Impaired Cognitive Function after Perineuronal Net Degradation in the Medial Prefrontal Cortex.

Perineuronal nets (PNNs) are highly organized components of the extracellular matrix that surround a subset of mature neurons in the CNS. These structures play a critical role in regulating neuronal plasticity, particularly during neurodevelopment. Consistent with this role, their presence is associated with functional and structural stability of the neurons they ensheath. A loss of PNNs in the prefrontal cortex (PFC) has been suggested to contribute to cognitive impairment in disorders such as schizophrenia. However, the direct consequences of PNN loss in medial PFC (mPFC) on cognition has not been demonstrated. Here, we examined behavior after disruption of PNNs in mPFC of Long-Evans rats following injection of the enzyme chondroitinase ABC (ChABC). Our data show that ChABC-treated animals were impaired on tests of object oddity perception. Performance in the cross-modal object recognition (CMOR) task was not significantly different for ChABC-treated rats, although ChABC-treated rats were not able to perform above chance levels whereas control rats were. ChABC-treated animals were not significantly different from controls on tests of prepulse inhibition (PPI), set-shifting (SS), reversal learning, or tactile and visual object recognition memory. Posthumous immunohistochemistry confirmed significantly reduced PNNs in mPFC due to ChABC treatment. Moreover, PNN density in the mPFC predicted performance on the oddity task, where higher PNN density was associated with better performance. These findings suggest that PNN loss within the mPFC impairs some aspects of object oddity perception and recognition and that PNNs contribute to cognitive function in young adulthood.

Perineuronal Nets Enhance the Excitability of Fast-Spiking Neurons.

Perineuronal nets (PNNs) are specialized complexes of extracellular matrix molecules that surround the somata of fast-spiking neurons throughout the vertebrate brain. PNNs are particularly prevalent throughout the auditory brainstem, which transmits signals with high speed and precision. It is unknown whether PNNs contribute to the fast-spiking ability of the neurons they surround. Whole-cell recordings were made from medial nucleus of the trapezoid body (MNTB) principal neurons in acute brain slices from postnatal day 21 (P21) to P27 mice. PNNs were degraded by incubating slices in chondroitinase ABC (ChABC) and were compared to slices that were treated with a control enzyme (penicillinase). ChABC treatment did not affect the ability of MNTB neurons to fire at up to 1000 Hz when driven by current pulses. However, f-I (frequency-intensity) curves constructed by injecting Gaussian white noise currents superimposed on DC current steps showed that ChABC treatment reduced the gain of spike output. An increase in spike threshold may have contributed to this effect, which is consistent with the observation that spikes in ChABC-treated cells were delayed relative to control-treated cells. In addition, parvalbumin-expressing fast-spiking cortical neurons in >P70 slices that were treated with ChABC also had reduced excitability and gain. The development of PNNs around somata of fast-spiking neurons may be essential for fast and precise sensory transmission and synaptic inhibition in the brain.

Dual-porosity hollow nanoparticles for the immunoprotection and delivery of nonhuman enzymes.

Although enzymes of nonhuman origin have been studied for a variety of therapeutic and diagnostic applications, their use has been limited by the immune responses generated against them. The described dual-porosity hollow nanoparticle platform obviates immune attack on nonhuman enzymes paving the way to in vivo applications including enzyme-prodrug therapies and enzymatic depletion of tumor nutrients. This platform is manufactured with a versatile, scalable, and robust fabrication method. It efficiently encapsulates macromolecular cargos filled through mesopores into a hollow interior, shielding them from antibodies and proteases once the mesopores are sealed with nanoporous material. The nanoporous shell allows small molecule diffusion allowing interaction with the large macromolecular payload in the hollow center. The approach has been validated in vivo using l-asparaginase to achieve l-asparagine depletion in the presence of neutralizing antibodies.

Prevalence and molecular epidemiological typing of penicillinase-producing Neisseria gonorrhoeae and their bla(TEM-135) gene variants in Nanjing, China.

BACKGROUND: This study aimed to investigate the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and their blaTEM-135 gene variant in 2007 and 2012 in Nanjing, China. In addition, molecular epidemiological typing of all isolates was performed to elucidate the genetic relationships of the PPNG strains. METHODS: A total of 199 and 77 N. gonorrhoeae isolates were collected at the National Center for STD Control in 2007 and 2012, respectively. Nitrocefin tests were performed to identify PPNG. Mismatch amplification mutation assay was used to identify blaTEM-135. All isolates were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST), and additionally, porB-based phylogenetic analysis was performed for the PPNG isolates. RESULTS: The total prevalence of PPNG isolates was 41% (114/276) and 58% (66/114) of these PPNG isolates possessed bla(TEM-135). In 2007, 45% (90/199) produced ß-lactamase, and of those PPNG, 58% (52/90) possessed bla(TEM-135). In 2012, 31% (24/77) were PPNG, and 58% (14/24) of those isolates contained bla(TEM-135). There were 162 NG-MAST STs among the 276 isolates, and 89 of those were novel STs. A strong association between specific NG-MAST STs and bla(TEM-135) was found, and the porB-based phylogenetic analysis showed a distant evolutionary relationship between isolates in 2007 and isolates in 2012. CONCLUSIONS: A high prevalence of PPNG and blaTEM-135 was found in Nanjing, China. bla(TEM-135) might be a precursor in the evolution into an extended-spectrum ß-lactamase that can degrade ceftriaxone, which stresses the need to continuously monitor PPNG, blaTEM-135, and additional evolving blaTEM gene variants.

Depletion of perineuronal nets enhances recognition memory and long-term depression in the perirhinal cortex.

Perineuronal nets (PNNs) are extracellular matrix structures surrounding cortical neuronal cell bodies and proximal dendrites and are involved in the control of brain plasticity and the closure of critical periods. Expression of the link protein Crtl1/Hapln1 in neurons has recently been identified as the key event triggering the formation of PNNs. Here we show that the genetic attenuation of PNNs in adult brain Crtl1 knock-out mice enhances long-term object recognition memory and facilitates long-term depression in the perirhinal cortex, a neural correlate of object recognition memory. Identical prolongation of memory follows localized digestion of PNNs with chondroitinase ABC, an enzyme that degrades the chondroitin sulfate proteoglycan components of PNNs. The memory-enhancing effect of chondroitinase ABC treatment attenuated over time, suggesting that the regeneration of PNNs gradually restored control plasticity levels. Our findings indicate that PNNs regulate both memory and experience-driven synaptic plasticity in adulthood.

[Current microbiological problems. Antibiotic resistance and therapeutic problems raised by Pseudomonas aeruginosa]. / Problèmes microbiologiques d'actualité. Résistance aux antibiotiques et problèmes thérapeutiques posés par Pseudomonas aeruginosa.

RESISTANCE: Pseudomonas aeruginosa is characterized by its low intrinsic susceptibility to many antibiotics and its capacity to acquire additional resistance mechanisms to usually active drugs. Some beta-lactam resistance mechanisms are well known (penicillinase production, cephalosporinase overproduction) and others have been recently identified, such as active efflux systems, which confer coresistance to quinolones, and new beta-lactamases which are limited to a few countries (extended-spectrum beta-lactamases, imipenemase). Ceftazidime remains the most active beta-lactam agent. ACTIVE DRUGS: Among aminoglycosides, amikacin and isepamicin are the most frequently active drugs. The use of fluoroquinolones is limited by a high incidence of acquired resistance. The percentage of resistant strains is highly variable according to countries, hospitals and wards. CLINICAL PRACTICE: Therapy, usually based on a beta-lactam-aminoglycoside combination, will be empirical at first, according to local epidemiological factors, site of infection and previously administered antibiotics, then re-evaluated according to susceptibility results.

Modified microbiological method for the screening of antibiotics in milk.

The Bacillus stearothermophilus disc assay is routinely used by the dairy industry to screen milk for antibiotic residues. Although the assay detects the presence of beta-lactam antibiotics, it does not distinguish cephalosporins from other beta-lactam antibiotics. In this study, the B. stearothermophilus disc assay was modified to allow it to distinguish parent ceftiofur from other antibiotics by incorporation of the enzymes penicillinase and cephalosporinase into the assay. The modified B. stearothermophilus disc assay involves determining the zone of inhibition of a sample on an agar plate after the plate was incubated at 65 degrees C for 2.5 to 3 h as well as determining the zone of inhibition after the sample was treated with penicillinase or cephalosporinase. Samples in which this zone diameter was > 19 mm and < or = 25 mm were interpreted using the data from the primary assay. Samples with zone diameters > 25 mm must be diluted 2- to 10-fold and reassayed to obtain a zone diameter > 19 and < or = 25 mm, for proper interpretation. Samples with zone diameters > or = 16 mm and < or = 19 mm must also be reassayed using dilute enzyme solutions for proper interpretation. When these modifications of the B. stearothermophilus disc assay are used, ceftiofur can be distinguished from ampicillin, amoxicillin, penicillin, cephapirin, cloxacillin, novobiocin, and pirlimycin for samples with zone diameters > or = 16 mm. This assay cannot, however, separate ceftiofur from cefazolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Naturally-occurring, osmo-remedial variants of Escherichia coli.

Two clones of Escherichia coli O27:K1:H31 and O2:H7, isolated from patients with urinary tract infection or bacteraemia, failed to grow in a synthetic minimal medium (MM) of low osmolality. They were considered to be osmo-remedial because they grew well when sufficient amounts of NaCl, mannitol or sucrose were added to raise the osmolality of the medium to > 300 mOsm/kg. The defect could also be corrected by nicotinamide or its precursors quinolinic and aspartic acids. Each clone had a unique DNA restriction enzyme profile, fimbriae and antibiotic susceptibility patterns. The osmo-remedial variants were unstable and underwent phenotypic modulation to form mixtures with osmo-tolerant forms when grown in MM. They tended to form satellites of small colonies around large colonies of osmo-tolerant cells on MM agar plates. The penicillin method of Davis was used to separate the two forms. Nicotinamide induced the expression of ompF when the osmo-remedial strains were grown under conditions of low osmolality. It is possible that the variants are defective in the synthesis of membrane-derived oligosaccharides or outer-membrane proteins, but this has yet to be determined.

Activity of sub-minimal inhibitory concentrations of aspoxicillin in prolonging the postantibiotic effect against Staphylococcus aureus.

Aspoxicillin, a newly developed acylureido-penicillin with a long half-life in mouse serum of 55 min, induced postantibiotic effects (PAEs) against Staphylococcus aureus Smith of 1.7 h in vitro and 5.2 h in vivo in a thigh infection model in neutropenic mice. The long serum half-life meant that in order to evaluate the in-vivo PAE, it was necessary to examine the contribution of the drug at a sub-minimal inhibitory concentration (sub-MIC). Growth suppression by sub-MICs of aspoxicillin was examined in vitro using either previously unexposed bacterial cells or cells which had been pre-exposed to twice the MIC of aspoxicillin for 2 h. At each sub-MIC tested, the duration of growth suppression for pre-exposed cells was longer than that for unexposed cells. In an attempt to eliminate the sub-MIC effect in vivo, penicillinase was injected into mice at the time after administration when the aspoxicillin serum concentration approached the MIC. The in-vivo PAE decreased to 2.7 h when penicillinase was injected. It was concluded that aspoxicillin induced a PAE in vivo which was additional to the effect of sub-inhibitory residual drug, but that sub-MIC levels of the drug were simultaneously involved in suppressing bacterial regrowth after the drug concentration decreased below the MIC. Similar postantibiotic sub-MIC effects may also occur with other long half-life antibiotics.

Evaluation of Stuart's transport medium containing penicillinase. An in vitro study. Brief report.

An in vitro study was performed to evaluate the value of penicillinase in Stuart's transport medium. Swabs were taken from serum broth cultures of Staphylococcus aureus, Escherichia coli and Proteus rettgeri, incubated with cefuroxime. Following 24 hours' storage in the transport medium, the recovery of P. rettgeri was significantly higher from Stuart's transport media containing penicillinase in a concentration of 100,000 units/ml, than from swabs stored in Stuart's medium without penicillinase. Although susceptible to cefuroxime, we did not find the same effect on S. aureus and E. coli, presumably because these strains had higher MIC values than the P. rettgeri strain tested.

Bactericidal effect and regrowth of Streptococcus faecalis exposed to amoxicillin following beta-lactamase.

Streptococcus faecalis usually requires high concentrations of penicillin or ampicillin to achieve killing (i.e. a high MBC/MIC ratio). However, most strains show the Eagle or paradoxical effect. We subjected 12 strains of S. faecalis to 0.1, 1, 10 and 100 micrograms/ml of amoxicillin. Turbidometry studies have shown that 3 h after the inactivation of amoxicillin by penicillinase, there was a longer effect for 1 micrograms/ml following beta-lactamase (12 h 31 min +/- 2 h 09 min) than for 10 micrograms/ml (7 h 0 min +/- 1 h 12 min) or 100 micrograms/ml (5 h 22 min +/- 0 h 52 min). After 3 h, the reduction of CFU/ml (inoculum 10(6) CFU/ml) was -1.8 +/- 0.6 for 1 micrograms/ml, -0.56 +/- 0.56 for 10 micrograms/ml and -0.21 +/- 0.20 for 100 micrograms/ml. The more rapid killing at 3 h was not the only reason for the longer effect following beta-lactamase observed with 1 micrograms/ml. Indeed, the growth curve obtained with an inoculum of 10(3) CFU/ml was 2 h delayed from the control curve (10(6) CFU/ml). In conclusion, a paradoxical effect (killing curves and effect following beta-lactamase) was observed for all S. faecalis strains included in this series.

[Penicillin-selective electrode based on a cellulose membrane with immobilized enzyme]. / Penitsillinselektivnyi élektrod na osnove tselliuloznoi membrany s immobilizovannym fermentom.

Modified cellulose films are recommended to be used as carriers in preparing enzymatic membranes for penicillin-selective electrodes. The results of laboratory testing of the enzymatic penicillin-selective electrode based on the cellulose membrane with immobilized penicillinase are presented. In principle it was shown possible to use the enzymatic electrode in determining penicillin concentration in solutions. Dependence of the electrode reading on the buffer capacity was revealed. Optimal characteristics of the enzymatic membranes and the life-expectancy of the enzymatic electrode were determined.

The effect of imipenem on strains of Enterobacteriaceae expressing Richmond & Sykes class I beta-lactamases.

Fourteen strains of Enterobacteriaceae producing Richmond & Sykes Class I beta-lactamase were studied. The ability of cefoxitin and imipenem to induce beta-lactamase production (reversible derepression) and to select stably derepressed mutants in these strains was assessed. beta-Lactamase induction by cefoxitin and imipenem was demonstrated by the disc diffusion technique in all strains. Cefoxitin selected stably derepressed mutants for all strains in broth cultures, but in an identical experiment imipenem did not. The susceptibility of each strain and its stably derepressed mutant (selected with cefoxitin) to a range of beta-lactam antibiotics was then ascertained. The stably derepressed mutants exhibited decreased susceptibility to all antibiotics tested except imipenem. The decrease in susceptibility varied between strains and between antibiotics but reached a maximum of a 256-fold decrease. The beta-lactamase activity of selected stably derepressed mutant strains showed at least a 600-fold increase in activity. Imipenem would therefore seem an appropriate choice for therapy of infections caused by this group of organisms, as it is active against derepressed mutants and unlikely to select any such strains during therapy.

Effect of clavulanic acid on inactivated amoxicillin excretion in urinary tract infection.

Oral amoxicillin was taken with and without clavulanic acid by normal subjects and by patients with chronic complicated urinary tract infection to examine the in vitro protective effect of clavulanic acid on amoxicillin degradation. When amoxicillin alone was taken, urinary excretion of the penicilloic acid of amoxicillin in bacteria-positive patients was higher than that in bacteria-negative patients and in normal subjects. There was no comparable change in urinary penicilloic acid excretion in the presence of clavulanic acid. There were significant in vitro protective effects of clavulanic acid on beta-lactamases in the urine.

Comparative assessment of in vitro inactivation of gentamicin in the presence of carbenicillin by three different gentamicin assay methods.

Inactivation of gentamicin (G) is known to occur secondarily to the formation of complexes with certain beta-lactam antibiotics. However, aminoglycosides in the presence of aminoglycoside-beta-lactam complexes may not be recognized uniformly by all assay methods. We tested this hypothesis by using mixtures of G plus carbenicillin (C), with and without the addition of penicillinase, in pooled sera under several in vitro conditions: at 25 and 35 degrees C and at low and high C concentrations. Samples were assayed for G with the EMIT and TDx systems, and a microbiological assay was performed with a strain of Klebsiella pneumoniae resistant to C. In the presence of C (500 micrograms/ml) at 35 degrees C, the initial G concentration of 5 micrograms/ml decreased markedly over 48 h as assessed by all three assay methods. However, significantly greater degradation was noted when samples were measured by microbiological assay and TDx than by EMIT. Differences between assays were less marked when mixtures were studied at a lower temperature and with a lower G to C ratio (5 micrograms of G plus 100 micrograms of C per ml). The addition of penicillinase to the antibiotic mixtures prevented the degradation of G over time when measured by all three assay systems. We concluded (i) that EMIT measures higher serum concentrations of G than do TDx or microbiological assays when complexes of G and C are present and (ii) that the addition of penicillinase to serum samples containing C and G would be effective in preventing G degradation during prolonged (greater than 24-h) periods between the time of sampling and assay.

Enzymatic conversion of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine to beta-lactam antibiotics.

Incubation of the unnatural tripeptide delta-(D-alpha-aminoadipyl)-L-cysteinyl-D-valine (DLD-ACV) with a partially purified extract of Cephalosporium acremonium resulted in the production of deacetoxycephalosporin C. The extract contained isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) but no penicillin epimerase activity, and was incubated aerobically in the presence of the components of the cyclase and expandase reaction mixtures (Fe++, ascorbate, dithiothreitol, alpha-ketoglutarate and ATP). The reaction was sensitive to penicillinase, indicating penicillin N to be an intermediate. However, when ring expansion was prevented by omission of alpha-ketoglutarate and ATP, no penicillin N was detected.

Controversies in antimicrobial susceptibility testing.

This article examines a number of areas of disagreement surrounding antimicrobial susceptibility testing and discusses some of the useful susceptibility testing techniques for which no standardized procedures have been established.