamicin in Pseudomonas aeruginosa strains in five areas of Japan between 1980 and 1983. Journal of Antimicrobial Chemotherapy 17, 755-762. Giamarellou, H., Bobey, D. G., Chalkipoulos, D., Petrikkos, G. & Daikos, G. K. (1983). Pseudomonas aeruginosa multi-resistant isolates in 1982 : A surveillance study. Proceedings 13th International Congress of Chemotherapy, Vienna. SE7, 2712-1.5.
134
R. J. Williams
Jaurin, B., Grundstrom, T., Edlund, T. & Normark, S. (1981). The E. coli B-lactamase attenuator mediates growth rate-dependent regulation. Nature 290, 221-225. Kauffman, C. A., Ramundo, N. C., Williams, S. G., Dey, C. R., Phair, J. P. & Watanakunakorn, C. (1978). Surveillance of gentamicin-resistant Gram-negative bacilli in a general hospital. Antimicrobial Agents and Chemotherapy 13, 918-923. Livermore, D. M. & Yang, Y-J. (1987). Beta-lactamase lability and inducer power of newer beta-lactam antibiotics in relation to their activity against beta-lactamase-inducibility mutants of Pseudomonas ueruginosu. Journal of Infectious Diseases 155, 775-782. O’Brien, T. F., Ross, D. G., Guzman, M. A., Medeiros, A. A., Hedges, R. W. & Botstein, D. (1980). Dissemination of an antibiotic resistance plasmid in hospital patient flora. Antimicrobial Agents & Chemotherapy 17, 537-543. Pitton, J-S. (1981). Survey of frequency of sensitivity and resistance to aminoglycosides in Switzerland. Journal of Antimicrobial Chemotherapy 8 (Suppl. A), 83-87. Price, K. E., Kresel, P. A., Farchione, L. A., Siskin, S. B. & Karpow, S. A. (1981). Epidemiological studies of aminoglycoside resistance in USA. Journal of Antimicrobial Chemotherapy 8 (Suppl. A), 89-105. Sinclair, M. I. & Holloway, B. W. (1982). A chromosomally located transposon in Pseudomonas ueruginosu. Journal of Bacteriology 151, 569-579. Weinstein, R. A., Nathan, C., Gruensfelder, R. & Kabins, S. A. (1980). Endemic aminoglycoside resistance in Gram-negative bacilli : Epidemiology and mechanisms. Journal of Infectious Diseases, 141 338-345. Whitaker, S., Hajipieris, P. & Williams, J. D. (1983). Distribution and type of beta-lactamase amongst 1,000 Gram-negative rod bacteria. Proceedings 13th International Congress of Chemotherapy, Vienna. PS 2.5/l-3. Williams, R. J., Lindridge, M. A., Said, A. A., Livermore, D. M. & Williams, J. D. (1984~). National survey of antibiotic resistance in Pseudomonas ueruginosu. Journal of Antimicrobial Chemotherapy 14, 9-16. Williams, R. J., Livermore, D. M., Lindridge, M. A., Said, A. A. & Williams, J. D. (19846). Mechanisms of beta-lactam resistance in British isolates of Pseudomonas ueruginosu. Journal of Medical Microbiology 17, 283-293.
A), 13&l 34
Epidemiology of antibiotic resistance Gram-negative bacteria Rosamund Department
in
J. Williams
of Medical
Microbiology
The London Hospital Medical College, Turner Street, London El 2AD Introduction The epidemiology of an infectious disease may be usefully evaluated in terms of the determinants, occurrence, distribution and control of the disease in a defined population. This review is intended to examine antibiotic resistance in Gram-negative bacteria in these terms, drawing examples from the opportunist Gram-negative rods which cause infections in hospitalized patients. The determinants of antibiotic resistance are genes which code for proteins which directly or indirectly result in the resistance of the cell to specific antibiotics. The gene products most clearly understood are the enzymes, such as beta-lactamases and aminoglycoside-modifying enzymes, which destroy or modify the antibiotic and thus prevent it from reaching its target site in the cell. Other mechanisms that the cell can employ to render it resistant include the synthesis of an altered target site, e.g. a change in a single ribosomal protein results in failure of streptomycin to bind to the ribosome, or synthesis of an altered cell wall or cell membrane structure, e.g., changes in porin proteins in the outer membrane of Escherichia coli cell walls result in reduced susceptibility to beta-lactams. The occurrence of antibiotic resistance in opportunist Gram-negative rods has been estimated for various antibiotics and bacterial species in many different localities. Table I shows the results of several surveys of gentamicin resistance in Pseudomonas aeruginosa carried out in the last 10 years. From these data one may conclude that gentamicin resistance is much more common among P. aeruginosa isolates in Japan and Greece than it is in isolates from Switzerland, UK or USA. However large surveys such as these are only capable of giving a rather general view of the situation. For example, in the UK multi-centre survey (Williams et al., 1984a) four of the 24 centres had no gentamicin-resistant isolates while in one centre the rate 01954701/88/02A130+05
SO,.OO/O
0
130
1988 The Hospital
Infection
Society
Resistance
in Gram-negative
bacteria
131
was 10%. Thus although national trends are interesting it is important to discern local differences in resistante rates especially because these may be indicators of antibiotic abuse *outbreaks of infection with resistant strains. Table Date of survey
1977-79 1981 1982 1983 1976 197677 1982-83 *Multi-centre
I. Aminoglyco#de
Location
-Worldwide* Switzerland J”aEt* Ohio, !JSA Chfe;$o, USA
resistance
in Pseudomonas
aeruginosa
Number of isolates examined
% isolates resistant to gentamicin
Authors
657 2235 1866 153 870 693 145
29 5.9 5.5 37 11.6 7.5 58
Price et al. (1981) Pitton (1981) Williams et al. (19840) Furusawa et al. (1986) Kauffman et al. (1978) Weinstein et al. (1980) Giamarellou et al. (1983)
surveys.
The large numbers of isolates collected in many surveys make analysis of the mechanisms of resistance an enormous task and survey data often fail to reveal whether the mechanism of resistance was the same in all isolates or in different localities. To gain a fuller understanding of the epidemiology of antibiotic resistance it is necessary to consider the occurrence and distribution of the determinants of resistance. This is complicated by the fact that the resistance genes may be located on plasmids or on the bacterial chromosome, or on transposons which are capable of integrating into plasmids and into the chromosome. The position of the genes affects their stability and transmissibility to other bacterial and human hosts. The frequency of occurrence and transmissibility of plasmids varies between different bacterial species. This was clearly exemplified by a survey of ampicillin resistance in hospital Gram-negative rods (Whitaker, Hajipieris & Williams, 1983). These workers showed that 48% of E. coli isolates were ampicillin resistant and in 90% of resistant isolates, the resistance was plasmid-encoded. In contrast all isolates of P. aeruginosa were resistant to ampicillin but in only two isolates could plasmid-mediated beta-lactamases be detected. Plasmid-mediated resistance may spread by three main routes. First, the resistant strain can spread from person-to-person carrying its plasmid with it. This route was exemplified by an outbreak described by Falkiner and his colleagues (1982) in which a multi-resistant P. aeruginosa spread to infect several patients in a leukaemia ward. The isolates from the patients and some from the environment were shown to be the same by several epidemiological typing methods, and all carried the same plasmid which encoded resistance to carbenicillin and aminoglycosides. The plasmid itself was non-transmissible and thus could only spread through the population as the bacterial host spread.
132
R. J. Williams
Second, the resistance genes can spread by means of plasmid transfer to another bacterial host of the same or different species. This may occur within a single human host or in environmental reservoirs. It is an important route of spread of resistance in hospitals because it allows rapid dissemination of resistance genes and they may be acquired by bacterial species that are particularly well-adapted to a pathogenic role. The dissemination of a single resistance plasmid in this manner was described by O’Brien and his colleagues (1980) when a plasmid carrying a gentamicin-modifying enzyme was identified in several different species of Enterobacteria. Third, resistance genes on transposons may move between plasmids or from a plasmid to the chromosome. For example, a move from a non-transmissible to a transmissible plasmid may result in greater spread of the resistance determinant. A move to the chromosome may result in greater stability and conservation of the resistance gene. For example, the genetic determinant of PSE4, the most common plasmid-mediated beta-lactamase in UK isolates of P. ueruginosa (Williams et al., 19846) has been shown to be on a chromosomally-integrating transposon (Sinclair & Holloway, 1982) and this may account for its frequency in this species. Despite the importance of resistance plasmids and the concern over the spread of ‘infectious resistance’ among hospital Gram-negative rods, it is important to remember that plasmid-mediated resistance is rare in some species of opportunists, such as P. aeruginosa. In a survey of beta-lactam resistance in P. ueruginosu (Williams et al., 1984b) less than one quarter of the resistant isolates produced plasmid-mediated beta-lactamases. By far the most common mechanism of resistance did not involve beta-lactamase and did not appear to be plasmid-mediated. This mechanism has been termed ‘intrinsic resistance’ and probably involves a decrease in the permeability of the outer membrane of the cell wall (Williams et al., 19846). Resistance determinants which are incorporated in the chromosome provide the cell with a stable mechanism of resistance that is less likely to be lost when antibiotic selection pressure is removed, and the epidemiology of the resistance reflects the epidemiology of the bacterial species itself. In recent years there has been concern over the emergence of resistance to and the failure of treatment with some of the newer beta-lactams. The bacterial species involved in treatment failures (P. ueruginosu, Enterobucter spp., Proteus vulgaris, Providenciu stuurtii, Morganella morguni and Serrutiu murcescens) are all capable of producing inducible, chromosomallymediated beta-lactamases. Production of the enzymes is normally under repressor control but in the presence of a beta-lactam inducer, repression is lifted and enzyme synthesis proceeds at a high level (Jaurin et al., 1981). Although induction may occur during therapy, the resistant isolates which emerged after therapy were no longer inducible but were permanently switched on to high level production. Such organisms are known as stablyderepressed mutants, and they are at a considerable advantage if their
Resistance in Gram-negative
bacteria
133
enzymes can hydrolyse the beta-lactams used in therapy. It appears that many of the newer beta-lactams, althnugh almost totally stable to hydrolysis by plasmid-mediated beta-lactmases, nevertheless have a high affinity for and are susceptible to hydrolysis by chromosomal enzymes (Livermore & Yang, 1987). It appears that stably-derepressed mutants arise only in bacterial species which have inducible chromosomal beta-lactamases. More than 90% of clinical isolates of P. aeruginosa, Proteus vulgaris, Providencia stuartii and Morganella morgani have been shown to produce inducible beta-lactamases, and among 48 Enterobacter cloacae isolates three were stably derepressed, 38 were inducible and 20 of these also produced plasmid-mediated beta-lactamases (Yane, personal communication). These species, even in their ‘susceptible’ form, present a challenge to antimicrobial chemotherapy but the broad cross-resistance exhibited by stably-derepressed mutants leaves our antibiotic armamentarium seriously depleted. There is no evidence to suggest that stably-derepressed mutants have lost their virulence or their ability to spread and thus they have the potential to cause outbreaks of infection in the same manner as their less resistant counterparts. rods is influenced by the location of the genetic determinants of resistance within the cell and by the epidemiology of the host bacterium. It is important to recognize that opportunist Gram-negative rods do not all behave in the same way in terms of sites both of the body and of the environment which they prefer to colonize. They also differ in the frequency with which they contain resistance plasmids and their ability to receive and disseminate plasmids. Plasmid-mediated resistance provides a mechanism by which strains can acquire multiple resistance and can disseminate it rapidly to other species in the patient and in the environment. Chromosomally-mediated resistance is a more stable property of the cell that may be considered as the ‘irreducible minimum’ which will be difficult or impossible to eradicate. It is in the face of chromosomal mechanisms such as stable derepression of beta-lactamase production that alternative therapeutic strategies may have to be considered.
References I. B. R., Rennie, R. P. & Duncan, N. H. (1981). A long-term study of Duncan, gentamicin-resistant Pseudomonas aeruginosa in a general hospital. Journal of Antimicrobial Chemotherapy 7, 147-l 55. Falkiner, F. R., Jacoby, G. A., Keane, C. T. & McCann, S. R. (1982). Amikacin, gentamicin and tobramycin resistant Pseudomonas aeruginosa in a leukaemic ward. Epidemiology and genetic studies. Journal of Hospital Infection 3, 253-261. Furusawa, T., Uete, T., Kawada, T., Kuwahara, M. & Okuma, A. (1986). Resistance to cefsulodin and gentamicin in Pseudomonas aeruginosa strains in five areas of Japan between 1980 and 1983. Journal of Antimicrobial Chemotherapy 17, 755-762. Giamarellou, H., Bobey, D. G., Chalkipoulos, D., Petrikkos, G. & Daikos, G. K. (1983). Pseudomonas aeruginosa multi-resistant isolates in 1982 : A surveillance study. Proceedings 13th International Congress of Chemotherapy, Vienna. SE7, 2712-1.5.
134
R. J. Williams
Jaurin, B., Grundstrom, T., Edlund, T. & Normark, S. (1981). The E. coli B-lactamase attenuator mediates growth rate-dependent regulation. Nature 290, 221-225. Kauffman, C. A., Ramundo, N. C., Williams, S. G., Dey, C. R., Phair, J. P. & Watanakunakorn, C. (1978). Surveillance of gentamicin-resistant Gram-negative bacilli in a general hospital. Antimicrobial Agents and Chemotherapy 13, 918-923. Livermore, D. M. & Yang, Y-J. (1987). Beta-lactamase lability and inducer power of newer beta-lactam antibiotics in relation to their activity against beta-lactamase-inducibility mutants of Pseudomonas ueruginosu. Journal of Infectious Diseases 155, 775-782. O’Brien, T. F., Ross, D. G., Guzman, M. A., Medeiros, A. A., Hedges, R. W. & Botstein, D. (1980). Dissemination of an antibiotic resistance plasmid in hospital patient flora. Antimicrobial Agents & Chemotherapy 17, 537-543. Pitton, J-S. (1981). Survey of frequency of sensitivity and resistance to aminoglycosides in Switzerland. Journal of Antimicrobial Chemotherapy 8 (Suppl. A), 83-87. Price, K. E., Kresel, P. A., Farchione, L. A., Siskin, S. B. & Karpow, S. A. (1981). Epidemiological studies of aminoglycoside resistance in USA. Journal of Antimicrobial Chemotherapy 8 (Suppl. A), 89-105. Sinclair, M. I. & Holloway, B. W. (1982). A chromosomally located transposon in Pseudomonas ueruginosu. Journal of Bacteriology 151, 569-579. Weinstein, R. A., Nathan, C., Gruensfelder, R. & Kabins, S. A. (1980). Endemic aminoglycoside resistance in Gram-negative bacilli : Epidemiology and mechanisms. Journal of Infectious Diseases, 141 338-345. Whitaker, S., Hajipieris, P. & Williams, J. D. (1983). Distribution and type of beta-lactamase amongst 1,000 Gram-negative rod bacteria. Proceedings 13th International Congress of Chemotherapy, Vienna. PS 2.5/l-3. Williams, R. J., Lindridge, M. A., Said, A. A., Livermore, D. M. & Williams, J. D. (1984~). National survey of antibiotic resistance in Pseudomonas ueruginosu. Journal of Antimicrobial Chemotherapy 14, 9-16. Williams, R. J., Livermore, D. M., Lindridge, M. A., Said, A. A. & Williams, J. D. (19846). Mechanisms of beta-lactam resistance in British isolates of Pseudomonas ueruginosu. Journal of Medical Microbiology 17, 283-293.