Cheminformatics Study on Structural and Bactericidal Activity of Latest Generation ß-Lactams on Widespread Pathogens.

Raman spectra of oxacillin (OXN), carbenicillin (CBC), and azlocillin (AZL) are reported for the first time together with their full assignment of the normal modes, as calculated using Density Functional Theory (DFT) methods with the B3LYP exchange-correlation functional coupled to the 6-31G(d) and 6-311+G(2d,p) basis sets. Molecular docking studies were performed on five penicillins, including OXN, CBC, and AZL. Subsequently, their chemical reactivity and correlated efficiency towards specific pathogenic strains were revealed by combining frontier molecular orbital (FMO) data with molecular electrostatic potential (MEP) surfaces. Their bactericidal activity was tested and confirmed on a couple of species, both Gram-positive and Gram-negative, by using the disk diffusion method. Additionally, a surface-enhanced Raman spectroscopy (SERS)-principal component analysis (PCA)-based resistogram of A. hydrophila is proposed as a clinically relevant insight resulting from the synergistic cheminformatics and vibrational study on CBC and AZL.

A simple and sensitive AuNPs-based colorimetric aptasensor for specific detection of azlocillin.

A new colorimetric biosensor for specific detection of azlocillin was developed by using DNA aptamer as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric indicator. In the absence of azlocillin, the AuNPs were protected by the aptamer and stabilized at high NaCl concentrations, displaying a red solution. In the presence of azlocillin, the aptamer reacts specifically with azlocillin, resulting in the aggregation of AuNPs and an apparent red to blue color change. The characteristic change can be easily observed by the naked eye and quantitatively detected by an ultraviolet-visible (UV-Vis) spectrometer. Under the optimal conditions, the absorbance variation at 522 nm (ΔA522) of AuNPs changed proportionally with increasing concentration of azlocillin, which exhibited a linear relationship in the concentration range of 50 nM to 500 nM, with a detection limit of 11.6 nM. Furthermore, the aptasensor was successfully used to detect azlocillin in milk and tap water samples, with recoveries ranging from 97.64% to 102.21% and a relative standard deviation (RSD) less than 3.81%.

The threat of persistent bacteria and fungi contamination in tuberculosis sputum cultures.

BACKGROUND: Tuberculosis (TB) sputum culture contaminants make it difficult to obtain pure TB isolates.We aimed to study and identify persistent TB sputum culture contaminants post the standard laboratory pre-culture sample decontamination techniques. METHODS: This was a longitudinal study of TB sputum culture contamination for a cohort of TB patients on standard treatment at: baseline, during TB treatment and post TB treatment. Sputum samples were decontaminated with 1.5%NaOH and neutralized using 6.8 Phosphate buffer solution.Sputum was then inoculated into MGIT (mycobactrial growth indicator tube) supplemented with 0.8ml PANTA. A drop of each positive MGIT culture was sub cultured onto blood agar and incubated for 48 hours at 35 -37OC.Any growth was identified using growth characteristics and colony morphology. RESULTS: From October 2017 through May 2019;we collected 8645 sputum samples of which 8624(99.8%) were eligible and inoculated into MGIT where 2444(28.3%)samples were TB culture positive and 255(10.4%)were positive for contaminants: 237 none-tuberculosis bacteria, 12 fungi and 6 mixed(none-tuberculous bacteria+fungi). There was no statistically significant difference between none tuberculosis bacteria and fungi in the treatment (OR=1.4,95%CI:0.26-7.47,p=0.690) and the post treatment TB phases(OR=2.02,95%CI:0.38-10.79,p=0.411)Vs baseline. CONCLUSION: None-tuberculous bacteria and fungi dominate the plethora of TB sputum culture contamination and persist beyond the standard laboratory pre-culture decontamination algorithm.

Population pharmacokinetics and dosing optimization of azlocillin in neonates with early-onset sepsis: a real-world study.

OBJECTIVES: Nowadays, real-world data can be used to improve currently available dosing guidelines and to support regulatory approval of drugs for use in neonates by overcoming practical and ethical hurdles. This proof-of-concept study aimed to assess the population pharmacokinetics of azlocillin in neonates using real-world data, to make subsequent dose recommendations and to test these in neonates with early-onset sepsis (EOS). METHODS: This prospective, open-label, investigator-initiated study of azlocillin in neonates with EOS was conducted using an adaptive two-step design. First, a maturational pharmacokinetic-pharmacodynamic model of azlocillin was developed, using an empirical dosing regimen combined with opportunistic samples resulting from waste material. Second, a Phase II clinical trial (ClinicalTrials.gov: NCT03932123) of this newly developed model-based dosing regimen of azlocillin was conducted to assure optimized target attainment [free drug concentration above MIC during 70% of the dosing interval ('70% fT>MIC')] and to investigate the tolerance and safety in neonates. RESULTS: A one-compartment model with first-order elimination, using 167 azlocillin concentrations from 95 neonates (31.7-41.6 weeks postmenstrual age), incorporating current weight and renal maturation, fitted the data best. For the second step, 45 neonates (30.3-41.3 weeks postmenstrual age) were subsequently included to investigate target attainment, tolerance and safety of the pharmacokinetic-pharmacodynamic model-based dose regimen (100 mg/kg q8h). Forty-three (95.6%) neonates reached their pharmacokinetic target and only two neonates experienced adverse events (feeding intolerance and abnormal liver function), possibly related to azlocillin. CONCLUSIONS: Target attainment, tolerance and safety of azlocillin was shown in neonates with EOS using a pharmacokinetic-pharmacodynamic model developed with real-world data.

Azlocillin can be the potential drug candidate against drug-tolerant Borrelia burgdorferi sensu stricto JLB31.

Lyme disease is one of most common vector-borne diseases, reporting more than 300,000 cases annually in the United States. Treating Lyme disease during its initial stages with traditional tetracycline antibiotics is effective. However, 10-20% of patients treated with antibiotic therapy still shows prolonged symptoms of fatigue, musculoskeletal pain, and perceived cognitive impairment. When these symptoms persists for more than 6 months to years after completing conventional antibiotics treatment are called post-treatment Lyme disease syndrome (PTLDS). Though the exact reason for the prolongation of post treatment symptoms are not known, the growing evidence from recent studies suggests it might be due to the existence of drug-tolerant persisters. In order to identify effective drug molecules that kill drug-tolerant borrelia we have tested two antibiotics, azlocillin and cefotaxime that were identified by us earlier. The in vitro efficacy studies of azlocillin and cefotaxime on drug-tolerant persisters were done by semisolid plating method. The results obtained were compared with one of the currently prescribed antibiotic doxycycline. We found that azlocillin completely kills late log phase and 7-10 days old stationary phase B. burgdorferi. Our results also demonstrate that azlocillin and cefotaxime can effectively kill in vitro doxycycline-tolerant B. burgdorferi. Moreover, the combination drug treatment of azlocillin and cefotaxime effectively killed doxycycline-tolerant B. burgdorferi. Furthermore, when tested in vivo, azlocillin has shown good efficacy against B. burgdorferi in mice model. These seminal findings strongly suggests that azlocillin can be effective in treating B. burgdorferi sensu stricto JLB31 infection and furthermore in depth research is necessary to evaluate its potential use for Lyme disease therapy.

In vitro selection of DNA aptamers and their integration in a competitive voltammetric biosensor for azlocillin determination in waste water.

The uncontrolled usage of veterinary antibiotics has led to their widespread pollution in waterways and milk products. Potential impact of antibiotic residues on the environment and human health such as increased antibiotic resistance of microorganisms and triggering allergic reactions in humans have been reported. In this work, we developed a highly selective and sensitive voltammetric aptasensor for on-step, sensitive and low cost detection of azlocillin antibiotic, one of the broad spectrum ß-lactam antibiotics. The successful selection of DNA aptamers against azlocillin was accomplished using systemic evolution of ligands by exponential enrichment (SELEX) method. Fluorescence-binding assays showed dissociation constant of 55 nM for one of the selected aptamers (Az9). This aptamer was used to construct a competitive voltammetric aptasensor for azlocillin. A limit of detection of 1.2 pg/mL as well as remarkable selectivity against potential interfering agents, including amoxicillin, were achieved. This signal-off competitive sensor takes 30-50 min to complete the quantification of the target antibiotic. The sensor was challenged by detecting the target directly in complex environments such as tap and waste water where good recovery percentages were achieved.

A fast and simple FIA-chemiluminescence method for the evaluation of Roselle flowers as scavenger of the free radicals generated by UV irradiated antibiotics.

This work proposes a new method for the in vitro evaluation of the effect of UV irradiation on the production of free radicals and other reactive species during the photodecomposition of drugs. The method was based on the UV irradiation of antibiotics molecules to generate excited states that undergo to homolytic bond cleavages. These reactive species can be detected by their ability to oxidize the luminol, producing the electronically excited aminophtalate, which decays to the ground state releasing electromagnetic radiation in the visible zone of the spectrum. This method was applied to penicillin G, nafcillin, azlocillin and neomycin dissolved in water. It was found that the intensity of the luminol chemiluminescence emission (CL) was proportional to the concentration and dependent on the molecular structure of these drugs. Under the optimized conditions, it was found that penicillin and azlocillin were the most susceptible to photodegradation, while neomycin sulfate was the less affected by the UV light. It was observed that the addition to the antibiotics dissolutions of a hydro-alcoholic extract of petals of calyxes of Roselle reduced the CL intensity, indicating that the extract was able to scavenge the free radicals in the irradiated drugs. This result suggest that its addition to the antibiotics can help in the protection against the radicals formed during the exposition to solar light of patients treated with topic similar antibiotics.

Enhanced antibacterial effect of azlocillin in conjugation with silver nanoparticles against Pseudomonas aeruginosa.

In recent years, the problems associated with bacterial resistance to antibiotics caused nanodrugs to be considered as a new way for infectious diseases treatment. The main purpose of this study was to develop a new agent against Pseudomonas aeruginosa, a very difficult bacterium to treat, based on azlocillin antibiotic and silver nanoparticles (AgNPs). Azlocillin was conjugated with AgNPs by chemical methods and its antimicrobial activity was studied against P. aeruginosa using well diffusion agar method. Then, minimum inhibitory concentration and minimum bactericidal concentration of the new conjugate was specified with macro-dilution method. The animal study showed the considerable enhanced antibacterial effect of azlocillin in conjugation with AgNPs against P. aeruginosa in comparison with azlocillin alone, AgNPs alone and azlocillin in combination with AgNPs.

Crystal structures of penicillin-binding protein 3 in complexes with azlocillin and cefoperazone in both acylated and deacylated forms.

Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of ß-lactam-based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of ß-lactams to PBP3. Comparison of wild-type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin-binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.

Interactions of some commonly used drugs with human α-thrombin.

Adverse side effects of drugs are often caused by the interaction of drug molecules to targets other than the intended ones. In this study, we investigated the off-target interactions of some commercially available drugs with human α-thrombin. The drugs used in the study were selected from Super Drug Database based on the structural similarity to a known thrombin inhibitor argatroban. Interactions of these drugs with thrombin were initially checked by in silico docking studies and then confirmed by thrombin inhibition assay using a fluorescence microplate-based method. Results show that the three commonly used drugs piperacillin (anti-bacterial), azlocillin (anti-bacterial), and metolazone (anti-hypertensive and diuretic) have thrombin inhibitory activity almost similar to that of argatroban. The Ki values of piperacillin, azlocillin, and metolazone with thrombin are .55, .95, and .62 nM, respectively. The IC50 values of piperacillin, azlocillin, and metolazone with thrombin are 1.7, 2.9, and 1.92 nM, respectively. This thrombin inhibitory activity might be a reason for the observed side effects of these drugs related to blood coagulation and other thrombin activities. Furthermore, these compounds (drugs) may be used as anti-coagulants as such or with structural modifications.

Uni-molecular detection and quantification of selected ß-lactam antibiotics with a hybrid α-hemolysin protein pore.

Single-nanopores have recently been used to electrically detect a wide range of analytes. Similarly, using electrophysiology, we demonstrate how a system comprised of an ion channel formed by α-hemolysin (α-HL) and single-cyclic γ-cyclodextrin (γ-CD) molecule permits the detection of, and differentiation between three different antibiotics from the ß-lactam family. Specifically, histograms of the time between the successive binding events, and the residence time distributions of the antibiotic in the γ-CD molecular adapter vary with the antibiotic type. The results show that the association times of amoxicillin, azlocillin, and ampicillin are τ(on) = 2.1 ± 0.2, 2.2 ± 0.3, and 3.1 ± 0.4 ms, respectively. Interestingly, we found that the residence time of the bulkier and negatively charged azlocillin (τ(off) = 0.008 ± 0.0005 ms) is much less than that of ampicillin (τ(off) = 0.07 ± 0.005 ms) and amoxicillin (τ(off) = 0.1 ± 0.02 ms), even though the γ-CD-α-HL complex is anionic selective. The data were also used to estimate the standard free energy of binding between ampicillin to γ-CDs binding (-12 kJ mol(-1) [corrected]). The difference in association times might be due to γ-CDs-imposed steric hindrance or an energetically more expensive desolvation step for the antibiotics to gain access to the binding site in the CD. We suggest that this technique may be used to detect other analytes used in pharmaceutical applications.

Reduction of contamination of mycobacterial growth indicator tubes using increased PANTA concentration.

We assessed the effect of a double concentration of supplemental polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) added to the Mycobacterial Growth Indicator Tube (MGIT) on contamination and positivity rates in 216 sputum cultures. Contamination rates were respectively 12.9% and 5.5% for samples processed using standard and double PANTA concentrations (P = 0.0001, McNemar's test). Thirty-five per cent of cultures performed using standard PANTA and 36.5% of those performed using two-fold PANTA concentrations were positive for Mycobacterium tuberculosis, compared to 25.9% of cultures inoculated on Ogawa medium. These results suggest that the use of MGIT with 2× PANTA may be useful in reducing culture contamination without reducing the diagnostic yield.

Contrasting patterns of phylogeographic relationships in sympatric sister species of ironclad beetles (Zopheridae: Phloeodes spp.) in California's Transverse Ranges.

BACKGROUND: Comparative phylogeography of sympatric sibling species provides an opportunity to isolate the effects of geography and demographics on the evolutionary history of two lineages over the same, known time scale. In the current study, we investigated the phylogeographic structure of two zopherid beetle species, Phloeodes diabolicus and P. plicatus, where their ranges overlap in California's Transverse Ranges. RESULTS: Although P. diabolicus and P. plicatus share similar habitats with largely overlapping distributions, the results of this study revealed different evolutionary histories for each species since divergence from their most recent common ancestor. In general, P. plicatus had higher genetic diversity, and more among population isolation than P. diabolicus. The mismatch distributions indicated that one major difference between the two species was the timing of population expansion. This result was consistent with genetic patterns revealed by the Phist values and genetic diversity. Lastly, there were no parallel genetic breaks at similar geographic barriers between the species. CONCLUSIONS: Our data revealed that differential demographics rather than geography were responsible for the genetic patterns of the two species.

Pleistocene climatic cycling drives intra-specific diversification in the intermediate horseshoe bat (Rhinolophus affinis) in Southern China.

The repeated formation and loss of land-bridges during the Pleistocene have had lasting impacts on population genetic structure. In the tropics, where island populations persisted through multiple glacial cycles, alternating periods of isolation and contact are expected to have driven population and taxonomic divergence. Here, we combine mitochondrial and nuclear sequence data with microsatellites to dissect the impact of Pleistocene climate change on intra-specific diversification in the horseshoe bat Rhinolophus affinis. This taxon shows considerable morphological and acoustic variation: two parapatric subspecies (himalayanus and macrurus) occur on mainland China and a third (hainanus) on Hainan Island. Our phylogeographic reconstruction and coalescent analyses suggest the island subspecies formed from an ancestral population of himalayanus via two colonization events c. 800,000 years before present. R. a. hainanus then recolonized the mainland, forming macrurus and thus a secondary contact zone with himalayanus. Finally, macrurus recolonized Hainan following the LGM. We found that all three biological events corresponded to known periods of land-bridge formation. Evidence of introgression was detected between macrurus and both its sister taxa, with geographical proximity rather than length of separation appearing to be the biggest determinant of subsequent genetic exchange. Our study highlights the important role of climate-mediated sea level changes have had in shaping current processes and patterns of population structure and taxonomic diversification.

Molecular taxonomy, phylogeny and evolution in the family Stichopodidae (Aspidochirotida: Holothuroidea) based on COI and 16S mitochondrial DNA.

The Stichopodidae comprise a diverse assemblage of holothuroids most of which occur in the Indo-Pacific. Phylogenetic analyses of mitochondrial gene (COI, 16S rRNA) sequence for 111 individuals (7 genera, 17 species) clarified taxonomic uncertainties, species relationships, biogeography and evolution of the family. A monophyly of the genus Stichopus was supported with the exception of Stichopus ellipes. Molecular analyses confirmed genus level taxonomy based on morphology. Most specimens harvested as S. horrens fell in the S. monotuberculatus clade, a morphologically variable assemblage with others from the S. naso clade. Taxonomic clarification of species fished as S. horrens will assist conservation measures. Evolutionary rates based on comparison of sequence from trans-ithmian Isostichopus species estimated that Stichopus and Isostichopus diverged ca. 5.5-10.7Ma (Miocene). More recent splits were estimated to be younger than 1Ma.

Phylogeography and recolonization of the Swiss Alps by the Valais shrew (Sorex antinorii), inferred with autosomal and sex-specific markers.

Using one male-inherited, one female-inherited and eight biparentally inherited markers, we investigate the population genetic structure of the Valais shrew (Sorex antinorii) in the Swiss Alps. Bayesian analysis on autosomal microsatellites suggests a clear genetic differentiation between two groups of populations. This geographically based structure is consistent with two separate postglacial recolonization routes of the species into Switzerland from Italian refugia after the last Pleistocene glaciations. Sex-specific markers also confirm genetic structuring among western and eastern areas, since very few haplotypes for either Y chromosome or mtDNA genome are shared between the two regions. Overall, these results suggest that two already well-differentiated genetic lineages colonized the Swiss Alps and came into secondary contact in the Rhône Valley. Low level of admixture between the two lineages is likely explained by the mountainous landscape structure of lateral valleys orthogonal to the main Rhône valley.

Human oral drugs absorption is correlated to their in vitro uptake by brush border membrane vesicles.

Brush border membrane vesicles (BBMV) were prepared from the rabbit small intestine for testing drug absorption potency through the enterocyte's apical membrane, which is an important compartment for drug oral absorption. Some modifications have been made to the traditional vesicle assay for adapting it to the 96-well plate format. The accumulation of 23 reference drugs was measured, and the data showed a good correlation with human oral absorption with a correlation coefficient R=0.853 (P<0.001), with the exception of a few false positive results. As the measured drug absorption may contain a membrane/protein binding component as well as drug uptake into vesicles, these two fractions can be discriminated by changing extravesicular osmolarity using different mannitol concentrations. This model can be applied for evaluating drug absorption rate/mechanisms, and helping drug selection in early drug research and development.

[Meningoencephalitis caused by Pseudomonas cepacia]. / Meningoencefalitis por Pseudomonas cepacia.

A case of meningoencephalitis of bacterial etiology caused by Pseudomonas cepacia was described. The strain was received at the Reference Laboratory of Bacterial Acute Respiratory Infections of "Pedro Kouri" Institute of Tropical Medicine, where its microbiological identification was confirmed. This isolation was a finding in an adult immunocompetent patient. The evolution was favourable with no sequelae for his future life. Pseudomona cepacia has been associated with respiratory infections in patients with cystic fibrosis. Patients with Pseudomonas cepacia may be asymptomatic or present fatal acute and fulminant infection.

Structure-activity relationships of different beta-lactam antibiotics against a soluble form of Enterococcus faecium PBP5, a type II bacterial transpeptidase.

Penicillin-binding proteins (PBPs) catalyze the essential reactions in the biosynthesis of cell wall peptidoglycan from glycopeptide precursors. beta-Lactam antibiotics normally interfere with this process by reacting covalently with the active site serine to form a stable acyl-enzyme. The design of novel beta-lactams active against penicillin-susceptible and penicillin-resistant organisms will require a better understanding of the molecular details of this reaction. To that end, we compared the affinities of different beta-lactam antibiotics to a modified soluble form of a resistant Enterococcus faecium PBP5 (Delta1-36 rPBP5). The soluble protein, Delta1-36 rPBP5, was expressed in Escherichia coli and purified, and the NH(2)-terminal protein sequence was verified by amino acid sequencing. Using beta-lactams with different R1 side chains, we show that azlocillin has greater affinity for Delta1-36 rPBP5 than piperacillin and ampicillin (apparent K(i) = 7 +/- 0.3 microM, compared to 36 +/- 3 and 51 +/- 10 microM, respectively). Azlocillin also exhibits the most rapid acylation rate (apparent k(2) = 15 +/- 4 M(-1) s(-1)). Meropenem demonstrates an affinity for Delta1-36 rPBP5 comparable to that of ampicillin (apparent K(i) = 51 +/- 15 microM) but is slower at acylating (apparent k(2) = 0.14 +/- 0.02 M(-1) s(-1)). This characterization defines important structure-activity relationships for this clinically relevant type II transpeptidase, shows that the rate of formation of the acyl-enzyme is an essential factor determining the efficacy of a beta-lactam, and suggests that the specific side chain interactions of beta-lactams could be modified to improve inactivation of resistant PBPs.

[Bacterial meningitis and Pseudomonas aeruginosa: apropos of a case]. / Meningitis bacteriana y Pseudomonas aeruginosa: a propósito de un caso.

Meningitis caused by gram-negative bacilli increased since the 1970, with a higher incidence in small children. Within this group of infections, the meningitis caused by Pseudomonas sp is rare. The case of a 54-year-old patient with a clinical picture of meningitis is reported. Pseudomonas aeruginosa was isolated from the cerebrospinal fluid. The meningitis caused by Pseudomonas aeruginosa should be taken into consideration because of the severity of the clinical picture and the high mortality and increasing strain resistance.