L Gutmann

Association of two resistance mechanisms in a clinical isolate of Enterobacter cloacae with high-level resistance to imipenem.

Carbapenem resistance was studied in a clinical isolate of Enterobacter cloacae, strain 201 (MIC of imipenem and meropenem, 16 micrograms/ml). This strain was analyzed comparatively with the carbapenem-susceptible parent strain 200, an equally susceptible revertant, 201-Rev, and in vitro-selected mutants with different levels of carbapenem resistance. All strains produced similarly high amounts of the same cephalosporinase (pIapp = 8.8). Strain 201 apparently lacked two major outer membrane proteins of ca. 37 and 38 kDa, while 201-Rev produced only the 37-kDa protein. The permeability coefficient, determined with cephaloridine, was reduced up to ninefold in the resistant strains which also showed a substantial reduction in the uptake of [14C]meropenem. The introduction of the plasmid-borne ampD gene (whose product decreases the expression of ampC) resulted in almost complete cessation of cephalosporinase production in all strains and a substantial decrease in the MICs of the carbapenems which remained, however, 8- to 16-fold higher than those determined for the susceptible strains containing the ampD gene. This residual resistance was attributed to reduced outer membrane permeability. The contribution of cephalosporinase production was verified in a reverse experiment, in which the introduction of ampC into a low-level cephalosporinase producer resulted in a fourfold increase in the carbapenem MICs. From these results, we infer that reduced outer membrane permeability and high-level cephalosporinase production can operate in conjunction in clinical isolates of E. cloacae to confer imipenem resistance. Images

Penicillin-Binding Protein 5 Sequence Alterations in Clinical Isolates of Enterococcus faecium with Different Levels of β-Lactam Resistance

The low-affinity penicillin-binding protein (PBP) 5 is the main beta-lactam target and is responsible for resistance to this class of antibiotics in Enterococcus faecium. The PBP 5 variants of 15 clinical isolates (including 8 resistant to vancomycin) with different levels of beta-lactam resistance were analyzed. Most of the highly beta-lactam-resistant isolates produced small quantities of PBP 5 of low affinity. This was associated with particular amino acid substitutions: an Ala or Ile for Thr-499, a Glu for Val-629, and a Pro for Ser-667. A change of Met-485 to Thr or Ala (adjacent to the conserved SDN box) was observed in isolates with MICs of ampicillin of 64 or 128 microg/mL, respectively. In the 2 most resistant isolates, with MICs of ampicillin of 256 microg/mL, an additional Ser was present just after Ser-466. Thus, particular point mutations in PBP 5 and combinations thereof may lead to high-level beta-lactam resistance in E. faecium.

SHV-5, a novel SHV-type beta-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams.

SHV-5 (pI 8.2), a novel broad-spectrum beta-lactamase encoded by a ca. 150-kilobase plasmid, was found in Klebsiella pneumoniae 160. SHV-5 beta-lactamase caused decreased susceptibility to most penicillins, cephalosporins, and monobactams, except imipenem and compounds which have a C6 or C7 alpha-methoxy substituent. beta-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) inhibited its activity and showed a synergistic effect when associated with different hydrolyzable beta-lactam compounds. Hybridization studies suggested that this enzyme may be related to, or derived from, the SHV enzyme. Increased MICs of cephamycins and temocillin associated with a decreased synergistic effect of the inhibitors on K. pneumoniae 160 might be linked to a decrease in two outer membrane proteins. Images

Synergistic effect of amoxicillin and cefotaxime against Enterococcus faecalis.

The antibacterial efficacy of the combination of amoxicillin and cefotaxime was assessed against 50 clinical strains of Enterococcus faecalis. For 48 of 50 strains, the MIC of amoxicillin that inhibited 50% of isolates tested decreased from 0.5 microgram/ml (range, 0.25 to 1 microgram/ml) to 0.06 microgram/ml (range, 0.01 to 0.25 microgram/ml) in the presence of only 4 micrograms of cefotaxime per ml. Alternatively, the MIC of cefotaxime that inhibited 50% of isolates tested decreased from 256 micrograms/ml (range, 8 to 512 micrograms/ml) to 1 micrograms/ml (range, 0.5 to 16 micrograms/ml) in the presence of only 0.06 microgram of amoxicillin per ml. For JH2-2, a reference strain of E. faecalis, the MICs of amoxicillin, cefotaxime, and amoxicillin in the presence of cefotaxime (4 micrograms/ml) were 0.5, 512, and 0.06 microgram/ml, respectively. By using a penicillin-binding protein (PBP) competition assay, it was shown that with cefotaxime, 50% saturation of PBPs 2 and 3 was obtained at very low concentrations (< 1 microgram/ml), while 50% saturation of PBPs 1, 4, and 5 was obtained with > or = 128 micrograms/ml. With amoxicillin, 50% saturation of PBPs 4 and 5 was obtained at 0.12 and 0.5 microgram/ml, respectively. Therefore, the partial saturation of PBPs 4 and 5 by amoxicillin combined with the total saturation of PBPs 2 and 3 by cefotaxime could be responsible for the observed synergy between these two compounds.

Novel gyrA point mutation in a strain of Escherichia coli resistant to fluoroquinolones but not to nalidixic acid.

We have previously described a clinical isolate of Escherichia coli (Q2) that is highly resistant to fluoroquinolones (MIC of ciprofloxacin, 16 micrograms/ml) but susceptible to nalidixic acid (MIC of nalidixic acid, 4 micrograms/ml) (N. Moniot-Ville, J. Guibert, N. Moreau, J.F. Acar, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 35:519-523, 1991). Transformation of strain Q2 with a plasmid carrying the wild-type gyrA gene from E. coli K-12(pAFF801) resulted in a 32-fold decrease in the MIC of ciprofloxacin, suggesting that at least one mutation in gyrA was involved in the resistance of Q2. Intragenic gyrA fragments of 668 and 2,500 bp from strain Q2 were amplified by the polymerase chain reaction. We sequenced the 668-bp fragment and identified a single novel point mutation (transition from G to A at position 242), leading to an amino acid substitution (Gly-81 to Asp) in the gyrase A subunit. We constructed hybrid plasmids by substituting either the 668-bp fragment or the 2,500-bp fragment from Q2 DNA, both of which contained the gyrA point mutation, for the corresponding fragments in wild-type gyrA (2,625 bp) of E. coli K-12. When introduced into E. coli KNK453 (gyrA temperature sensitive), both plasmids conferred an eightfold increase in the MIC of ciprofloxacin, but only a twofold increase in the MIC of nalidixic acid. When introduced into E. coli Q2, neither plasmid conferred any change in the MICs of ciprofloxacin or nalidixic acid, suggesting that only the point mutation found in gyrA was involved in the resistance that we observed.

A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one‐step mutation

High-level carbapenem-resistant (CpmR) mutants, with MICs for imipenem and carbapenem of greater than 128 micrograms/ml, were selected in vitro from four carbapenem-susceptible (CpmS) clinical isolates of Bacteroides fragilis. The CpmS strains produced very low levels of beta-lactamase activity, which was increased approx. 50- to 100-fold in the CpmR mutants. Isoelectric focussing and enzyme kinetic analysis (Km and Vrel) of the carbapenemases from the CpmR mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584-2593) hybridized with DNA from the CpmR mutants, their CpmS parental strains as well as clinical CpmR isolates, but not from randomly chosen carbapenem-susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.

In Vitro Exchange of Fluoroquinolone Resistance Determinants between Streptococcus pneumoniae and Viridans Streptococci and Genomic Organization of the parE-parC Region in S. mitis

Transfer of fluoroquinolone (FQ) resistance determinants between Streptococcus pneumoniae and viridans streptococci was explored by transformation in vitro. One-step FQ-resistant parC mutants were selected, and resistance could be transferred from DNA from S. oralis, S. mitis, S. sanguis, and S. constellatus to S. pneumoniae, with frequencies of 10(-3) to <10(-7) in correlation with the homologies of their quinolone resistance determining region sequences (95%, 91%, 85%, and 81%, respectively). Reciprocal transfers of mutated parC from DNA from S. pneumoniae to S. mitis and S. oralis were also observed. Simultaneous transfer of mutated parC and gyrA genes from S. mitis to S. pneumoniae yielded high-level-resistant pneumococcal transformants in one step at low frequencies. The parE-parC region of the type strain S. mitis 103335T had >90% homology with that of S. pneumoniae. The efficient interspecific transfer of quinolone resistance determinants in vitro leads us to anticipate their dissemination in the clinical setting.

Nucleotide sequence of the SHV-5 beta-lactamase gene of a Klebsiella pneumoniae plasmid.

The nucleotide sequence of the SHV-5 beta-lactamase gene, subcloned from a plasmid of Klebsiella pneumoniae, was determined. The amino acid changes thought to be responsible for the extended substrate profile of SHV-5 are Gly----Ser234 and Glu----Lys235. SHV-5 is identical to SHV-4, except for Leu----Arg201, which accounts for the difference in apparent pI of the two enzymes.

Mechanism of resistance to vancomycin in Enterococcus faecium D366 and Enterococcus faecalis A256.

The role of the glycopeptide-inducible proteins of Enterococcus faecium D366 (39.5 kilodaltons) and Enterococcus faecalis A256 (39 kilodaltons) in the mechanism of resistance to vancomycin and teicoplanin was examined. Crude cell walls from noninduced cells or from induced cells treated with sodium dodecyl sulfate to remove the inducible proteins were shown to bind vancomycin, in contrast to cell walls containing the cytoplasmic membrane-associated induced proteins, which did not bind vancomycin. Cytoplasmic membranes from vancomycin-induced cells did not inactivate (bind) vancomycin or teicoplanin, but they could protect the glycopeptides from being bound to the synthetic pentapeptide. This protection could be competitively abolished by D-alanyl-D-alanine. A decrease in glycopeptide binding to the pentapeptide was observed in a time-dependent fashion after treatment of the pentapeptide with the cytoplasmic membranes from induced cells. We hypothesize that the inducible proteins are responsible for glycopeptide resistance due to the binding to, and subsequent enzymatic modification of, the pentapeptide precursor of peptidoglycan, which is considered to be the natural target of glycopeptides. Images

Inducible carboxypeptidase activity in vancomycin-resistant enterococci.

Vancomycin was found to coinduce DD-carboxypeptidase activity, together with resistance, in eight low- or high-level glycopeptide-resistant strains of enterococci. The constitutively resistant mutant (MT10) of a low-level-resistant strain of Enterococcus faecium (D366) spontaneously expressed a level of carboxypeptidase similar to that of the induced strain D366. Pentapeptide, UDP-MurNac-pentapeptide, as well as D-alanyl-D-alanine were in vitro substrates for the carboxypeptidase which was not inhibited by penicillin. The level of vancomycin resistance correlated roughly with the level of carboxypeptidase activity. We infer from these results that the carboxypeptidase is one component of the glycopeptide resistance mechanism. Images