Biomedical subjects

B C Hill

Publications and source records attributed to B C Hill.

At least 37 records · Page 2Linked to original sources

PubMed1995

High-yield purification of cytochrome aa3 and cytochrome caa3 oxidases from Bacillus subtilis plasma membranes.

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.

Bacillus subtilis↗

PubMed1995

Effects of bipolar point and line stimulation in anisotropic rabbit epicardium: assessment of the critical radius of curvature for longitudinal block.

Excitation front shape and velocity were studied in anisotropic perfused rabbit epicardium stained with potentiometric fluorescent dye. In the combined results from all experiments, convex excitation fronts produced by stimulation with a single electrode propagated longitudinally 13.3% slower than flat excitation fronts produced by stimulation with a line of electrodes. For transverse propagation, the two stimulation methods produced similar flat excitation fronts and velocities. The critical excitation front radius of curvature for longitudinal block (Rcr), calculated from excitable media theory, was 92 microns in control hearts. In hearts exposed to diacetyl monoxime (20 mmol/L), which decreases inward sodium current, Rcr was 175 microns. The slower longitudinal propagation velocity of convex fronts versus flat fronts and the theoretically predicted critical radius of curvature may be important for propagation and block of ectopic depolarizations in the heart.

Action Potentials↗

PubMed1994

The room temperature reaction of carbon monoxide and oxygen with the cytochrome bd quinol oxidase from Escherichia coli.

When grown under O2-limited conditions, Escherichia coli expresses a cytochrome bd quinol oxidase that has an unusually high affinity for O2. We have studied the reaction of cytochrome bd with CO and O2 by rapid-reaction spectrophotometry. The reduced enzyme forms a photosensitive ferrocytochrome d-CO complex, and following photolysis, CO recombines with the reduced enzyme with a bimolecular rate of 8 x 10(7) M-1 s-1. Reaction of CO-bound enzyme with O2 gives a CO off-rate of 1.6 s-1. The O2 reaction is followed by a flow-flash procedure in which CO-ligated enzyme is mixed with O2, and the reaction commenced by photolysis of cytochrome d-CO. In the presence of O2, two processes are resolved on a time-scale of 300 microseconds. The absorbance at 645 nm first increases at a rate that is dependent on O2 concentration with a value of 2 x 10(9) M-1 s-1. The second phase results in decreased absorbance at 645 nm and increased absorbance at 680 nm. The rate of the second process is independent from O2 concentration above 50 microM O2 and reaches a first-order limit of 1 x 10(4) s-1. A model for the reaction of the cytochrome bd quinol oxidase with O2 is proposed in which an initial ferrocytochrome d-oxy adduct forms, and then decays to a ferryl-oxo species. The oxidation of the low-spin cytochrome b component of the oxidase, monitored at 560 nm, occurs at the same time as the ferryl species forms. We suggest that the suitability of the cytochrome bd quinol oxidase to function at low O2 concentration is conferred by its rapid rate of binding O2.

Carbon Monoxide↗

PubMed1994

The pathway of CO binding to cytochrome c oxidase. Can the gateway be closed?

Addition of cyanide to the CO complex of cytochrome oxidase reduces the apparent photosensitivity of the Fe-CO bond. This effect is not seen with azide, or when cyanide is added to ferromyoglobin-CO. It is proposed that cyanide binds to CuB, and restricts the passage of CO out of the protein. This restriction favors geminate recombination of CO and ferrocytochrome a3, thereby lowering the apparent quantum yield for CO photolysis. The apparent Kd of cyanide for CuB is 15.4 mM. These data support a direct role for CuB in ligand binding by cytochrome c oxidase.

Animals↗

PubMed1994

Modeling the sequence of electron transfer reactions in the single turnover of reduced, mammalian cytochrome c oxidase with oxygen.

Single-turnover studies of the reaction of reduced cytochrome oxidase with oxygen has led to a mechanistic model that specifies a linear sequence of electron transfer from cytochrome c to O2 via the four redox active metals of mitochondrial cytochrome oxidase. Fully reduced oxidase initially forms a dioxygen adduct within which two electrons are transferred at a rate of 6 x 10(4) s-1 to form a peroxy adduct. This two-electron step results in single-electron oxidation of cytochrome alpha 3 and cytochrome alpha. Cytochrome alpha is then re-reduced by CuA in an intramolecular reaction. Subsequent reoxidation of cytochrome alpha occurs at a rate nearly 100-fold slower than its initial oxidation. Oxidation of CuB in this sequence has been the most difficult to determine and here is specified as the third electron transfer step. This reaction sequence is unchanged in the presence of tightly bound cytochrome c, although cytochrome c is rapidly oxidized via CuA and cytochrome alpha. The linear model advanced here has CuA as the acceptor of electrons from cytochrome c, with cytochrome alpha serving as a bridge to deliver electrons from CuA (and cytochrome c) to the binuclear center, cytochrome alpha 3-CuB.

Animals↗

PubMed1994

Epidemic gram-negative bacteremia in a neonatal intensive care unit in Guatemala.

BACKGROUND: Nosocomial bloodstream infection is an important cause of morbidity and mortality among neonates. From September 1 through December 5, 1990 (epidemic period), gram-negative bacteremia developed in 26 neonates after their admission to the neonatal intensive care unit (NICU) of Hospital General, a 1000-bed public teaching hospital in Guatemala with a 16-bed NICU. Twenty-three of the 26 patients (88%) died. METHODS: To determine risk factors for and modes of transmission of gram-negative bacteremia in the NICU, we conducted a cohort study of NICU patients who had at least one blood culture drawn at least 24 hours after admission to the NICU and performed a microbiologic investigation in the NICU. RESULTS: The rate of gram-negative bacteremia was significantly higher among patients born at Hospital General, delivered by cesarian section, and exposed to selected intravenous medications and invasive procedures in the NICU during the 3 days before the referent blood culture was obtained. During the epidemic period, the hospital's chlorinated well-water system malfunctioned; chlorine levels were undetectable and tap water samples contained elevated microbial levels, including total and fecal coliform bacteria. Serratia marcescens was identified in 81% of case-patient blood cultures (13/16) available for testing and from 57% of NICU personnel handwashings (4/7). Most S. marcescens blood isolates were serotype O3:H12 (46%) or O14:H12 (31%) and were resistant to ampicillin (100%) and gentamicin (77%), the antimicrobials used routinely in the NICU. CONCLUSIONS: We hypothesize that gram-negative bacteremia occurred after invasive procedures were performed on neonates whose skin became colonized through bathing or from hands of NICU personnel.

Bacteremia↗

PubMed1994

Virtual electrode effects in myocardial fibers.

The changes in transmembrane potential during a stimulation pulse in the heart are not known. We have used transmembrane potential sensitive dye fluorescence to measure changes in transmembrane potential along fibers in an anisotropic arterially perfused rabbit epicardial layer. Cathodal or anodal extracellular point stimulation produced changes in transmembrane potential within 60 microns of the electrode that were positive or negative, respectively. The changes in transmembrane potential did not simply decrease to zero with increasing distance, as would occur with a theoretical fiber space constant, but instead became reversed beyond approximately 1 mm from the electrode consistent with a virtual electrode effect. Even stimulation from a line of terminals perpendicular to the fibers produced negative changes in transmembrane potential for cathodal stimulation with the largest negative changes during a 50-ms pulse at 3-4 mm from the electrode terminals. Negative changes as large as the amplitude of the action potential rising phase occurred during a 50-ms pulse for 20-volt cathodal stimulation. Switching to anodal stimulation reversed the directions of changes in transmembrane potential at most recording spots, however for stimulation during the refractory period negative changes in transmembrane potential were significantly larger than positive changes in transmembrane potential. Anodal stimulation during diastole with 3-ms pulses produced excitation in the region of depolarization that accelerated when the stimulation strength was increased to > 3 times the anodal threshold strength. Thus, virtual electrode effects of unipolar stimulation occur in myocardial fibers, and for sufficiently strong stimuli the virtual electrode effects may influence electrical behavior of the myocardium.

Animals↗

PubMed1993

Quenching of intrinsic fluorescence of yeast cytochrome c peroxidase by covalently- and noncovalently-bound quenchers.

The intrinsic steady-state fluorescence of the heme enzyme cytochrome c peroxidase (CCP) has been characterized as a probe of its structure in solution. The fluorescence is dominated by tryptophan emission, which has a quantum yield of 7% relative to the tryptophan standard N-acetyltryptophanamide, and an emission maximum at 324 nm indicative of a relatively hydrophobic environment for the fluorescent residues. These fluorescence properties are consistent with the known structure of CCP; six of the seven tryptophan residues are well within quenching distance for efficient Förster energy transfer to the heme, so that the intrinsic fluorescence arises largely from Trp101 which is approximately 26 A from the heme and partially buried. Quenching studies using Cs+, I-, and acrylamide are also consistent with this picture, since the charged species are poor quenchers, but acrylamide, which can penetrate the protein matrix, is a more effective quencher. The intrinsic fluorescence of two CCP derivatives with the quencher pentaammineruthenium(III) covalently attached to His6 and His60 has also been characterized. The His60 derivative, shown by X-ray analysis to be essentially structurally identical to native CCP, is 17% less fluorescent than native CCP, consistent with the quenching expected from distance calculations and the assignment of Trp101 as the major fluorescent center. The observed quenching of 38% in the second derivative is close to that predicted for ruthenation of His6 assuming that Trp101 is the major fluorophore. The fluorescence of compound I of CCP is also reported. This species, which has a ferryl (FeIV = O) heme and a protein radical purportedly on Trp191, exhibits 9% higher fluorescence than native CCP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗

PubMed1993

The reaction of Bacillus subtilis aa3-600 oxidase with oxygen: an aa3-oxidase lacking the CuA site.

The reaction of reduced cytochrome aa3-600 nm oxidase from Bacillus subtilis with oxygen has been measured. The reaction of fully reduced oxidase with oxygen was studied by the flow-flash method which employs the photosensitive CO complex to protect the enzyme from oxygen until the reaction is initiated by a photolytic flash. The rate of the reaction, measured in the Soret region, is oxygen concentration dependent at low levels of oxygen and becomes independent from oxygen above 200 microM. The reaction reaches a rate limit of 9500 s-1 at high oxygen. This type of oxygen concentration dependence indicates that the oxidation reaction is preceded by another event, probably an oxygen combination reaction. When compared to mammalian cytochrome oxidase the Bacillus oxidase exhibits a simpler reaction profile. This simpler observed reaction profile can be accounted for by the absence of CuA in the Bacillus oxidase using the same model developed for the single turnover of mammalian cytochrome oxidase.

Bacillus subtilis↗

PubMed1993

Kinetic and ligand binding evidence for two heme A-based terminal oxidases in plasma membranes from Bacillus subtilis.

Detergent-solubilized plasma membranes from Bacillus subtilis have been characterized for their cytochrome oxidase content. Triton X-100-solubilized membranes show high O2 turnover with ascorbate plus TMPD. Reduced-oxidized difference spectroscopy of ascorbate-TMPD-reduced membranes reveals the presence of cytochrome c and cytochrome a. An additional, b-type cytochrome appears when the membranes are reduced with dithionite. Time-resolved difference spectra taken during reduction by ascorbate-TMPD reveal two kinetic forms of heme A-containing cytochromes. There is a high-turnover form that is rapidly reduced upon anaerobiosis, and a second type which is only slowly reduced upon anaerobiosis. The slowly reduced oxidase is distinguished by an alpha-band blue-shifted to 600 nm relative to the 603-nm position observed for high-turnover oxidase. Addition of CO to ascorbate-TMPD-reduced membranes gives a spectrum typical of ferrocytochrome a3-CO, and the intensity corresponds to the total ferrocytochrome a3 concentration. Photolysis of ascorbate-TMPD-reduced, CO-bound membranes indicates that both species are photosensitive with similar rates of recombination. Addition of CO to dithionite-reduced membranes shows an additional CO reactive center that has a spectrum characteristic of cytochrome o. Cyanide blocks complete reduction of high-turnover oxidase by ascorbate plus TMPD, but does not appear to effect slowly reduced oxidase. These results indicate the presence of two different types of cytochrome aa3 oxidase in plasma membranes of B. subtilis.

Ascorbic Acid↗

PubMed1993

The sequence of electron carriers in the reaction of cytochrome c oxidase with oxygen.

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochrome c by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochrome c, to CuA, to cytochrome a, and then to the binuclear (i.e., cytochrome a3-CuB) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochrome c to CuA 5-7 A, cytochrome c to cytochrome a 20-25 A, CuA to cytochrome a 14-16 A and cytochrome a to cytochrome a3-CuB 8-10 A. It is proposed that the step from cytochrome a to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.

Animals↗

PubMed1993

Characterization of glycopeptide-resistant enterococci from U.S. hospitals.

We examined 105 clinical isolates of glycopeptide-resistant enterococci collected from 31 U.S. hospitals in 14 states during May 1988 to July 1992. The isolates included 82 Enterococcus faecium, 8 E. faecalis, 6 Enterococcus spp., 5 E. gallinarum, 3 E. casseliflavus, and 1 E. raffinosus. The isolates were categorized into the following four phenotypes of glycopeptide resistance on the basis of their MIC patterns: (i) 70 VanA (vancomycin [Vm] MIC, > or = 64 micrograms/ml; teicoplanin [Tei] MIC, 16 to > or = 128 micrograms/ml), (ii) 26 VanB (Vm MIC, 16 to 1,024 micrograms/ml; Tei MIC, < or = 2 micrograms/ml), (iii) 5 VanC (Vm MIC, 4 to 16 micrograms/ml; Tei MIC, < or = 2 micrograms/ml) in E. gallinarum, and (iv) 3 E. casseliflavus and 1 E. raffinosus isolates for which Vm MICs were 4 to 16 micrograms/ml and Tei MICs were < or = 1 micrograms/ml were called unclassified. Of the 101 isolates with the VanA, VanB, and VanC phenotypes, 99 were confirmed by production of a specific 1,030-, 433-, or 796-bp polymerase chain reaction product, respectively, and hybridization with the respective gene probe. The vanA gene was also detected in the E. raffinosus isolate for which the Vm MIC was 16 micrograms/ml and the Tei MIC was 1 microgram/ml. The vanA gene was located on either a 34- or a 60-kb plasmid in all of the U.S. isolates examined. Pulsed-field gel electrophoresis demonstrated both intrahospital and interhospital diversity among Vmr enterococci in the United States and was more useful than plasmid analysis for epidemiologic studies.

Anti-Bacterial Agents↗

PubMed1993

Optical recordings of the effect of electrical stimulation on action potential repolarization and the induction of reentry in two-dimensional perfused rabbit epicardium.

BACKGROUND: Prolonged membrane depolarization induced by an electric shock in the heart may produce propagation block leading to repetitive beats. We studied prolonged depolarization and its role in repetitive beats in a thin epicardial layer of endocardially prefrozen arterially perfused rabbit heart. METHODS AND RESULTS: A laser scanner recorded optical action potentials at 63 sites within a 1-cm2 area on the left ventricle of hearts stained with potentiometric fluorescent dye. Pacing (S1) produced propagation across the myofibers; then, a 3-millisecond shock (S2) given in the S1 refractory period produced an electric field that decreased in strength with distance along the fibers. The S2 strengths at the center of the scanned region (C) were 2.1 +/- 0.2 or 5.6 +/- 0.3 V/cm (mean +/- SD, n = 4). Repetitive beats occurred in 50% of hearts when C was 2.1 V/cm and in 100% of hearts when C was 5.6 V/cm. With each occurrence of repetitive beats, prolonged depolarization of the shocked action potential occurred within 1 mm of the S2 electrode when C was 2.1 V/cm and within 3 mm when C was 5.6 V/cm. Transient block immediately after S2 occurred between tissue with prolonged depolarization (S2 strength, 6 to 9 V/cm) and tissue without prolonged depolarization (S2 strength, 1 to 3 V/cm). Propagation in the scanned region after S2 occurred first on the side of the block distal to the S2 electrode, propagated from the most recovered to the least recovered tissue, and then turned toward the S2 electrode. When C was 5.6 V/cm, reentry by retrograde propagation near the S2 electrode produced repetitive beats. The center of the reentrant circuit exhibited further transient block and small depolarizations associated with the circulating activation. CONCLUSIONS: Prolonged depolarization occurs where the S2 strength is more than 6 V/cm, block occurs between regions of prolonged depolarization and no prolonged depolarization, and reentry occurs around the block. Shock-induced prolonged depolarization can be proarrhythmic and may account for electrically induced arrhythmias.

Action Potentials↗

PubMed1993

Optical measurements of transmembrane potential changes during electric field stimulation of ventricular cells.

We evaluated transmembrane potential changes at the ends of isolated rabbit ventricular myocytes during defibrillation-strength shocks given in the cellular refractory period. The myocytes were stimulated (S1 pulse) to produce an action potential. Then a constant-field shock (S2 pulse) with an electric field of 20 or 40 V/cm was given at an S1-S2 interval of 50 msec. The cells were stained with potentiometric dye (di-4-ANEPPS), and the cell end facing the S2 anode or cathode was illuminated with a laser while the fluorescence was recorded. During S2, the cell end facing the S2 cathode became more positive intracellularly, whereas the cell end facing the S2 anode became more negative intracellularly. The S2-induced transmembrane potential change at the cell end (delta Vm) was determined relative to the amplitude of the S1-induced action potential (APA) in each recording (i.e., delta Vm/APA). In Tyrode's solution containing 4.5 mM potassium, delta Vm/APA for 40-V/cm S2 was 1.36 +/- 0.34 at the cell end facing the S2 cathode and -1.65 +/- 0.61 at the cell end facing the S2 anode (n = 9). For the 20-V/cm S2, delta Vm/APA was 0.61 +/- 0.33 at the cell end facing the S2 cathode and -0.71 +/- 0.33 at the cell end facing the S2 anode (n = 6). The delta Vm/APA was not significantly influenced by 20 mM diacetyl monoxime. These results indicate that large delta Vm values occurred at the ends of the cells during S2. The calculated values of delta Vm, assuming a nominal APA of 130 mV, were 177 and -214 mV for the 40-V/cm S2 and 79 and -93 mV for the 20-V/cm S2. The delta Vm was correlated with cell size (r > or = 0.95) and agreed with values predicted by the S2 electric field strength multiplied by half of the cell length to within 27%. When the potassium concentration was increased to 20 mM, delta Vm/APA for 40 V/cm S2 increased 85% and 67% at the cell ends facing the S2 cathode and anode, respectively (n = 9, p < 0.005 versus 4.5 mM potassium), consistent with reduced APA. Thus, with normal or elevated extracellular potassium, transmembrane potential changes at the ends of cells during defibrillation-type stimulation are large enough to produce activation or recovery of voltage-dependent ion channels and may produce the effects responsible for defibrillation.

Animals↗

PubMed1992

A cluster of vancomycin-resistant Enterococcus faecium in an intensive care unit.

OBJECTIVE: To describe the epidemiology of a cluster of vancomycin-resistant Enterococcus faecium (VAREC) in a cardiothoracic surgery intensive care unit. DESIGN: A case series of patients identified through review of surveillance data on nosocomial infections, review of microbiologic records, and culture survey of patients in the unit. RESULTS: Six patients in the cardiothoracic surgery intensive care unit had VAREC with identical antimicrobic susceptibility patterns over a 6-month period. Four patients were identified with VAREC through prospective surveillance and 2 through retrospective review. Prior vancomycin use was seen more commonly in patients with VAREC (6/6, 100%) than in those without VAREC (3/12, 25%) (Fisher's exact test, p = .01). Six of the 7 patients with prior infection developed VAREC (85.7%). A prior nosocomial infection and prior exposure to vancomycin were found to be important variables in a logistic regression analysis. VAREC also was isolated from the environment. A combination of cohorting of patients and staff, and modifications of standard contact isolation practices eliminated the presence of VAREC from the cardiothoracic surgery intensive care unit. CONCLUSIONS: The results suggest that prior administration of vancomycin, especially in the patient who develops nosocomial infection, can influence the acquisition of vancomycin-resistant enterococci and that VAREC may be transmitted from patient to patient. Using a modification of the standard infection control practice of isolation, we were able to control the spread of this resistant strain of E faecium.

Cross Infection↗

PubMed1992

Infections and pseudoinfections due to povidone-iodine solution contaminated with Pseudomonas cepacia.

In 1989 we investigated the first instance of Pseudomonas cepacia infections due to intrinsic contamination of a povidone-iodine product. Six patients in a Texas pediatric facility had P. cepacia infection or pseudoinfection (three, peritonitis; one, pseudoperitonitis; and two, pseudobacteremia). Epidemiological studies showed one risk factor for infection of peritoneal fluid with P. cepacia: performance of peritoneal dialysis in the dialysis unit with use of one lot of povidone-iodine later found to be intrinsically contaminated (4/5 vs. 0/16, P = .001). Blood cultures yielded P. cepacia after nurses wiped the tops of blood culture bottles with the povidone-iodine solution before inoculation. P. cepacia was cultured from three povidone-iodine containers used at the hospital and from four containers of the same lot obtained from other health-care facilities in Texas and California. Isolates from patients and the povidone-iodine had similar antibiograms, identical plasmid profiles, and identical DNA banding patterns on the basis of results of ribonucleotide typing. This investigation demonstrates that intrinsic contamination of povidone-iodine solution with P. cepacia can result in infections in addition to colonization and/or pseudoinfection.

Bacteremia↗

PubMed1992

d-Sotalol has opposite effects from encainide and propafenone on the proportion of episodes of ventricular tachycardia that are sustained in an experimental substrate for reentry.

Conversion of sustained ventricular tachycardia (VT) to nonsustained VT may be a potent mode of antiarrhythmic drug action, whereas a drug's conversion of nonsustained VT to sustained VT could produce serious clinical complications. We tested the effects of two class Ic drugs [encainide (1, 2, and 4 microM) and propafenone (0.1, 0.3, and 0.6 microM)) and a class III drug (d-sotalol (25, 50, and 100 microM)] on the proportion of VT episodes that were sustained (duration greater than 1 min) in an acute in vitro model of reentrant VT in left ventricular (LV) epicardium: a Langendorff-perfused rabbit heart whose LV endocardial and midwall cells have been killed by a selective freezing procedure. Multiple VT episodes were generated by increasing the stimulation rate until self-sustained activity occurred. Some episodes spontaneously terminated in less than 1 min; others lasted longer and were terminated by transient cooling of the heart. Both encainide and propafenone increased the fraction of episodes of sustained VT in all preparations; the increase was significant in 4 of the 5 encainide preparations (p less than 0.02) and 4 of the 5 propafenone preparations (p less than 0.003). d-Sotalol, on the other hand, decreased the fraction of sustained VT in all preparations; the decrease was significant (p less than 0.002) in 4 of the 5 preparations. All 3 drugs increased the basic cycle length of pacing at which VT was induced and the cycle time of the resulting VT. These results of this new assay of drug action may be related to the drugs' different mechanisms of prolonging refractoriness.

Animals↗

PubMed1992

Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.

Autoradiography↗