We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 ,ug/ml; range, 0.1 to 0.7 ,ug/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, c0.06 ,ug/ml) for comparison. The resistant strains did not produce detectable I-lactamase activity, otherwise modify penicillin G, or bind less total penicillin.Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3.Concurrent with an epidemic of meningococcal disease in Spain from 1978 to 1985, the National Reference Laboratory began surveillance which included screening for penicillin G susceptibility. Of 3,264 strains isolated from blood or cerebrospinal fluid during those years, only one resistant isolate was observed in the final year (12). Subsequently, in the first six months of 1986, 9 (5%) of 168 invasive isolates were found to be relatively resistant to penicillin G. More worrisome is the decreased susceptibility of these strains to other P-lactams (2). The emergence of relatively penicillin-Gresistant strains in a short period of time suggests the possibility of a mutational event and dispersion of a clone or acquisition of a mobile genetic element harboring resistance. We sought to define the mechanism of resistance in these straihs. Possibilities include the production of ,B-lactamase, other enzymatic modification of the penicillin G molecule, acqcuisition of relative impermeability to penicillin G, or insensitivity of the cellular targets, the penicillin-binding proteins (PBPs). Each mechanism has different implications as to the activity of alternative antibiotics against these strains. MATERIALS AND METHODSStrains. All 16 resistant (defined as those growing on media containing 0.1 jig of penicillin G per ml) strains were isolated from cerebrospinal fluid or blood during 1985 and 1986 in Spain and were identified as Neisseria meningitidis by standard methods (9); 11 were serogroup B, 4 were serogroup C, and 1 was autoagglutinable. Twelve strains identified as susceptible (no growth on media containing 0.1 jig of penicillin G per ml) isolated in the same geographic area of Spain were used for comparisons; 8 were serogroup B, and 4 were serogroup C. All strains were stored in 50% tryptic soy broth (Difco Laboratories, Detroit, Mich.) containing 10% glycerol at -70°C.Enzymatic modification. P-Lactamase activity was sought by two methods; the rapid acidimetric assay with penicillin * Corresponding author. G (Sigma Chemical Co., St. Louis, Mo.) as substrate and phe...

A 10-year-old boy presented with nuchal rigidity and cerebrospinal fluid (CSF) leukocytosis initially and again on day 6 of intravenous cefuroxime therapy (200 mg/kg/day). Both CSF specimens yielded nontypable beta-lactamase-negative Haemophilus influenzae that were susceptible by disk tests but relatively resistant to cefuroxime (MIC, 8- to 16-fold greater than that of control isolates). To define the mechanism of resistance, the cefuroxime resistance marker was transformed to a susceptible H. influenzae recipient; inactivation and permeability of beta-lactam substrate were tested and the penicillin-binding protein (PBP) profiles were examined. Inactivation of beta-lactam substrate was not detected and reduced permeability was not found. However, reduced beta-lactam binding to PBPs 4 and 5 was observed; 18- to 27-fold more penicillin and 2.5-to 4-fold more cefuroxime was required to saturate or block 50% of the binding sites of these PBPs, respectively. Thus, reduced affinity of PBPs 4 and 5 for beta-lactam substrate appears to be the mechanism of cefuroxime resistance in this strain. The reduced affinity of these targets appears to have contributed to the bacteriologic and clinical failure in this patient.

Penicillin-binding protein (PBP) alterations have been associated with non-p-lactamasemediated ampicillin resistance in Haemophilus injhenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. injluenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 pg ml-I for the susceptible isolates at an inoculum of 100000 c.f.u. when tested by broth dilution, and was 0-5 pg.ml-I for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 pg mi-' when tested by broth dilution, and from 1.5 to 16 ug ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in oivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillinsusceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two ofthe resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics. 'These results suggest ( a ) that care should be taken in interpreting changes in PBP profiles for species that demonstrate variability such as H . influenzae, and (b) that the decreased binding affinity of PBP 5 is a consistent finding associated with multiple ampicillin-resistant wild-type isolates.

Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-4-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.of Haemophilus influenzae.