Edwards, Richard
(1995)
Mechanisms of resistance to β-lactam antibiotics in Bacteroides species.
PhD thesis, University of Nottingham.
Abstract
Mechanisms responsible for resistance to β-Iactam antibiotics were investigated in clinical isolates of Bacteroides spp., the most common anaerobic Gram negative pathogen. Among 108 isolates of Bacteroides spp. obtained from clinical material in Nottingham, 69 (64%) were identified as Bacteroides fragilis. Approximately one-fifth of the Bacteroides spp. produced elevated levels of β-Iactamases, and many of these strains showed increased resistance to β-Iactam antibiotics usually regarded as β-Iactamase stable.
Four β-Iactamase types were identified: Type one was represented by zinc dependent metallo-β-lactamases that hydrolysed cefoxitin, latamoxef and imipenem. Type two displayed intermediate to high specific activity in tests with nitrocefin as substrate and hydrolysed cefoxitin and latamoxef, but not imipenem. Type three exhibited intermediate levels of specific activity and caused reduced susceptibility to cefoxitin, latamoxef and imipenem, although they hydrolysed these antibiotics inefficiently. Type four probably represented enhanced production of the β-Iactamases characteristic of most bacteroides strains. Organisms producing this enzyme normally remained susceptible to cefoxitin, latamoxef and imipenem, and the enzyme was fully susceptible to β-Iactamase inhibitors, including clavulanic acid.
Several strains were detected that exhibited reduced susceptibility to imipenem that was not related to metallo-β-Iactamases production. In contrast, one strain that produced a metallo-β-lactamase remained fully sensitive to imipenem as judged by conventional titration. Investigation of isolates producing metallo-β-lactamase and several similar ones encountered in an earlier study showed a correlation between the degree of resistance to imipenem and specific imipenemase activity.
In an attempt to elucidate reduced susceptibilty to imipenem that was not related to metallo-β-lactamase production, cell envelope properties and penicillin-binding proteins (PBPs) of selected strains were investigated. Studies of outer membrane proteins and lipopolysaccharide composition of B.fragilis strains unexpectedly showed that metallo-β-Iactamase producers displayed unusual cell envelope profiles. Those strains that showed reduced susceptibility that was not associated with β-Iactamase appeared to be normal, although two of these strains displayed abnormally high crypticity values.
Between three and six PBPs were visualised in tests with 3H-benzylpenicillin. PBPs 1 to 3 were present in all strains, but were observed in imipenem-resistant strains only when tests were carried out in the presence of β-lactamase inhibitors. Competitive binding experiments indicated that imipenem showed affinity for the high molecular weight PBPs of sensitive B.fragilis. The low molecular weight PBPs 4 to 6 were detected intermittently and only in certain strains, notably those that exhibited reduced susceptibility to imipenem. PBP 6 was found only in strains showing non-enzymic resistance.
The results suggest that several different types of resistance to β-lactam antibiotics are circulating in clinical isolates of Bacteroides spp. in Nottingham: firstly, strains that produce a metallo-β-lactamase that hydrolyses 'β-lactamase-stable' compounds, including imipenem; secondly, strains producing enzymes that hydrolyse cefoxitin and latamoxef, but not imipenem; thirdly, strains that possess a permeability barrier that affects imipenem as well as other β-Iactam antibiotics; fourthly, strains with altered penicillin-binding proteins; and fifthly strains that produce raised amounts of 'normal' β-lactamase. The latter strains slowly hydrolyse imlpenem and other β-lactam compounds and this may be a factor in the reduced susceptibility to these agents.
Clinical isolates that possess these resistance mechanisms may appear susceptible according to conventional break-point criteria in vitro. Never the less, they exhibit considerably reduced susceptibility to β-Iactam agents and the therapeutic implications of this need investigating. At present such strains represent a relatively small proportion of clinical isolates of Bacteroides spp., but the prevalence of resistance needs to be carefully monitored.
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