Class of 2012 Abstract / Specific Aims: To assess the chemical stability of ampicillin for injection in normal saline at pH values ranging from 5 to 6. Methods: A stability-indicating high performance liquid chromatography (HPLC) method was developed and used to determine the stability of ampicillin for injection in normal saline following buffering with sodium acetate and acid adjustment with HCl at pH values of 5, 5.5, and 6. To confirm that the assay was stability-indicating, ampicillin trihydrate reference standard (1 mg/mL) was exposed to alkali, acid, and oxidative stress conditions and analyzed by HPLC for the presence of degradation products. Analysis was performed on a reverse-phase (C-18) column with a mobile phase consisting of water, acetonitrile, 1 M monobasic potassium phosphate, and 1 N acetic acid (909:80:10:1). Other HPLC parameters were: flow rate 1 mL/min; detection wavelength 254 nm; injection volume 20 μL; column temperature 30 ̊C. The method was evaluated for linearity, precision, and accuracy. The chemical stability of ampicillin for injection (18 mg/mL) in normal saline and sodium acetate (pH adjusted at values of 5, 5.5, and 6) was assessed at baseline (t=0), 7, 11, 17, 31, and 44 hours and compared to a control solution (no pH adjustment). Measurements at each time interval were performed in triplicate. Main Results: Ampicillin trihydrate reference standard (1 mg/mL) was adequately separated from degradation products following exposure to alkali, acid, and oxidative stress conditions. After 16 hours, a precipitate was observed in the solution at pH 6, and therefore stability is not reported. All other solutions (pH 5, pH 5.5, and control) were stable for at least 24 hours at room temperature and yielded t90 values of 110, 64.2, and 27.5 hours, respectively. Conclusions: Adjustment of intravenous ampicillin solutions to pH values of 5 or 5.5 significantly increased stability. Ampicillin appears to be most stable at a pH near its isoelectric point (pH 5).

by Ling Kin Wah, Thomas. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 103-117). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xv / LIST OF FIGURES --- p.xix / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.2 / Chapter 1. --- Structure of the bacterial cell envelope --- p.2 / Chapter 2 . --- The β-lactam antibiotics --- p.4 / Chapter 3. --- Mode of action of β-lactam antibiotics --- p.5 / Chapter 4. --- Penicillin-binding proteins (PBPs) --- p.6 / Chapter 5. --- Mechanisms of bacterial resistance to β-lactam antibiotics --- p.7 / Chapter 5.1 --- Non-enzymatic resistance --- p.7 / Chapter 5.1.1 --- Alteration in cell permeability --- p.8 / Chapter 5.1.2 --- Alteration of the target site --- p.9 / Chapter 5.1.3 --- Tolerance and persistence --- p.9 / Chapter 5.2 --- Enzyme-mediated resistance --- p.12 / Chapter 6. --- Transfer of resistance --- p.13 / Chapter 7. --- β-lactamases --- p.16 / Chapter 7.1 --- History --- p.16 / Chapter 7.2 --- Classification of β-lactamases --- p.17 / Chapter 7.2.1 --- Richmond and Sykes scheme --- p.17 / Chapter 7.2.2 --- Matthew scheme --- p.18 / Chapter 7.2.3 --- Bush scheme --- p.19 / Chapter 7.3 --- β-lactamases of Gram-negative bacteria --- p.19 / Chapter 7.3.1 --- Chromosomally-mediated β-lactamases --- p.19 / Chapter 7.3.2 --- Plasmid-mediated β-lactamases --- p.20 / Chapter 7.4 --- β-lactamase inhibitors --- p.25 / Chapter 7.5 --- Regulation of β-lactamase production --- p.28 / Chapter 7.5.1 --- β-lactamase induction --- p.28 / Chapter 7.5.2 --- Mutation to constitutive enzyme production --- p.29 / Chapter 7.5.3 --- β-lactam induced β-lactamase production --- p.30 / Chapter 8. --- Emergence of resistance due to production of β-lactamases --- p.31 / Chapter 8.1 --- Resistance in staphylococci --- p.32 / Chapter 8.2 --- Resistance in haemophili and gonococci --- p.33 / Chapter 8.3 --- Resistance in Enterobacteriaceae (non E. coli) --- p.34 / Chapter 8.4 --- Distribution of β-lactamases in E. coli --- p.35 / MATERIALS AND METHODS / Chapter 1. --- Bacterial strains --- p.38 / Chapter 1.1 --- Standard organisms --- p.38 / Chapter 1.2 --- Clinical isolates --- p.38 / Chapter 2. --- Antibiotics --- p.39 / Chapter 3. --- "Media, chemicals and culture conditions" --- p.39 / Chapter 4. --- Bacterial identification and viable bacterial count --- p.39 / Chapter 5. --- Antibiotic sensitivity testing --- p.40 / Chapter 5.1 --- Disk diffusion --- p.40 / Chapter 5.2 --- Determination of minimal inhibitory concentration (MIC) --- p.40 / Chapter 6. --- Plasmid analysis --- p.41 / Chapter 6.1 --- Transfer of drug resistance plasmids --- p.41 / Chapter 6. 2 --- Molecular studies of plasmids --- p.42 / Chapter 6.2.1 --- Extraction of plasmid DNA --- p.43 / Chapter 6.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 6.2.3 --- Molecular size determination --- p.44 / Chapter 7 . --- DNA hybridization --- p.44 / Chapter 7.1 --- DNA blotting --- p.44 / Chapter 7.1.1 --- Colony blotting --- p.45 / Chapter 7.1.2 --- Southern blotting --- p.45 / Chapter 7.2 --- Labeling of oligonucleotide probe --- p.46 / Chapter 7.3 --- Hybridization --- p.47 / Chapter 7.4 --- Autoradiography --- p.47 / Chapter 7.5 --- Re-use of blots --- p.48 / Chapter 8. --- Detection and screening for classification of β-lactamases --- p.48 / Chapter 8.1 --- Detection of β-lactamases --- p.48 / Chapter 8.1.1 --- Acidimetric --- p.48 / Chapter 8.1.2 --- Chromogenic substrate --- p.49 / Chapter 8.1.2.1 --- Whole cell --- p.49 / Chapter 8.1.2.2 --- Cell extract and filtrate --- p.49 / Chapter 8.2 --- Screening for classification of β-lactamases --- p.49 / Chapter 9. --- "Preparation, purification, qualitative and quantitative analyses of the β-lactamase from transconjugants TU117, TB117 and the recipient K12" --- p.51 / Chapter 9.1 --- Large scale preparation of enzyme --- p.51 / Chapter 9.2 --- Gel filtration --- p.52 / Chapter 9.3 --- Preparative isoelectric focusing (PIEF) --- p.53 / Chapter 9.4 --- Protein determination --- p.55 / Chapter 9.5 --- Qualitative analyses and characterization of β-lactamases --- p.56 / Chapter 9.5.1 --- Analytical isoelectric focusing --- p.56 / Chapter 9.5.1.1 --- Semi-quantitative determination of β-lactamases --- p.56 / Chapter 9.5.1.2 --- Polyacrylamide gel preparation --- p.57 / Chapter 9.5.1.3 --- Isoelectric focusing --- p.58 / Chapter 9.5.1.4 --- pH measurement --- p.58 / Chapter 9.5.1.5 --- Gel development and recording --- p.59 / Chapter 9.5.1.5.1 --- Nitrocefin staining --- p.59 / Chapter 9.5.1.5.2 --- Silver staining --- p.59 / Chapter 9.5.1.6 --- Isoelectric point (pI) determination --- p.60 / Chapter 9.5.2 --- Spectrophotometric assay of β-lactam substrates --- p.60 / Chapter 9.5.2.1 --- Absorption spectra of β-lactam antibiotics --- p.60 / Chapter 9.5.2.2 --- The molar extinction coefficient of β-lactam substrates --- p.60 / Chapter 9.5.2.3 --- Measurement of β-lactamase hydrolytic activities --- p.61 / Chapter 9.5.2.4 --- Determination of enzyme kinetics --- p.61 / Chapter 9.5.3 --- Molecular weight determination of proteins --- p.62 / Chapter 9.5.3.1 --- SDS-polyacrylamide gel preparation --- p.62 / Chapter 9.5.3.1.1 --- Resolving gel --- p.62 / Chapter 9.5.3.1.2 --- Stacking gel --- p.63 / Chapter 9.5.3.2 --- Electrophoresis --- p.63 / Chapter 9.5.3.3 --- Staining and recording --- p.64 / Chapter 9.5.3.4 --- Molecular weight determination --- p.64 / RESULTS / Chapter 1. --- Collection of organisms --- p.65 / Chapter 2 . --- Identification of organisms --- p.65 / Chapter 3. --- Antibiotic sensitivity testing --- p.66 / Chapter 4. --- Genetic and molecular studies of ampicillin- resistant plasmids --- p.68 / Chapter 4.1 --- Transfer of ampicillin-resistant factor --- p.68 / Chapter 4.1.1 --- E. coli K12 14R525 as recipient --- p.68 / Chapter 4.1.2 --- other Enterobacteriaceae --- p.68 / Chapter 4.2 --- Plasmid studies of E. coli --- p.69 / Chapter 5. --- Detection and identification of β-lactamases --- p.69 / Chapter 5.1 --- Analytical IEF --- p.70 / Chapter 5.2 --- DNA hybridization --- p.70 / Chapter 5.2.1 --- Colony blot hybridization --- p.70 / Chapter 5.2.2 --- Southern blot hybridization --- p.71 / Chapter 6. --- Characterization of TEM-1 producing E. coli --- p.71 / Chapter 6.1 --- Susceptibility testing --- p.71 / Chapter 6.2 --- Enzyme kinetic study --- p.72 / Chapter 6.2.1 --- Absorption spectra and molar extinction coefficient of β-lactam antibiotics --- p.72 / Chapter 6.2.2 --- Comparison of the substrate profiles --- p.73 / Chapter 6.3 --- Correlation of MICs to β-lactamase activities --- p.73 / Chapter 7. --- "Isolation, quantitation and characterization of β-lactamases isolated from three E. coli strains" --- p.74 / Chapter 7.1 --- Preparation of β-lactamases --- p.75 / Chapter 7. 2 --- Purification of β-lactamases --- p.76 / Chapter 7.2.1 --- Gel-filtration chromatography --- p.76 / Chapter 7.2.2 --- Preparative isoelectric focusing --- p.77 / Chapter 7.3 --- Characterization of the purified β-lactamases --- p.78 / Chapter 7.3.1 --- Isoelectric point --- p.78 / Chapter 7.3.2 --- Molecular weight assessment --- p.79 / Chapter 7.3.3 --- Enzyme kinetic study --- p.79 / DISCUSSION / Chapter 1. --- Epidemiology of ampici11in (or amoxycillin)- resistant E. coli --- p.81 / Chapter 2. --- Distribution of β-lactamases in ampicillin- resistant E. coli --- p.84 / Chapter 3. --- Correlation between level of resistance and β-lactamase activity --- p.86 / Chapter 4. --- Plasmid-mediated TEM-1 β-lactamase --- p.89 / Chapter 4.1 --- Transfer of resistance --- p.89 / Chapter 4 .2 --- Identification of β-lactamases by DNA hybridization --- p.91 / Chapter 5. --- Mechanism of high-level resistance --- p.93 / Chapter 5.1 --- Selection of resistant strains --- p.93 / Chapter 5.2 --- β-lactamases preparation and purification --- p.95 / Chapter 5.3 --- Hyperproduction of β-lactamase --- p.97 / SUMMARY AND CONCLUSIONS --- p.102 / REFERENCES --- p.103 / APPENDICES / Chapter 1. --- TABLES --- p.118 / Chapter 2. --- FIGURES --- p.153

Ampicillin-sulbactam was compared with cefamandole in the treatment of bacterial pneumonia in elderly patients. Clinical improvement ard hospital discharge occurred in 25 of 26 patients in the ampicillin-sulbactam group vs. 20 of 23 in the cefamardole group (P >.06). Etiologic agents isolated from adequate sputum samples were more likely to be eradicated with ampicillin-sulbactam (24/26) than with cefamandole (15/23) (P <.05). The most common organisms isolated from elderly patients with pneumonia included Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. M. catarrhalis was eradicated from sputum in 2 of 5 patients treated with cefamandole, and these isolates had relatively high minimal inhibitory concentrations (8 to 16 μg/mL).

Deoxyribonucleic acid (DNA) aptamers are oligonucleotides with high specificity and affinity for non-nucleotide targets ranging from molecular species to cellular proteins. Their high affinity, rapid synthesis, and the ease with which they can be chemically modified to include convenient chemical groups (e.g. amine group on 5’ end) make them excellent adaptable ligands for use in colloidal drug delivery vehicles for both uptake and release of therapeutic agents. This work uses pre-identified aptamers for vascular endothelial growth factor (VEGF) to investigate the design of one such vehicle for controlled uptake and release of target therapeutics and analyzes the ability of particle-immobilized aptamers to bind both nucleotide and non-nucleotide targets. Aptamer sequences are immobilized on colloidal microspheres and binding activity of both the primary DNA and protein targets are directly monitored using flow cytometry. Additionally, the dual nature of aptamer-target binding is further investigated by evaluating the effects of simultaneous and serial incubation of the primary targets. Finally, the ability to recover the functionality of the aptamer is evaluated after displacement of the primary DNA target through DNA mediated interactions. It has been shown that the nature of aptamer-target interactions are complex in nature, requiring optimization for each species incorporated into a delivery vehicle; however, partial recovery of aptamer functionality was achieved after hybridization with the primary DNA target.

A ampicilina é um antibiótico semi-sintético, derivado da penicilina, que exibe amplo espectro de ação, sendo largamente comercializada no Brasil, onde são encontrados cerca de doze produtos sob a forma de comprimidos. O presente trabalho teve como propósito avaliar, do ponto de vista biofarmacêutico, quatro marcas comerciais (A, B, C e D) de comprimidos de ampicilina 500 mg, sendo que, para um dos laboratórios foram analisados dois lotes diferentes (AI e A2). Foram executadas análises físicas e físico-químicas in vitro (aspecto, variação de peso, diâmetro, espessura e dureza, friabilidade, tempo de desagregação, teste e perfil de dissolução e teor de ampicilina), com a finalidade de detectar-se possíveis falhas em relação a formulação e/ou tecnologia utilizada na fabricação dos produtos. Executou-se, também, um estudo de bioequivalência entre as formulações A2 e B, a fim de se verificar se ambas podem ser consideradas intercambiáveis. Para tanto, realizou-se um estudo cruzado aleatório em dois períodos, utilizando-se dezesseis voluntários sadios, sendo o fármaco quantificado na urina pelo método espectrofotométrico descrito inicialmente por SMITH et al. (1967). Os parâmetros farmacocinéticos avaliados foram a quantidade total de ampicilina excretada na urina (Du&#8734;) e a velocidade máxima de excreção [(dDu/dt)max]. Os resultados das análises físicas e físico-químicas indicaram que o produto A1 apresentou diversas falhas, sendo estas constatadas, também, para C e D. Ao contrário, A2 e B não registraram qualquer discrepância. Na avaliação da bioequivalência entre as formulações A2 e B, obteve-se um intervalo de confiança de 93 a 110% para Du&#8734; e 91 a 132% para (dDu/dt)max. De acordo com as normas internacionalmente aceitas é possível concluir que as formulações podem ser consideradas bioequivalentes e, portanto, intercambiáveis. / Ampicillin is a semi-synthetic penicillin-derived antibiotic that has a broad action spectrum. In Brazil, where it is largely marketed, about twelve different tablets brands can be found. The aim of the present work was to evaluate from the biopharmaceutic point of view four ampicillin 500mg-tablet brands (A, B, C and D). Two different A-brand lots (AI and A2) were analyzed. Physical and in vitro physical-chemical analysis (aspect, weight variation, diameter, thickness and hardness, friability, disintegration time, dissolution test and profile and ampicillin content) were carried out in order to detect possible manufacturing failures as to formulation and/or technology used. A bioequivalence study between A2 and B was also carried out in order to check if both of them could be interchangeable. For such an aleatory two-period cross-over study was done by quantifying through spectrophotometry (SMITH et al., 1967) the content of ampicillin in sixteen healthy volunteers\'urine. The total ampicillin amount excreted in urine (Du&#8734;) and maximum rate of excretion [(dDu/dt)max] were taken as pharmacokinetics parameters. The physical and physical-chemical analyses results indicated critical failures in A1 brand. Similarly several failures were also detected in C and D. Brands A2 and B instead showed no discrepancies. Confidence intervals of 93 to 110% for Du&#8734; and 91 to 132% for (dDu/dt)max, were found through bioequivalence evaluation between A2 and B formulations. According to international accepted standards both the fomulations can be considered bioequivalent, therefore interchangeable.

HDGF (hepatoma-derived growth factor) is a novel growth factor,identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. HDGF, a novel defined growth factor with mitogenic effect, has homology protein sequence as HMG (high mobility group) protein and their three dimension structures appeared to be similar to each other. Recently, elevated HDGF expression was found in developing kidneys but less was found in adult kidney. In addition, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. HDGF also plays a role in cell-cell interaction and cell migration. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs)proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. HDGF is sciential factor in stimulating DNA replication and cell proliferation of vascular smooth muscle cell.In this study,we used E. coli strain BL21 (DE3) to express the recombinant protein hepatoma derived growth factor(HDGF). To find out the optimal production conditions,we studied on the different temperature and fermentor to calculate all cost .

A ampicilina é um antibiótico semi-sintético, derivado da penicilina, que exibe amplo espectro de ação, sendo largamente comercializada no Brasil, onde são encontrados cerca de doze produtos sob a forma de comprimidos. O presente trabalho teve como propósito avaliar, do ponto de vista biofarmacêutico, quatro marcas comerciais (A, B, C e D) de comprimidos de ampicilina 500 mg, sendo que, para um dos laboratórios foram analisados dois lotes diferentes (AI e A2). Foram executadas análises físicas e físico-químicas in vitro (aspecto, variação de peso, diâmetro, espessura e dureza, friabilidade, tempo de desagregação, teste e perfil de dissolução e teor de ampicilina), com a finalidade de detectar-se possíveis falhas em relação a formulação e/ou tecnologia utilizada na fabricação dos produtos. Executou-se, também, um estudo de bioequivalência entre as formulações A2 e B, a fim de se verificar se ambas podem ser consideradas intercambiáveis. Para tanto, realizou-se um estudo cruzado aleatório em dois períodos, utilizando-se dezesseis voluntários sadios, sendo o fármaco quantificado na urina pelo método espectrofotométrico descrito inicialmente por SMITH et al. (1967). Os parâmetros farmacocinéticos avaliados foram a quantidade total de ampicilina excretada na urina (Du&#8734;) e a velocidade máxima de excreção [(dDu/dt)max]. Os resultados das análises físicas e físico-químicas indicaram que o produto A1 apresentou diversas falhas, sendo estas constatadas, também, para C e D. Ao contrário, A2 e B não registraram qualquer discrepância. Na avaliação da bioequivalência entre as formulações A2 e B, obteve-se um intervalo de confiança de 93 a 110% para Du&#8734; e 91 a 132% para (dDu/dt)max. De acordo com as normas internacionalmente aceitas é possível concluir que as formulações podem ser consideradas bioequivalentes e, portanto, intercambiáveis. / Ampicillin is a semi-synthetic penicillin-derived antibiotic that has a broad action spectrum. In Brazil, where it is largely marketed, about twelve different tablets brands can be found. The aim of the present work was to evaluate from the biopharmaceutic point of view four ampicillin 500mg-tablet brands (A, B, C and D). Two different A-brand lots (AI and A2) were analyzed. Physical and in vitro physical-chemical analysis (aspect, weight variation, diameter, thickness and hardness, friability, disintegration time, dissolution test and profile and ampicillin content) were carried out in order to detect possible manufacturing failures as to formulation and/or technology used. A bioequivalence study between A2 and B was also carried out in order to check if both of them could be interchangeable. For such an aleatory two-period cross-over study was done by quantifying through spectrophotometry (SMITH et al., 1967) the content of ampicillin in sixteen healthy volunteers\'urine. The total ampicillin amount excreted in urine (Du&#8734;) and maximum rate of excretion [(dDu/dt)max] were taken as pharmacokinetics parameters. The physical and physical-chemical analyses results indicated critical failures in A1 brand. Similarly several failures were also detected in C and D. Brands A2 and B instead showed no discrepancies. Confidence intervals of 93 to 110% for Du&#8734; and 91 to 132% for (dDu/dt)max, were found through bioequivalence evaluation between A2 and B formulations. According to international accepted standards both the fomulations can be considered bioequivalent, therefore interchangeable.

Purpose: Use of ampicillin in outpatient parenteral antimicrobial therapy (OPAT) has historically been complicated by frequent dosing and short beyond use dates. However historic stability data relied on inaccurate testing methods. The purpose of this study is to evaluate the stability of ampicillin using high-pressure liquid chromatography (HPLC), the gold standard, in a real-world OPAT dosing model using continuous infusion at room temperature over 24 hours immediately following preparation compared to batches stored under refrigeration for 24 hours, 72 hours, and 7 days. Methods: An HPLC method was developed and validated as stability – indicating according to guidance in USP general Chapter . Method development included linearity, precision, accuracy, repeatability and forced degradation. Four batches were prepared using 4 different lots from 2 different manufacturers for each storage condition (immediate, 24 hours, 72 hours, and 7 days). Three 2-gram vials were each reconstituted with 10 mL of sterile water for injection (SWFI) and added to 250 mL of normal saline by a licensed pharmacist and stored in a laboratory refrigerator (2 – 8oC). A pump system was used to continuously circulate the solutions through medical grade tubing at room temperature. One milliliter aliquots were removed from each batch at time 0, 4 hours, 8 hours, 12 hours and 24 hours and analyzed for ampicillin concentration using the aforementioned HPLC method. The samples were filtered prior to analysis using a 0.22-micron syringe filter and analyzed in triplicates along with freshly prepared calibration samples (24 – 12 mg/mL). Peak area was used to determine percent recovery for each sample. Results: Each batch was assayed for initial concentration (20.34 – 21.50 mg/mL) upon preparation, and percent recovery was compared to that initial concentration thereafter. Acceptable recovery was defined as 90 – 110% of initial concentration. On the day of product preparation (immediate use), the average percent recovery over 24 hours was 96.4%. The other average percent recoveries were as follows: 95.8% (24-hour storage), 94.6% (72-hour storage) and 90.3% (7-day storage). These data represent the average percent recovery for all time points during the 24 hours sampling (n = 60 for each experiment). When evaluating individual time points, the percent recovery remained above 90% for all batches and time points except for the 7-day storage experiment. Under 7-day storage conditions, the percent recovery fell below 90% after 4 hours of circulation through the medical grade tubing. Furthermore, 95% confidence interval for percent recovery for ampicillin in the samples stayed within 90 – 110% of the initial concentration for the duration of the experiment for all test groups except 7-day storage. Conclusions and Relevance: Ampicillin can be prepared and stored in a refrigerator for up to 72-hours prior to continuously infusing at room temperature over 24 hours with less than a 10% loss of potency over the dosing period. This model supports twice weekly OPAT delivery of ampicillin.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science (medicine).' Johannesburg 1996 / Fifty-two strains of Haemophilus ducreyi from various geographic regions of southern Africa (Botswana, Lesotho, Namibia, Gauteng, Natal and Transkei) isolated between 1988 to 1994 were tested for susceptibilities to five antimicrobial agents and characterized according to their plasmid content and ampicillin- resistance genes. / IT2019

Philosophiae Doctor - PhD / Background: Extended-spectrum beta-lactamase-producing (ESBL) and carbapenemaseproducing Gram-negative bacilli showing resistance to cephalosporins and carbapenems respectively, have been reported from several countries globally and recently among Libyan combatants who have been transferred to European countries for advanced medical care. However, there is a lack of data about their presence in Misrata and in Libya in general. This is the first documented study aimed at investigating the prevalence and resistance mechanisms of ESBL and carbapenemase-producing K. pneumoniae isolates from Misrata. Materials and Methods: Two hundred Gram-negative bacillus isolates were collected and identified from hospitals and pathology laboratories in Misrata. Following antimicrobial susceptibility screening, those showing resistance to cephalosporin and carbapenem were tested for ESBL activity using the Modified double disc synergy test, Sensititer ESBL confirmatory MIC plates and MAST AmpC detection sets D52C and D68C. Carbapenemase activity was detected using RAPIDEC CARBA NP test, Modified Hodge test (MHT), carbapenem inactivation methods (CIM), carbapenem combined test (CCT), and by MAST carba puls set. ESBL and carbapenemases genes were detected using multiplex PCR. Results: K. pneumoniae was the predominant species (85/200) of the 14 species identified, with 56 (65.8%) showing carbapenem resistance, 16 (18.8%) were cephalosporin-resistant carbapenem-susceptible and 13 (15.2%) were susceptible to all antibiotics except ampicillin. OXA-48 was the only carbapenemase detected, with SHV, TEM and CTX-M group 1 found in almost all carbapenem and cephalosporin resistant K. pneumoniae. Rep-PCR analysis revealed multiple clones and some K. pneumoniae strains were genetically related or indistinguishable despite differences in ESBL genes or carbapenemase activity. Conclusion: The findings of this study show that carbapenemase- and ESBL-producing K. pneumoniae are prevalent in Misrata and emphasize the urgent need for optimized infection control and antibiotic stewardship programmes in the Libyan hospitals to prevent further spread of these organisms.