In vivo protection of penicillin-susceptible Bacteroides melaninogenicus from penicillin by facultative bacteria which produce beta-lactamase

We investigated the possibility that beta-lactamase producing strains of Klebsiella pneumoniae and Staphylococcus aureus can protect organisms of the Bacteroides melaninogenicus group from penicillin. A mixed infection was induced in mice in the form of a subcutaneous abscess involving a penicillin-susceptible encapsulated B. melaninogenicus, and a beta-lactamase producing strain of either K. pneumoniae or S. aureus. The infected animals were treated for 7 days with single or combined antimicrobial therapy. The single agents used were penicillin, clavulanic acid, metronidazole, and gentamicin. The antimicrobial combinations were penicillin and clavulanic acid, penicillin and gentamicin, and metronidazole and gentamicin. Administration of a single agent was effective in treating abscesses caused by susceptible organisms. The only effective therapy for mixed infections was by combination therapy of penicillin and clavulanic acid or metronidazole and gentamicin. This study supports the hypothesis that beta-lactamase producing facultative bacteria may shield their anaerobic counterparts from penicillin therapy, thereby contributing to the persistence of the infection.     

Quantitative Nasal Culture: a Tool in Antibiotic Research

The use of the quantitative nasal culture was investigated as a means of evaluation of new antimicrobial drugs in man. Cyclacillin was somewhat more active in vitro than penicillin G against penicillin G-resistant organisms. Cyclacillin was highly effective in suppressing staphylococci susceptible to penicillin G in nasal carriers but did not suppress staphylococci resistant to penicillin G. Although in previous studies by others cyclacillin was effective in treating mice infected with penicillin G-resistant staphylococci, in the present studies cyclacillin was not effective in suppressing nasal penicillin G-resistant staphylococci in man at doses which markedly suppressed penicillin G-sensitive organisms.     

I. Clinical evaluation

A study was undertaken to evaluate the therapy of streptococcal pharyngitis. The compliance of 118 patients with beta-hemolytic streptococcal pharyngitis to follow-up was 72%. Of 74 patients checked by means of urine tests 66 took their oral medication. No differences were detected in the clinical and bacteriological results (>98% streptococcal eradication) after the 7th or 10th day of therapy after taking either cephalexin or penicillin.It was concluded that: (a) for effective surveillance and follow-up special attention should be given to the uncooperative segment of the patient population; (b) a seven-day course of penicillin may be satisfactory in the eradication of BHS from the throat; and (c) cephalexin appears to be an effective alternative to penicillin for the treatment of streptococcal pharyngitis.     

Book Reviews

Book Review in this Article.
Cerebral Energy Metabolism and Metabolic Encephalopathy D. W. McCandless.
Microcomputers in the Neurosciences edited by G. Kerkut.
Myelin edited by P. Morell.
The Enzymes of Biological Membranes, Vol. 1. Membrane Structure and Dynamics (2nd edition) edited by A. N. Martonosi.     

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.     

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.     

Resistance to Penicillin in Mutants of a Penicillinase-Negative Organism, Staphylococcus aureus H

Penicillin-resistant mutants of Staphylococcus aureus H were similar to the parent in their response to penicillin though proportionately more penicillin was required for a given effect. The mutants did not inactivate penicillin. Most of the penicillin-binding sites (presumed to be murein transpeptidase molecules) bound penicillin rapidly when exposed to a very low concentration of penicillin (0.1 mug/ml), and yet the mutants retained some functional murein transpeptidase even in the presence of 500 mug of penicillin per ml. An hypothesis based on (i) functional versus nonfunctional transpeptidase molecules and (ii) variations in accessibility to penicillin can explain these findings.     

Antibiotics in management of staphylococcal endocarditis; with special reference to increasing bacterial resistance

Eighteen patients with staphylococcal endocarditis were observed at the Los Angeles County Hospital over a 3-year period (1947-49, inclusive). Twelve died. Bacterial sensitivity studies were carried out in 15 of the cases, and there was resistance to penicillin in ten. Aureomycin was effective in two cases of Staphylococcus aureus endocarditis in which there was no response to penicillin therapy. In one case of Staphylococcus aureus endocarditis the organism was resistant to penicillin and developed increasing resistance to aureomycin.     

ANTIBIOTICS IN MANAGEMENT OF STAPHYLOCOCCAL ENDOCARDITIS—With Special Reference to Increasing Bacterial Resistance

Eighteen patients with staphylococcal endocarditis were observed at the Los Angeles County Hospital over a 3-year period (1947-49, inclusive). Twelve died.Bacterial sensitivity studies were carried out in 15 of the cases, and there was resistance to penicillin in ten.Aureomycin was effective in two cases of Staphylococcus aureus endocarditis in which there was no response to penicillin therapy.In one case of Staphylococcus aureus endocarditis the organism was resistant to penicillin and developed increasing resistance to aureomycin.     

Allergy to penicillin: fable or fact?

OBJECTIVE--To assess whether, on the basis of one blood test, penicillin allergy might be excluded sufficiently for general practitioners to give oral penicillin to patients claiming a history of penicillin allergy. DESIGN--Prospective study of patients referred by general practitioners. SETTING--Outpatient allergy clinic in a district general hospital. PATIENTS--175 referred patients who gave a history of immediate type reaction to penicillin, of whom 144 attended as requested and 132 completed the investigations. MAIN OUTCOME MEASURES--History and examination, serum radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin, and oral challenge with penicillin. RESULTS--Of 132 patients, four were confirmed to have penicillin allergy by the radioallergosorbent test and 128 had an oral penicillin challenge without ill effect. CONCLUSIONS--Most patients who gave a history of penicillin allergy are not so allergic, and their actual allergic state should be substantiated whenever feasible. For patients reporting minor or vague reactions negative findings with a radioallergosorbent test to phenoxymethylpenicillin and benzylpenicillin provide sufficient evidence to give oral penicillin safely.     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Serum angiotensin-converting enzyme. Isolation and relationship to the pulmonary enzyme.

Angiotensin-converting enzyme from rabbit serum was purified almost 60,000-fold to apparent homogeneity by a procedure exploiting its affinity for antibodies prepared against the enzyme from lung. The pure serum and pulmonary enzymes exhibited identical behavior during gel filtration, sucrose gradient centrifugation, and disc gel electrophoresis in the reduced, denatured state. Their catalytic properties with hippurylhistidylleucine, angiotensin I, and bradykinin as substrates were similar and their reactivity with antilung enzyme antibody was indistinguishable as examined by immunodiffusion, inhibition dose-response curves, and radioimmunoassay. Their content of fucose, mannose, galactose, and N-acetylglucosamine was also comparable; however, N-acetylneuraminic acid was much more abundant in the serum glycoprotein. This difference may reflect selective removal of sialic acid-deficient enzyme molecules from the circulation by the hepatic lectin which has been postulated to initiate the catabolic phase for plasma glycoproteins (Ashwell, G., and Morell, A.G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 91-128).     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.     

Minimum amount of penicillin prophylaxis required to control Streptococcus pyogenes epidemic in closed community.

The prophylaxis required to control an epidemic of Streptococcus pyogenes throat infection in a junior detention centre has been reported. In a further epidemic an attempt was made to determine the minimum amount of penicillin required to control the outbreak. Oral penicillin (0.5 g) given as a single daily dose for 10 days to all boys after entry proved effective. The added risk of relatively deprived adolescent boys developing rheumatic fever is stressed.     

Prevention of pneumococcal infection in children with homozygous sickle cell disease.

The efficacy of prophylactic penicillin and of 14 valent pneumococcal vaccine in preventing pneumococcal infection in homozygous sickle cell (SS) disease was investigated in 242 children aged 6 months to 3 years at entry. In the first five years of the trial there were 11 pneumococcal infections in the pneumococcal vaccine treated group, 10 by serotypes present in the vaccine. Type 23 accounted for five of these, and there was evidence of higher infection rates in those given the vaccine before age 1. No pneumococcal isolations occurred in the penicillin group while receiving penicillin, although four isolations occurred within one year of stopping penicillin. Probably the most effective prophylaxis against pneumococcal infection requires penicillin beyond the age of 3. The age at which pneumococcal vaccine should be given must await further data on antibody response and clinical efficacy in these patients.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.     

Rapid detection of a chromosomally mediated penicillin resistance-associated ponA mutation in Neisseria gonorrhoeae using a real-time PCR assay

The recent emergence of a decreased susceptibility of Neisseria gonorrhoeae strains to penicillin in New Caledonia has lead clinicians to operate a change in the treatment strategy. In addition, this important health issue has emphasized the need for a rapid means of detecting penicillin resistance in N. gonorrhoeae in order to select an effective treatment and limit the spread of resistant strains. In recent years, the use of fluorescence resonance energy transfer on the LightCycler has proven to be a valuable tool for the screening of mutations occurring in the genome of various microorganisms. In this study, we developed a real-time PCR assay coupled with a fluorometric hybridization probes system to detect a penicillin resistance-associated mutation on the N. gonorrhoeae ponA gene. Following an extensive evaluation involving 136 isolates, melting curve analysis correctly evidenced a 5 degrees C T(m) shift in all N. gonorrhoeae strains possessing this mutation, as determined by conventional sequencing analysis. Moreover, the mutation profiles obtained with the real-time PCR showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Overall, our molecular assay allowed an accurate and reproducible determination of the susceptibility to penicillin corresponding to a mutation present in all chromosomally mediated resistant strains of N. gonorrhoeae.     

Active site mutations of recombinant deacetoxycephalosporin C synthase

Site-directed mutagenesis of active site residues of deacetoxycephalosporin C synthase active site residues was carried out to investigate their role in catalysis. The following mutations were made and their effects on the conversion of 2-oxoglutarate and the oxidation of penicillin N or G were assessed: M180F, G299N, G300N, Y302S, Y302F/G300A, Y302E, Y302H, and N304A. The Y302S, Y302E, and Y302H mutations reduced 2-oxoglutarate conversions and abolished (<2%) penicillin G oxidation. The Y302F/G300A mutation caused partial uncoupling of penicillin G oxidation from 2-oxoglutarate conversion, but did not uncouple penicillin N oxidation from 2-oxoglutarate conversion. Met-180 is involved in binding 2-oxoglutarate, and the M180F mutation caused uncoupling of 2-oxoglutarate from penicillin oxidation. The N304A mutation apparently enhanced in vitro conversion of penicillin N but had little effect on the oxidation of penicillin G, under standard assay conditions.