Inactivation Of Penicillin Research Articles

The occurrence of blaTEM, blaSHV and blaOXA genotypes in Extended-Spectrum β-Lactamase (ESBL)-producing Pseudomonas aeruginosa strains in Southwest of Iran

Pseudomonas aeruginosa is considered as a potential nosocomial pathogen, which shows a great tendency for infecting immune-compromised individuals, particularly burn patients. Producing extended spectrum beta-lactamases (ESBL) enzymes by these bacteria resulting inactivation of penicillins and cephalosporins which renders drug resistance. The aims of this study were to assess the antimicrobial susceptibility pattern and the prevalence of resistance determinants in ESBL producing P. aeruginosa in hospitalized patients. A total of 165 isolates of P. aeruginosa which included in this study isolated from teaching Shiraz hospitals, southwest of Iran. Antimicrobial susceptibility of isolates to antibiotics was determined using disk agar diffusion test. For detecting ESBL producing bacteria, all P. aeruginosa isolates that were resistant to Cefotaxime and/or Ceftazidime were examined for resistant to combined disks of Ceftazidime + Clavulanic acid and/or Cefotaxim + Clavulanic acid. The presence of antibiotic resistance determinants were investigated by PCR method. The results revealed that 99 (60%) of the isolates were multiple-drug resistant (MDR) and 21 (12.7%) of the isolates were ESBL positive. Among 21 ESBL-producing strains, 19 (90.5%), 11 (52.4%) and 7 (33.3%) strains were carrying blaOXA-10, blaSHV and blaTEM gene, respectively. The association between beta-lactamase genes with origin of isolates and drug resistance in this study indicates that further work with larger sample size is necessary to find out the actual involvement of P. aeruginosa strains carried these genes in the course of infections in burn patients.

Microbiological Features of Upper Respiratory Tract Infections in Bulgarian Children for the Period 1998-2014.

Across the globe, upper respiratory tract infections (URTIs) are the most prevalent cause of morbidity in childhood. The aim of our study is to analyze the incidence and etiology of bacterial URTIs in Bulgarian children, as well as the increasing antimicrobial resistance to the most common etiologic agents over a period of 17 years. Retrospective study. The study material comprised the data from 4768 patients (aged 1-16 years) with URTI during the period from 1998-2014. Specific microbiology agent detection was performed by culture examination. Susceptibilities to the investigated pathogens were determined by the disk diffusion method and minimal inhibitory concentration according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). Polymerase chain reaction was used to detect the presence of β-lactam resistance genes. We identified the following as the most common URTI bacterial pathogens: Streptococcus pneumoniae (40.94%), Streptococcus pyogenes (34.16%), Haemophilus influenzae (44.23%), Moraxella catarrhalis (39.19%) and Staphylococcus aureus (23.88%). In more than 70% of cases, a polymicrobial etiology was found. The most commonly affected individuals were pre-school-aged children, which accounted for more than 36% of all patients. During the study period, a dramatic increase in resistance to antibiotic agents was observed. The most frequent types of resistance were the enzymatic inactivation of penicillins and cephalosporins (close to 100% in staphylococci and moraxellae) and inducible macrolide-lincozamide resistance (about 20% of Gram-positive cocci). Due to mandatory immunization against pneumococci and H. influenzae in Bulgaria and the vast expanding resistance to the most popular antimicrobial agents changes in the etiology of URTI have recently been noted. Regular analysis of this etiological dynamic and the antimicrobial resistance of respiratory pathogens is important for choosing the correct therapy and successful treatment.

A simple method for the production of high titre penicillinase.

Abstract The culture filtrates of certain penicillinase-constitutive mutant strains of B. cereus, grown overnight in casein hydrolysate and citrate, can serve, without further treatment, as a source of stable, high-titre penicillinase suitable for inactivation of penicillin in sterility tests, blood cultures, etc.

Inactivation of Penicillin G in Milk Using Hydrogen Peroxide

Milk antibiotic residues have been a public concern in recent years. The Grade A Pasteurized Milk Ordinance mandates that raw Grade A milk will test negative for β-lactam antibiotic residues before processing. The purpose of this research was to investigate the ability of various levels of peroxide and heat to inactivate penicillin G in raw milk. Whole milk spiked to a mean of 436±15.1 (standard error of the mean) ppb of potassium penicillin G was treated with hydrogen peroxide at levels of 0.0, 0.09, 0.17, and 0.34%. Samples at each peroxide level (n=6 per treatment) were treated as follows: 1) incubated at 54.4°C for 3h, 2) pasteurized at 62.8°C for 30min, 3) incubated and pasteurized as in treatments 1 and 2, or 4) received no further treatment. A β-lactam competitive microbial receptor assay was used for quantification of penicillin G. Concentrations of penicillin in selected samples were determined by HPLC for a comparison of test methods. Treatments were evaluated relative to their ability to reduce milk penicillin G levels to below the safe level of 5ppb.The 0.09% hydrogen peroxide level was ineffective for all treatments. Hydrogen peroxide at 0.17% lowered the mean penicillin G (± SEM) from 436±15.1 to 6±1.49ppb using the incubated and pasteurized heat treatment. The 0.34% concentration of hydrogen peroxide was the most effective, inactivating penicillin G to a level well below the safe level of 5ppb with the pasteurized heat treatment, with or without incubation.

Tonsillar Microbial Flora: Comparison of Recurrent Tonsillitis and Normal Tonsils

Tonsillar microbial flora was studied in cultures of tonsillar core specimens from 34 patients tonsillectomized due to recurrent group A streptococcal pharyngotonsillitis (n=17) or sleep apnoea (n=17). Patients in the sleep apnoea subgroup, who had no history of recurrent tonsillitis and manifested no tonsillar hypertrophy at ENT examination, served as controls. Tonsillar core specimens were cultured for semi-quantitative estimation of growth of aerobic, anaerobic and facultative organisms. The recurrent tonsillitis and apnoea subgroups did not differ significantly in the mean number of isolates per patient, either of aerobic spp. (3.8 vs. 4.3) or anaerobic spp. (5.2 vs. 4.7). Nor did the two subgroups differ significantly in the proportion of patients whose specimens manifested beta-lactamase producers (71% vs. 59%), in the isolation frequency of viridans (alpha) streptococci, or in the occurrence of semi-quantitative growth estimates of 3-4+ for aerobic, anaerobic or beta-lactamase-producing spp. Thus, the study provided no support for the hypothesis that inactivation of penicillin V by beta-lactamase-producing bacteria in oral or throat flora, or the eradication of viridans streptococci with their GAS-inhibitory capacity, is an important factor with regard to recurrent group A streptococcal tonsillitis. Other possible explanations, such as poor antibiotic penetration at the site of infection, are discussed.

The stability analysis and modeling of pH- and ionic strength inactivation of penicillin G acylase obtained from various species of Escherichia coli

A modified model was proposed for the pH-inactivation rate constant of penicillin G acylase obtained from various species of Escherichia coli. A new approach was applied and a new parameter called steric factor defined to obtain the more precise description of the pH-inactivation rate constant. The parameter is used as the measure of the conformational rigidity of the enzyme with respect to pH. The proposed model accuracy was verified and compared with the reported models. The model can be used for pH-inactivation studies of other biocatalysts by determining its parameters. Also, the effect of the ionic strength on the stability of the enzymatic solution of penicillin G acylase has been studied in NaCl and KCl solution. The inactivation reaction of the enzyme with respect to the ionic strength of the solution obeys first order irreversible mechanism. The rate constant of this reaction was determined as a function of the solution ionic strength and a mathematical description was developed. The results show the role of the ionic strength of the non-inhibitory salt solution on the stability of penicillin G acylase, especially for long-time storage and operation.

Detection of bacteria in the presence of antibiotics by using specific monoclonal antibodies to neutralize the antibiotics.

Inactivation of penicillin and gentamicin in cultures was achieved by using monoclonal antibodies against these antibiotics. A viridans group streptococcus (penicillin MIC, less than or equal to 0.06 micrograms/ml) and Escherichia coli ATCC 35218 (gentamicin MIC, less than or equal to 1 microgram/ml) were able to grow in broth containing 0.25 micrograms of penicillin per ml and 4 micrograms of gentamicin per ml, respectively, when the specific antibodies were added. This procedure may be useful to increase the yield of bacteria from body fluid specimens that contain antibiotics.

Beta-Lactamase production in experimental endocarditis due to aminoglycoside-resistant Streptococcus faecalis.

We used a beta-lactamase-producing (beta L+) strain of Streptococcus faecalis that also had high levels of resistance to all aminoglycosides to induce experimental endocarditis in rats. The rats were treated for five or 10 days with procaine penicillin, vancomycin, gentamicin, rifampin, or ciprofloxacin (alone or in various combinations), or with penicillin plus clavulanic acid. The levels of penicillin in serum and vegetations declined rapidly in the beta L+-infected rats treated with procaine penicillin alone, unlike the sustained levels of penicillin in either beta L- -infected rats treated with procaine penicillin or beta L+-infected rats treated with penicillin plus clavulanic acid. For the beta L+-infected rats, the enterococcal counts in vegetations were significantly reduced (greater than 3 log10 cfu/g) only by vancomycin and by penicillin plus clavulanic acid. The efficacy of the latter regimen probably resulted from the inhibition of penicillin inactivation by clavulanic acid in vegetations infected with the beta L+ strain. Our in vivo findings document the biologic significance of beta-lactamase production.

Hydroxylamine technique for in vitro prevention of penicillin inactivation of tobramycin.

Hydroxylamine was evaluated and found to be a highly effective agent for the in vitro prevention of penicillin inactivation of tobramycin. This inactivation reaction resulted in an underestimation of tobramycin concentrations and was dependent on time, temperature, amount and type of penicillin, and amount of tobramycin. Plasma samples containing tobramycin and three clinically relevant concentrations of ticarcillin, carbenicillin, azlocillin, or piperacillin were incubated with and without hydroxylamine, and tobramycin concentrations were monitored at 0, 12, 24, 48, and 72 h. The inactivation reaction was found to be completely inhibited by hydroxylamine (1 mg/ml) compared with a 27 to 50% loss of measured tobramycin concentration in the unprotected tobramycin-penicillin samples. Hydroxylamine did not interfere with the Emit enzyme immunoassay (Syva Co.) at either high or low tobramycin concentrations. Hydroxylamine was effective in inhibiting the tobramycin inactivation at both room and refrigerator temperatures and was 100% effective in protecting tobramycin on a 1:1 molar basis.

Transferable beta-lactamase. A new mechanism for in vitro penicillin resistance in Streptococcus faecalis.

Although enterococci are relatively resistant to penicillin, the mechanism of resistance is largely unknown and enzymatic inactivation does not play a role. In this study, an isolate of Streptococcus faecalis was found to have beta lactamase activity resulting in complete inactivation of penicillin. With a high inoculum, this strain was resistant to greater than 1,000 micrograms/ml of penicillin. Penicillin resistance and beta lactamase activity were transferred by conjugation at a high frequency to an enterococcal laboratory recipient strain together with two plasmids of molecular size 34 X 10(6) and 56 X 10(6), thus demonstrating the emergence of plasmid-mediated penicillin resistance in the genus Streptococcus.

Inactivation of penicillins by Thiol broth.

Thiol broth with sodium polyanetholesulfonate inactivated penicillin G, carbenicillin, nafcillin, oxacillin, and gentamicin, but had no effect on cephalothin, cefoxitin, clindamycin, chloramphenicol, erythromycin, and tetracycline. Only Thiol broth was capable of this inactivation, which was not influenced by the presence of blood.

Nafcillin-tolerant Staphylococcus epidermidis endocarditis

RESISTANCE of<i>Staphylococcus aureus and S epidermidis</i>organisms to penicillin is known to be mediated by several different mechanisms. Resistance due to production of β-lactamase (penicillinase) resulting in penicillin inactivation was recognized with<i>S aureus</i>in the early 1960s.¹"Intrinsic" resistance to the penicillinase-resistant penicillins (PRPs) has been observed in both<i>S aureus</i>and<i>S epidermidis</i>for more than ten years.²Recently, strains of S aureus described as "tolerant" to PRPs have been reported.³This tolerance is manifested by a wide disparity between the concentration of penicillin required to inhibit growth of the organism and that required for killing it. Routine antibiotic sensitivity testing shows these strains to be sensitive to PRPs. The minimal inhibitory concentration (MIC) is well within the therapeutic range of these drugs, but the minimal bactericidal concentration (MBC) is 32 times the MIC or greater, indicating a required therapeutic level not practically achievable

Nafcillin-Tolerant Staphylococcus epidermidis Endocarditis

RESISTANCE of<i>Staphylococcus aureus and S epidermidis</i>organisms to penicillin is known to be mediated by several different mechanisms. Resistance due to production of β-lactamase (penicillinase) resulting in penicillin inactivation was recognized with<i>S aureus</i>in the early 1960s.¹"Intrinsic" resistance to the penicillinase-resistant penicillins (PRPs) has been observed in both<i>S aureus</i>and<i>S epidermidis</i>for more than ten years.²Recently, strains of S aureus described as "tolerant" to PRPs have been reported.³This tolerance is manifested by a wide disparity between the concentration of penicillin required to inhibit growth of the organism and that required for killing it. Routine antibiotic sensitivity testing shows these strains to be sensitive to PRPs. The minimal inhibitory concentration (MIC) is well within the therapeutic range of these drugs, but the minimal bactericidal concentration (MBC) is 32 times the MIC or greater, indicating a required therapeutic level not practically achievable

Inactivation of penicillin G during experimental infection with Bacteroides fragilis.

An animal model implanted with intraperitoneal plastic reservoirs was used for study of the penetration of penicillin G into sites infected with Bacteroides fragilis. Penicillin G was given to rabbits, and its concentration in uninfected reservoirs and in those infected with B. fragilis was determined. The mean percentage penetration ([concentration in capsule divided by peak concentration in serum] X 100) of penicillin into uninfected capsules was 19.9%, whereas that into heavily infected capsules was 1.5%. The percentage penetration of radiolabeled penicillin into infected capsules was 12.5%, whereas the proportion of bioactive drug in the same capsules was again very low (1%). These results show that there is a modest reduction in penetration of penicillin into infected sites and a striking inactivation of the drug by B. fragilis in this experimental model.

Qualitative Study of the Paradoxical Zone Phenomenon of Penicillins against 17 Bacterial Species of Clinical Importance

Using a triple agar layer technique and enzymatic inactivation of penicillin, the occurrence of a paradoxical zone phenomenon (illustrated by a typical 'target' image around the reservoir of antibiotic) was determined for several bacterial species specially chosen with regards to their taxonomic position, clinical importance and penicillin susceptibility. Among gram-positive bacteria, a paradoxical zone was obtained for approximately 43% of the strains studied here (all 10 strains of Staphylococcus aureus, all 10 strains of Streptococcus faecalis, 7 of 10 strains of group B beta-hemolytic streptococci, 1 of 10 group A strains, 3 of 10 strains of alpha-hemolytic streptococci, 3 of 10 strains of Clostridium perfringens but for none of 10 strains each of Streptococcus pneumoniae, and Listeria monocytogenes). Among gram-negative bacteria, a target image was regularly obtained with Haemophilus influenzae (all 10 strains tested) and Proteus species (9 of 10 strains) but with none of the following species: Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis and Bacteroides fragilis. Therapeutic implications of these observations are difficult to assess, and need further investigation.

New Method for Detecting In Vitro Inactivation of Penicillins by Haemophilus influenzae and Staphylococcus aureus

New Method for Detecting In Vitro Inactivation of Penicillins by Haemophilus influenzae and Staphylococcus aureus

A Rapid, simple method for demonstrating synergy of amikacin and penicillin against various microorganisms.

A simple disk diffusion technique with use of triple-layer agar and enzymatic inactivation of penicillin demonstrated synergy between penicillins and aminoglycosides. The method was applied to measurement of the synergistic activity of amikacin and penicillin G against a strain of Streptococcus faecalis sensitive to both drugs, amikacin and ampicillin against a strain of S. faecalis sensitive to amplicillin but resistant to amikacin, and amikacin and carbenicillin against a strain of Proteus mirabilis sensitive to both drugs. The degree of synergy was related to the specific organism tested. For example, syngery of penicillin G and amikacin for S. faecalis could be demonstrated only when the organism was subjected first to the action of penicillin G alone. In addition to providing useful clinical information, the triple-layer technique appears to be a promising tool for the study and understanding of synergy between antimicrobial agents.

Screening and isolation of penicillinase inhibitor, KA-107.

It is known that penicillin resistance of bacteria is mainly caused by the inactivation of penicillin by penicillinase derived from such strains. We have developed a screening procedure for penicillinase inhibitors. Several microorganisms were found to produce such inhibitors, and from the culture filtrate of Streptomyces gedanensis ATCC 4880 a penicillinase inhibitor, named KA-107, was isolated. The characteristics of this inhibitor were revealed by an in vitro test by using penicillinase derived from penicillin resistant Staphylococcus aureus, FS-1277. When KA-107 was used in combination with penicillin-G, ampicillin, d- or l-phenethicillin, the growth inhibitory activity of these penicillins was maintained.

Kinetics of β-Lactamase Inactivation of Penicillins I: Effect of Side-Chain Structure, Ionic Strength, pH, and Temperature

The Staphylococcus aureus Tex-2 β-lactamase-catalyzed hydrolysis of several penicillins was shown to follow Michaelis–Menten kinetics. Penicillins with a lipophilic side chain had the highest affinity for the enzyme. Introduction of a polar group (either positively or negatively charged) into the side chain and/or a bulky diester group on the penicillin nucleus drastically lowered the binding. Of the penicillins studied, the α-aminopenicillins were the most susceptible. The effect of ionic strength on the staphylococcal β-lactamase hydrolysis of penicillin G and ampicillin was small, but the effect of pH was conspicuous. At 25°, a V-shaped pH–Km profile with a pH optimum at 6.8 was obtained for penicillin G, while a sigmoid pH–Km profile with bell-shaped pH–Vmax profile at 6.4 was obtained for the amphoteric ampicillin. The heats of activation of penicillin β-lactamase-catalyzed hydrolysis of penicillins was shown to vary with the enzyme species and with the substrate. The amphoteric penicillin had a much lower heat of activation value than that of the monobasic penicillin. For the S. aureus Tex-2 catalyzed hydrolysis of penicillin G and ampicillin, the heat of activation values were 7.18 and 5.17 kcal./mole at pH’s of 6.8 and 7.4, respectively, while a value of 5.10 kcal./mole was obtained for the Bacillus cereus B569 catalyzed hydrolysis of ampicillin at pH 6.2. The temperature optimum for the staphylococcal β-lactamase inactivation of ampicillin at pH 7.4 may be 35–37°.

Action of gentamicin on penicillin G-induced proteus spheroplasts.

The action of gentamicin on penicillin G-induced spheroplasts was studied in 4 Proteus strains (1 <i>Proteus mirabilis</i>, 2 <i>P. rettgeri</i>, 1 <i>P. vulgaris</i>). Although marked synergism could be demonstrated, this synergism between penicillin G and gentamicin was not increased by spheroplast formation. No greater bactericidal activity of gentamicin resulted when Proteus rods were transformed into spheroplasts by penicillin. Gentamicin disc sensitivity of Proteus spheroplasts after inactivation of penicillin was not better than of Proteus rods. Gentamicin and probably other aminoglycosides should not be recommended as drugs of choice for treatment of spheroplast or L-form infections.