Penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: kinetic characterization of its interactions with beta-lactams using electrospray mass spectrometry.

Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a) was overexpressed in Escherichia coli mostly in the soluble form accounting for approximately 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry. A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (ka) of PBP2a acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant Kd of 13.3 mM and the first-order rate constant k2 of 0.22 s-1. From these values, the second-order rate constant k2/Kd, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s-1 was obtained. The fairly high Kd value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a and methicillin revealed k2 of 0.0083 s-1 and Kd of 16.9 mM, resulting in an even smaller k2/Kd value of 0.49 M-1 s-1. The rate constants k3 for deacylation of the acyl-PBP2a complexes, the third step in the interactions, were measured to be <1.5 x 10(-)5 s-1. These results indicate that the resistance of PBP2a to penicillin inactivation is mainly due to the extremely low penicillin acylating rate in addition to the low association affinity, but not to a fast rate of deacylation. Acylation of PBP2a by a high-affinity cephalosporin, Compound 1, also followed a saturation curve of ka versus the compound concentration, from which k2 = 0.39 s-1, Kd = 0.22 mM, and k2/Kd = 1750 M-1 s-1 were obtained. The 100-fold increase in the k2/Kd value as compared with that of benzylpenicillin is mostly attributable to the decreased (60-fold) Kd, indicating that the cephalosporin fits much better to the binding pocket of the protein.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori.

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.

Cytotoxic activity in broth-culture filtrates of Campylobacter pylori.

Broth-culture filtrates of Campylobacter pylori induced non-lethal cytopathic effects in vitro in 7 of 9 mammalian cell lines tested. Transmission electronmicroscopy revealed that the response consisted of intracellular vacuolisation. Intestine 407 cells were among the most responsive and were used for routine assay. About 55% of isolates of C. pylori tested, originating from four geographic regions worldwide, produced cytotoxic activity. The activity was neutralisable by specific antisera to broth-culture filtrates or to sonicated bacteria but not by antisera to other bacterial preparations. Cytotoxic activity was heat-labile (70 degrees C for 30 min), was protease-sensitive and ammonium-sulphate precipitable. It did not pass through an ultrafiltration membrane with a nominal mol.-wt limit of 100 X 10(3). It was concluded that C. pylori can produce a factor that alters cultured cells in vitro. The relevance of this factor to the pathogenesis of gastritis associated with C. pylori remains to be determined.

Growth of Campylobacter pylori in liquid media.

Until recently, broth cultivation techniques for Campylobacter pylori were unavailable. We developed a method to cultivate bacterial cells within 24 h in liquid media. Cultivation in broth depended on the adequate dispersion of appropriate gases. A static broth at 37 degrees C in a GasPak jar (BBL Microbiology Systems, Cockeysville, Md.) with a CampyPak (BBL) envelope did not support growth after 5 days of incubation. A broth placed in a flask on a Gyrotory water bath shaker (150 rpm; New Brunswick Scientific Co., Inc., Edison, N.J.) fitted with a gassing hood connected to a gas mixture of 10% CO2, 5% O2, and 85% N2 supported good growth. An initial inoculum of 10(5), 10(3) to 10(4), or 10(2) CFU/ml resulted in greater than or equal to 10(8) CFU/ml after incubation for 24, 48, or 72 h, respectively. Under these conditions, the bacteria grew as motile, spiral bacilli rather than the oval and coccal bacilli occasionally reported. Several bases supported good growth when supplemented with serum. For the determination of basal growth conditions, brucella broth base was used. Fetal calf serum (1%) provided maximum growth. Vitox was not necessary for growth and did not augment growth. C. pylori grew over a wide optimal pH range of 5.5 to 8.5.

Establishment of gastric Campylobacter pylori infection in the neonatal gnotobiotic piglet.

Campylobacter pylori, a gram-negative microaerophilic bacterium, has been implicated in the genesis of human gastritis, dyspepsia, and gastroduodenal ulceration. Previous attempts to reproduce the diseases in conventional laboratory animal species have been unsuccessful. To determine if neonatal gnotobiotic piglets were susceptible to C. pylori, we orally challenged two litters (n = 17) with 10(9) CFU after pretreating them with cimetidine. Controls housed in separate units received nothing or peptone water alone. Piglets were examined 1, 2, 3, and 4 weeks after challenge. Colonization by the bacterium and inflammation of the gastric mucosa persisted throughout the study period. Organisms were revealed by Warthin-Starry silver stain to reside between the mucus layer and the gastric epithelium. Culturing of samples from sites along the gastrointestinal tract revealed that the bacterium colonized essentially only the gastric and proximal duodenal mucosae. Gross pathological changes were restricted to the stomachs of infected piglets and consisted of submucosal edema, increased gastric mucus production, and progressive development of mucosal lymphoid follicles. Microscopic lesions consisted of transient neutrophilic infiltrates followed by diffuse and follicular infiltrations of mononuclear leukocytes into the mucosa and submucosa. Alcian blue-periodic acid-Schiff stains suggested that the infection resulted in the depletion of mucopolysaccharide production by deep gastric glands. These data indicate that gnotobiotic piglets reproduce many of the features of diseases associated with C. pylori in humans.

Cutaneous infection in normal and immunocompromised mice.

Since a model of staphylococcal skin infection adequately reflecting human disease was unavailable, a self-limiting animal infection model specific for virulent Staphylococcus species was developed. A virulent strain of S. aureus, NCTC 9789 (ATCC 27700), was used to develop an infection model in adult, male CF-1 mice treated with 0 to 150 mg of cyclophosphamide (CY) per kg 4 days before challenge. Bacteria were inoculated onto the dorsal side of shaved mice at 0 to 10(6) CFU per mouse. Simultaneously, the skin was gently scraped to remove the superficial layers without drawing blood. The wound was occluded with impermeable film secured with surgical tape. At a CY dose of 50 mg/kg and an inoculum of 10(5) CFU, 89% of the mice (96 of 108) developed large abscesses (approximately 15-mm diameter). Mice which were not immunocompromised developed fewer abscesses (20 of 68). Generally, no abscesses formed when the mice were not wounded (1 of 62), occluded (0 of 89), or inoculated (11 of 50). The abscesses developed 24 to 48 h after challenge and persisted for 2 to 3 weeks. The challenge organism was isolated from the abscesses. The rates of abscess formation of three additional S. aureus strains varied widely in normal and CY-treated mice. Three strains of S. epidermidis, one of Micrococcus varians, and one of S. saprophyticus failed to cause abscesses. Bacterial proliferation studies demonstrated that a strain of S. aureus and a strain of S. epidermidis proliferated to the same levels 48 h after challenge. Immunosuppression and wounding had little effect on the levels of proliferation of S. aureus (P greater than 0.2). Without occlusion, however, S. aureus proliferated to significantly lower levels (P less than 0.005). This model may be be useful for screening topical anti-infective agents or studying the mechanisms of bacterial pathogenesis and host response.