A clinical isolate of Klebsiella ozaenae with transferable resistance to broad-spectrum cephalosporins produces a ,(-lactamase determined by plasmid pBP60. The P-lactamase had the same isoelectric point as SHV-1 (7.6). From heteroduplex analysis, an extensive homology between the two bla genes could be deduced; therefore, the new ,l-lactamase was designated SHV-2. Enzymatic studies revealed that SHV-2 was able to hydrolyze broad-spectrum cephalosporins due to an increased affinity of these compounds for the enzyme. The assumption that SHV-2 is a natural mutant of SHV-1 was strongly supported by the isolation of a laboratory mutant of SHV-1 that showed activities similar to those of SHV-2.The main indications for broad-spectrum cephalosporins are life-threatening infections with gram-negative bacteria like those of the family Enterobacteriaceae and Pseudomonas aeruginosa. These antibiotics are more or less stable to all 3-lactamases produced by these strains.

In this study, we analyzed the mechanisms of multiresistance for 22 clinical multiresistant and clonally different Pseudomonas aeruginosa strains from Germany. Twelve and 10 strains originated from cystic fibrosis (CF) and non-CF patients, respectively. Overproduction of the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM was studied. Furthermore, loss of OprD, alterations in type II topoisomerases, AmpC overproduction, and the presence of 25 acquired resistance determinants were investigated. The presence of a hypermutation phenotype was also taken into account. Besides modifications in GyrA (91%), the most frequent mechanisms of resistance were MexXY-OprM overproduction (82%), OprD loss (82%), and AmpC overproduction (73%). Clear differences between strains from CF and non-CF patients were found: numerous genes coding for aminoglycoside-modifying enzymes and located, partially in combination with ␤-lactamase genes, in class 1 integrons were found only in strains from non-CF patients. Furthermore, multiple modifications in type II topoisomerases conferring high quinolone resistance levels and overexpression of MexAB-OprM were exclusively detected in multiresistant strains from non-CF patients. Correlations of the detected phenotypes and resistance mechanisms revealed a great impact of efflux pump overproduction on multiresistance in P. aeruginosa. Confirming previous studies, we found that additional, unknown chromosomally mediated resistance mechanisms remain to be determined. In our study, 11 out of 12 strains and 3 out of 10 strains from CF patients and non-CF patients, respectively, were hypermutable. This extremely high proportion of mutator strains should be taken into consideration for the treatment of multiresistant P. aeruginosa.

The present study describes a database on the natural susceptibility of Listeria spp. to a wide range of antibiotics, which can be used to validate susceptibility testing results of these microorganisms.

Construction of a malE-ampD gene fusion allowed purification of biologically active fusion protein by affinity chromatography. The cloned malE-ampD gene fusion complemented a chromosomal ampD mutation. Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic murein turnover products in Escherichia coli. Being a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD is likely to be involved in recycling of the turnover products. It is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropeptides which may function as signals for beta-lactamase induction.

Isogenic mutants derived from quinolone-susceptible isolate WT by introducing gyrA (S83L, D87G) and parC (S80I, E84K) mutations associated with quinolone resistance were characterized with respect to quinolone resistance, growth rate, and degree of global supercoiling. The latter was determined by use of a pair of reporter plasmids carrying supercoiling-dependent promoters pgyrA and ptopA, respectively, transcriptionally fused to the reporter gene bla coding for TEM-1 ␤-lactamase. The quotient (Qsc) of the ␤-lactamase specific activity determined for a mutant carrying either plasmid was taken as a measure of the degree of global supercoiling. These Qsc data were comparable to results obtained from the separation of topoisomers of plasmid pBR322 on chloroquine-containing agarose gels and indicate a reduced degree of negative supercoiling in resistant mutants relative to the parent, WT. The S83L mutation in gyrA had the strongest influence on quinolone resistance while leaving other parameters nearly unaffected. The gyrA double mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation. Phenotypic expression of the parC mutation (S80I) was dependent on the presence of at least one gyrA mutation. Expression of high-level fluoroquinolone resistance (ciprofloxacin MIC, >4 g/ml) required a combination of the gyrA double mutation and one parC mutation (S80I or E84K). Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of a parC mutation affected neither the doubling time nor the degree of supercoiling, while the presence of the gyrA D87G mutation was associated with a significant reduction in the degree of DNA supercoiling.

About 70%o of all Enterobacter cloacae strains tested possessed one of two species-specific 3-lactamases. These enzymes, E. cloacae ,B-lactamase A and E. cloacae ,-lactamase B, with isoelectric points of 8.8 and 7.8, respectively, had the same pH and temperature optima. Both showed similar enzyme kinetics and were inhibited by cloxacillin but not by p-chloromercuribenzoate. E. cloacae 3-lactamase B appeared to be identical with the enzyme of E. cloacae P99. By a mutation in a regulatory gene, inducible enzyme production could be converted into constitutive expression. In E. cloacae, both enzymes did not hydrolyze thirdgeneration cephalosporins, but they were solely responsible for resistance toward these drugs. This was demonstrated by the characterization of Escherichia coli strains expressing an identical resistance pattern after transfer of the corresponding Enterobacter gene.The importance of plasmid-coded 1-lactamases for the resistance of gram-negative bacteria to ,-lactam antibiotics is well established (6-8, 12, 17, 21, 24). In contrast, the contribution of chromosomal lactamases to resistance remains unclear. One exception is Escherichia coli mutants in which constitutive expression of the chromosomal ampC gene contributes to ampicillin resistance (2,3). Recently, however, evidence has begun to accumulate that in certain Enterobacter cloacae strains, the chromosomal ,B-lactamases may play an unexpected role (1, 13,22,23,25,28). These enzymes are typical cephalosporinases which usually can be induced by penicillins and cephalosporins (4,22,23 (25) proposed that thirdgeneration cephalosporins are not inactivated by hydrolysis but by a kind of trapping mechanism, or that other intrinsic factors (13,28) are involved in this process.In this paper we give evidence that (i) there are mainly two chromosomal ,3-lactamases in E.cloacae strains, (ii) that resistance to thirdgeneration cephalosporins arises by mutation to constitutive production of the P-lactamases, and (iii) that these P-lactamases are solely responsible for the resistance. In addition, we provide enzymatic data to characterize these enzymes.MATERIAULS AND METHODS Bacteril sra. A total of 61 cefotaxime-susceptible E. cloacae strains, isolated in 1979 from clinical specimens, were used for the epidemiological study. Five of these strains (E. cloacae 314, 352, 150, 988, and 149)

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Leclercia adecarboxylata is an opportunistic human pathogen that phenotypically resembles Escherichia coli. The natural susceptibilities of 101 Leclercia strains to 70 antimicrobial agents were investigated. MICs were determined with a microdilution procedure in cation-adjusted Mueller-Hinton broth (all strains) and IsoSensitest broth (some strains). Natural susceptibility patterns were assessed using German (DIN) standards (when applicable). In addition, biochemical properties recommended for the phenotypic identification of L. adecarboxylata were evaluated, applying two commercially available identification systems for Enterobacteriaceae and seven conventional tests. L. adecarboxylata strains were naturally sensitive to tetracyclines, aminoglycosides, all but two beta-lactams, quinolones, folate pathway inhibitors, chloramphenicol, nitrofurantoin and azithromycin. They were naturally resistant to penicillin G, oxacillin, erythromycin, roxithromycin, clarithromycin, ketolides, lincosamides, streptogramins, linezolid, glycopeptides, rifampicin, fusidic acid and fosfomycin. There were only minor medium-dependent differences in susceptibility to most antibiotics. Lysine decarboxylase, malonate assimilation and acid production from arabitol and cellobiose, but not from adonitol and sorbitol, allowed definitive separation of L. adecarboxylata from E. coli. The results of this study form a database that can be applied to validate forthcoming antibiotic susceptibility tests of L. adecarboxylata, and might contribute to its reliable identification. Susceptibility patterns did not indicate obvious therapeutic difficulties for treatment of Leclercia infections. Special attention should be paid to biochemically aberrant leclerciae. Apart from biochemical features, fosfomycin susceptibility might be useful to differentiate between L. adecarboxylata and E. coli.