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Wie das pathogene Bakterium Neisseria meningitidis kolonisiert auch Neisseria lactamica als Kommensale den oberen Nasopharynx des Menschen. Penicillin G ist ein first-line-Therapeutikum gegen Meningokokkeninfektionen. Reduzierte Empfindlichkeit gegenüber Penicillin wird bei Meningokokken durch Mutationen im penA-Gen verursacht. Horizontaler Gentransfer zwischen den verschiedenen Neisseria spp. wurde auch für das penA-Gen beschrieben. Ziel dieser Arbeit war daher eine phänotypische und genotypische Analyse der Penicillinresistenz von N. lactamica. Aus den Versuchen sollten Prognosen über die zukünftige Resistenzentwicklung von Meningokokken abgeleitet werden. Die phänotypische Analyse von 123 N. lactamica-Stämmen (MIC [Minimum inhibitory concentration]-Bereich: 0,064 – 2,0 µg/ml, Median: 0,38 µg/ml) und 129 N. meningitidis- Stämmen (MIC-Bereich: 0,016 – 0,25 µg/ml, Median: 0,064 µg/ml) zeigte signifikant höhere MIC-Werte gegenüber Penicillin G bei den N. lactamica-Stämmen als bei den untersuchten Meningokokken. Bei Meningokokken sind Polymorphismen (fünf spezifische Mutationen betreffend) im penA-Gen (kodiert für das PBP2 (penicillin binding protein 2)) für verminderte Penicillinsensibilität verantwortlich, weshalb der betroffene Abschnitt des penA-Gens in allen N. lactamica-Stämmen und N. meningitidis-Stämmen untersucht und mit den bekannten Allelen der penA-Datenbank verglichen wurde. Bei den 123 N. lactamica-Stämmen konnten 60 verschiedene penA-Allele nachgewiesen werden, wovon 51 neu in die internationale penA-Datenbank eingefügt werden konnten. Im Gegensatz zu Meningokokken trugen die N. lactamica-Stämme entweder drei oder fünf der für intermediär resistente Meningokokken charakteristischen Mutationen im penA-Gen. N. lactamica-Stämme mit fünf Mutationen (MIC-Bereich: 0,25 – 2,0 µg/ml, Median: 0,5 µg/ml) zeigten signifikant höhere MIC-Werte als Stämme mit drei Mutationen (MIC-Bereich: 0,064 – 0,38 µg/ml, Median: 0,125 µg/ml), aber auch als Meningokokken mit fünf Mutationen (MIC-Bereich: 0,064 – 0,25 µg/ml, Median: 0,125 µg/ml). Eine phylogenetische Analyse aller in der penA-Datenbank hinterlegten Allele zusammen mit den 51 neuen dieser Studie ergab, dass die Allele mit fünf Mutationen unabhängig von der Spezies eine gemeinsame phylogenetische Linie bildeten, während sowohl die Allele mit drei Mutationen (N. lactamica) als auch die ohne Mutationen (N. meningitidis) jeweils eine separate phylogenetische Gruppe formten. Im Rahmen von in vitro-Transformationen mit chromosomaler DNA von N. lactamica konnte der MIC-Wert des Penicillin-sensiblen Meningokokkenstamms 14 in einem single-step-Ereignis durch Übernahme des betreffenden penA-Gens von N. lactamica erhöht werden. Allerdings konnten nur MIC-Werte erreicht werden, die mit intermediär-sensiblen Meningokokken vergleichbar waren und somit weit unter den MIC-Werten der benutzten N. lactamica-Stämme lagen. Dieser Befund legt nahe, dass erhöhte MIC-Werte bei N. lactamica wie auch bei Meningokokken mit Mutationen in der Transpeptidaseregion des PBP2 assoziiert sind. Jedoch sind die im Vergleich zu Meningokokken generell höheren MIC-Werte bei N. lactamica auf andere Faktoren zurückzuführen, die bei N. lactamica eine verminderte Empfindlichkeit gegenüber Penicillin bedingen. In den in vitro-Experimenten der vorliegenden Studie konnten diese Faktoren nicht auf Meningokokken übertragen werden. Demnach kann eine Co-Kolonisation mit N. lactamica zwar die MIC-Werte von Meningokokken erhöhen, das Erreichen von bei N. lactamica beobachteten Resistenzniveaus ist allerdings auf diesem Wege nicht möglich. Es ist somit nicht zu befürchten, dass Meningokokken – wie bei Pneumokokken beobachtet – über kommensale Spezies der gleichen Gattung eine massive Reduktion der Empfindlichkeit gegenüber Penicillin entwickeln werden. / Neisseria lactamica colonises the human upper nasopharynx together with the pathogen bacteria Neisseria meningitidis. Penicillin G remains a first line therapy against meningococcal disease. Reduced penicillin susceptibility in N. meningitidis is caused by mutations in penA-gene. Horizontal gene transfer between Neisseria spp. has been described for the penA-gene as well. The aim of this study was to provide a phenotypic and genotypic analysis of penicillin resistance in N. lactamica. Moreover, implications about future developments of penicillin resistance in meningococci should be derived. A phenotypic analysis of 123 N. lactamica-isolates (MIC [Minimum inhibitory concentration]-range: 0.064 – 2.0 µg/ml, median: 0.38 µg/ml) and 129 N. meningitidis-isolates (MIC-range: 0.016 – 0.25 µg/ml, median: 0,064 µg/ml) showed significantly higher MIC values in N. lactamica. Five specific polymorphisms in penA-gene (encoding for PBP2 (penicillin binding protein 2)) are responsible for reduced penicillin susceptibility in meningococci. Therefore the penA-gene of all isolates in this study was analysed and compared with the registered alleles in the penA-database. Sixty different penA-alleles were found in the 123 N. lactamica-isolates of this study. Fifty-one of these alleles could not be found in the penA-database and were added to the database during this study. Unlike N. meningitidis, the analysed N. lactamica-isolates harbored either three or five of the mutations in penA-gene, which are typical for intermediate susceptible meningococci. N. lactamica-isolates with five mutations (MIC-range: 0.25 – 2.0 µg/ml, median: 0.5 µg/ml) showed significantly higher MIC-values than N. lactamica-isolates with three mutations (MIC-range: 0.064 – 0.38 µg/ml, median: 0.125 µg/ml), but also than meningococci with five mutations (MIC-range: 0.064 – 0.25 µg/ml, median: 0.125 µg/ml). A phylogenetic analysis of the 51 new alleles in this study together with all alleles of the penA-database showed, that alleles with five mutations grouped together independently of the species they came from, whereas alleles with three mutations (N. lactamica) as well as alleles without mutations (N. meningitidis) formed separate phylogenetic lines. In vitro-transformations with chromosomal DNA from N. lactamica could raise the MIC-value of the susceptible meningococcus a14 in a single step event by receiving the penA-allele from N. lactamica. However, the MIC-values observed in these transformants were only comparable to those in intermediate susceptible meningococci but considerably below the values of the N. lactamica-isolates used for the transformation. The results of this study showed that high MIC-values in N. lactamica as well as in N. meningitidis are associated with mutations in the transpeptidase region of PBP2. However, the generally higher MIC-values in N. lactamica must be attributed to other factors. These factors could not be transferred to meningococci in this study. As a result of this study it can be stated that co-colonisation with N. lactamica might raise MIC-values in N. meningitidis. However, considerable reductions of penicillin susceptibility by horizontal gene transfer from commensal spezies of the same genus (as observed in pneumococci) cannot be expected for N. meningitidis.

The first part of this research was undertaken to assess the impact of documented penicillin allergy on the choice of antibiotics and the clinical and financial consequences of changes in prescribing patterns in an Australian teaching hospital. The medical records of all patients aged >/= 18 years admitted with community-acquired pneumonia (CAP) to Sir Charles Gairdner Hospital (SGGH) over a 15-week period were reviewed prospectively. The severity of patients' penicillin allergies was assessed using a structured questionnaire. The antibiotic cost was calculated using acquisition, delivery (labour and equipment) and laboratory monitoring costs. The appropriateness of antibiotic prescribing was assessed using the Therapeutic Guidelines: Antibiotic (TG:A). The antimicrobial selections and costs were then compared for those patients with (Group A) and without (Group B) penicillin allergy. 155 patients were reviewed (males 71, females 84) with an average age of 68 ± 18 years. Of these, 27 (17.4%) had documented penicillin allergies; of which 12 were classified as Severity I (e.g. anaphylaxis, urticaria), 12 as Severity II (e.g. rash, itch) and three as intolerance (e.g. GI upset). The current TG:A recommends cephalothin or cephazolin as the drugs of choice for mild to moderate CAP patients with a history of penicillin allergy. However, combinations of cephalothin intravenously and azithromycin orally were the most commonly prescribed antimicrobials for such patients. The TG:A recommends erythromycin plus cefotaxime or ceftriaxone as the first-line therapy for severe CAP patients with a documented penicillin allergy. Yet, combinations of intravenous cephalothin, erythromycin and gentamicin were the most frequently prescribed antimicrobials for such patients. / A history of penicillin allergy significantly (p<0.05) increased the cost of antibiotic treatment and total cost of admission. The adherence of antibiotic prescribing to the TG:A for patients with penicillin allergies is variable. Patients with labelled penicillin allergies had greater antibiotic costs and total cost of admission. Identifying patients with intolerance rather than allergies would reduce the total inpatient costs at SCGH by A$ 463.01 a year for mild to moderate CAP patients and A$ 39 614.54 a year for severe CAP patients. The second part of the project was a prospective study of patients admitted to SCGH who had a history of penicillin allergy, but were not suffering from CAP. This study was conducted in order to ensure that the pattern of penicillin allergies of patients admitted to the hospital could be adequately characterised. Over a 5-week period, all adult patients admitted without CAP to SCGH who claimed to have a history of penicillin allergy were interviewed with regard to their penicillin allergies. The standard of allergy documentation was also assessed for each patient. Of the 140 patients assessed (males 63, females 77, average age 61 ± 17 years), 108 (77.1%) were classified as allergic: 61 (56.5%) as Severity I and 47 (43.5%) as Severity 11, 26 (18.6%) as intolerant and the remaining six (4.3%) as not substantiated. / The standard documentation of the patients' penicillin allergies was poor - only 40 (38.6%) of either medical records or drug charts had the type of reaction and only five (3.6%) had the date of reaction. In general, penicillin allergies were poorly documented in both patients' medical records and on drug charts. Inadequate detail of reported reactions often made it difficult to assess their clinical significance. These findings prompted a recommendation that pharmacists should help to ensure accurate allergy documentation by evaluating patients and educating both patients and health care professionals.

Adverse drug reactions (ADRs) are known to be a major cause of morbidity and mortality. However, only a small proportion are reported. An increase in the number and quality of reports by improving ADR reporting systems in hospitals, could improve patient outcomes and save healthcare costs. The first part of this project was to review the ADR reporting systems in Australian hospitals and to determine factors contributing to the ADR reporting rate. Data were collected by a postal, self-administered questionnaire. Questionnaires were sent to 299 chief pharmacists of Australian hospitals listed in the Society of Hospital Pharmacists of Australia (SHPA) directory. The response rate was 49.5%. Seventy seven (60%) hospitals had a formal hospital policy for ADR reporting and 110 (85.3%) hospitals targeted all drugs to be reported. ADR reporting rates to ADRAC in 2000 (ADR reports per patient admission) were between zero and 1.09% (median=0.02%) with 7.1% of hospitals having a reporting rate of zero. A centralised ADR system and the existence of an ADR policy was not associated with higher reporting rates. The next part of the project was a survey of 803 Western Australian (WA) doctors and 1323 Australian hospital pharmacists to evaluate involvement in, understanding of and reasons for reporting ADRs. A postal, self-administered, anonymous questionnaire was sent to doctors at two tertiary hospitals in Perth and three regional hospitals in WA. A similar questionnaire was sent to all hospital pharmacists listed in the membership list of SHPA, as well as non-SHPA members in WA. Response rates obtained for the WA doctors survey was 35% (n=277) and 43% (n=574) for hospital pharmacists. Sixty four percent of doctors and 96% of hospital pharmacists knew how to report ADRs within the hospital while 57% and 98% (respectively) knew how to report ADRs to ADRAC. / Factors that would encourage respondents to report ADRs included serious reactions, unusual reactions, reaction to a new product and confidence in the diagnosis of the ADR. More than 70% of respondents agreed that an uncertain association between the ADR and the suspected drug, minor reactions and well known reactions were factors that would deter them from reporting ADRs. From a list of 14 hypothetical ADR questions, it was found that respondents were more likely to report serious and uncommon reactions. Finally, the incidence of cross-sensitivity between penicillin and other β-lactam antibiotics among patients experiencing penicillin allergy in Fremantle Hospital and Health Services (FHHS) was assessed, along with the appropriate documentation of penicillin allergy in the medical records. The study was a retrospective audit and review of medical records in FHHS (1994-2000). All medical records of patients experiencing penicillin allergy during admission, or causing admission to FHHS, (n=85) were reviewed and data on reactions to other β-lactams were recorded. The incidence of definite cross-sensitivity between penicillins and cephalosporins was 6%, consistent with the reported rate of cross-sensitivity. The documentation of penicillin allergy in the medical records was less than optimal, with alerts on 89% of medication charts and only 28% of medical records (front cover). Improvement in the documentation of ADRs in patients' medical records would likely decrease the risk of preventable adverse events.

This study describes the pharmacokinetic profile of procaine penicillin G after intraperitoneal (IP) administration in 8 lactating dairy cows. Procaine pencillin G (PPG, 21,000 IU/kg) was deposited into the abdominal cavity of each cow following an incision in the right paralumbar fossa. Blood and milk samples were taken over the following 10 days, at which point the cows were euthanized. Plasma, milk, muscle, liver, and kidney penicillin concentrations were determined by HPLC, with a limit of detection (LOD) of 5 ppb for plasma and milk samples. Noncompartmental methods were used to analyze plasma kinetics. The mean pharmacokinetic parameters (} s.d.) were: Cmax, 5.5 } 2.6 Êg/mL; Tmax, 0.75 } 0.27 h; AUC0-, 10.8 } 4.9 Êg*h/mL; MRT, 2.2 } 0.9 h. All milk from treated cows contained penicillin residues for a minimum of 3 milkings (31 h) and maximum of 5 milkings (52 h) after administration. Concentrations of penicillin G in all muscle, liver, and kidney samples taken 10 days post-administration were below the limit of detection. Necropsy examinations revealed foci of hemorrhage on the rumenal omentum of most cows but peritonitis was not observed. Systemic inflammation as determined by altered leukograms and fibrinogen was noted in one cow. The results of this study demonstrate that IP procaine penicillin G is absorbed and eliminated rapidly in lactating dairy cows.

This study describes the pharmacokinetic profile of procaine penicillin G after intraperitoneal (IP) administration in 8 lactating dairy cows. Procaine pencillin G (PPG, 21,000 IU/kg) was deposited into the abdominal cavity of each cow following an incision in the right paralumbar fossa. Blood and milk samples were taken over the following 10 days, at which point the cows were euthanized. Plasma, milk, muscle, liver, and kidney penicillin concentrations were determined by HPLC, with a limit of detection (LOD) of 5 ppb for plasma and milk samples. Noncompartmental methods were used to analyze plasma kinetics. The mean pharmacokinetic parameters (} s.d.) were: Cmax, 5.5 } 2.6 Êg/mL; Tmax, 0.75 } 0.27 h; AUC0-, 10.8 } 4.9 Êg*h/mL; MRT, 2.2 } 0.9 h. All milk from treated cows contained penicillin residues for a minimum of 3 milkings (31 h) and maximum of 5 milkings (52 h) after administration. Concentrations of penicillin G in all muscle, liver, and kidney samples taken 10 days post-administration were below the limit of detection. Necropsy examinations revealed foci of hemorrhage on the rumenal omentum of most cows but peritonitis was not observed. Systemic inflammation as determined by altered leukograms and fibrinogen was noted in one cow. The results of this study demonstrate that IP procaine penicillin G is absorbed and eliminated rapidly in lactating dairy cows.

The order of events in the Deacetoxycephalosporin C/Deacetylcephalosporrn C Synthetase (DAOC/DAC Synthetase) catalysed ring expansion of penicillin N to deacetoxycephalosporin C has been investigated by the use of labelled/unlabelled penicillin N mixed competitive kinetic isotope effect experiments, in which the labelled penicillin N substrates were either labelled in the pro <strong>R</strong>- and pro <strong>S</strong>-methyl groups or at C-3. In addition, to assisting in the determination of the position of the first irreversible event in this reaction, deuteration at C-3 gave rise to a bifurcation of the natural biosynthetic pathway which led to enhanced production of the shunt metabolite, (2<strong>R</strong>,3<strong>S</strong>,6<strong>R</strong>,7<strong>R</strong>)-l-aza-3- methyl-3-hydroxy-7-[(5<strong>R</strong>)-5-amino-5-carboxy-pentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylate. The biosynthetic precursor to the 3<strong>S</strong>-hydroxycepham shunt metabolite has been investigated and the origin of the 3<strong>S</strong>-hydroxyl oxygen atom has been determined by the use of labelling studies with ¹⁸O₂ and shown to be derived from molecular oxygen. ¹³C-labelling studies are described which indicate that the ring expansion process is stereospecific to within the limits of the detection system employed. These experiments confirm earlier investigations but, in addition to improving upon the assessment of the degree of stereospecificity, have shown that the 3<strong>S</strong>- hydroxycepham shunt metabolite is produced with the same stereospecificity as that observed for the usual biosynthetic products, DAOC and DAC. Chapter 5 describes an investigation of the anomalous C-2 deuterium exchange detected in DAOC produced by incubation of di-(²H₃-methyl)-penicillin N with DAOC/DAC synthetase. The preliminary results from this study indicate that initially exchange occurs stereospecifically with the pro <strong>R</strong> C-2 deuterium atom being replaced by a hydrogen atom. The origins of the unusual tripeptides <strong>L</strong>-α-aminoadipyl-<strong>L</strong>-serinyl-<strong>D</strong>-valine (<strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ASV), α-aminoadipyl-serinyl-isodehydrovaline (ASdV) and α-aminoadipyl-cysteinyl- β-hydroxyvaline (AC-[β-OH]-V) isolated from Penicillium chrysogenum and Cephalosporium acremonium, have been examined by the use of variously ¹³C-labelled <strong>L</strong>,<strong>L</strong>,<strong>D</strong>-α-aminoadipyl-cysteinyl-valine (<strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ACV) and <strong>D</strong>,<strong>L</strong>,<strong>D</strong>-α-aminoadipyl- cysteinyl-valine (<strong>D</strong>,<strong>L</strong>,<strong>D</strong>-ACV) tripeptide isotopomers. The initial results obtained from this investigation may be considered as circumstantial evidence that ASdV is formed by the action of IPNS upon <strong>L</strong>,<strong>L</strong>,<strong>D</strong>-ACV. Finally, various substrate analogues have been prepared and evaluated as substrates for the ring expansion and hydroxylation activities of the bifunctional DAOC/DAC synthetase enzyme.