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Social experiences during early life are thought to be critical for proper social and emotional development. Conversely, social insults during development causes long-lasting behavioral abnormalities later in life. However, how juvenile social deprivation influences social and emotional behaviors remains poorly understood. Here, we show that juvenile social isolation induces a shift in microbial ecology that negatively impacts social and emotional behaviors in adulthood. These behavioral changes, which occur during this critical period are transferable to antibiotic pre-treated mice by fecal microbiota transplant. In addition, juvenile social isolation decreases the expression of oxytocin receptor (OXTR) in the medial prefrontal cortex (mPFC), and increases the amounts of fecal propionic acid (PA), a short-chain fatty acid derived from gut micobiota. Accordingly, infusion with an OXTR antagonist (OXTR-A, l-368,899) specifically in the mPFC or supplementation of PA both can cause social deficits and anxiety-like behaviors in group housed mice. Collectively, our findings reveal that juvenile social experience regulates prefrontal cortical OXTR expression through gut microbiota-produced PA and that is essential for normal social and emotional behaviors, thus providing a cellular and molecular context to understand the consequences of juvenile social deprivation.

Neuropsychiatric dysfunction and reactive microglia are hallmarks of high-fat diet (HFD)-induced obesity, yet whether these reactive microglia contribute to HFD-induced obesity-related behavioral abnormalities and the underlying mechanisms remain unclear. Here, we show that HFD feeding causes social deficits and anxiety-like behaviors with impaired neuronal activity and alters the gut microbiota, particularly by depleting Lactobacillus reuteri (L. reuteri), in mice. The profiles of microbiome and metabolome in HFD-fed mice predict that specific microbial taxa and their metabolites regulate HFD-induced obesity-related behavioral abnormalities. Oral treatment with the L. reuteri reduces microglial activation and increases dendritic spine density, thus ameliorates social deficits and anxiety in HFD-fed mice. HFD-fed mice that are administered L. reuteri are also found to accumulate butyrate in their gut, sera and brain. Moreover, supplementation of butyrate improves behavioral abnormalities and modulates microglial homeostasis in HFD-fed mice. In addition, selectively removal of microglia through a pharmacologic approach can rescue dendritic spine loss and increase neuronal activity that profoundly alleviates social deficits and anxiety arising from HFD-induced obesity. Overall, this study reveals an unexpected pivotal role of gut commensal-derived butyrate in HFD-induced social deficits and anxiety-like behaviors through regulation of microglial homeostasis and identifies a potential probiotic treatment for HFD-induced obesity-related behavioral abnormalities.

Autophagy is a physiological process governing proteostasis and implicated in many diseases. Enhanced ROS levels was known to be associated with lysosomal autophagy and therefore a lyso-specific ROS probe could find applications in autophagy related studies. Following the probe-design principle of bridging-group modification, we previously developed a ROS probe capable of detect a broader range of reactive species than the existing probes. Additional merits of the probe include ratiometry-capability, minimal probe-product spectral crosstalk, resistance toward autoxidation at lysosomal acidity, and structural simplicity. It is the aim of this manuscript to render this probe lyso-specificity and examine its potentials to monitor autophagy-related ROS generation in HeLa cells treated by H2O2 or 6-OHDA.

Obesity is one of the most serious public health challenges in the world. Obesity during early life has been associated with an increased risk of neurodevelopmental disorders, including deficits in learning and memory, yet the underlying mechanisms remain unclear. Here, we show that early life high-fat diet (HFD) feeding impairs hippocampus-dependent contextual/spatial learning and memory, and alters the gut microbiota, particularly by depleting Akkermansia muciniphila (A. muciniphila), in mice. Transplantation of the HFD microbiota confers hippocampus-dependent learning and memory deficits to mice fed a chow diet. Oral treatment of HFD-fed mice with the gut commensal A. muciniphila corrects gut permeability, reduces hippocampal microgliosis and proinflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6) expression, and restores neuronal development and synapse plasticity, thus ameliorates defects in learning and memory. Interestingly, treatment of mice with lipopolysaccharide (LPS) mimics HFD-induced hippocampus-dependent cognitive impairment in chow-fed mice. In line with these findings, pharmacologic blockade of Toll-like receptor 4 (TLR4) signalling or antibiotics treatment both effectively prevent hippocampus-dependent learning and memory deficits in HFD-fed mice. Collectively, our findings demonstrate an unexpected pivotal role of gut microbiota in HFD-induced cognitive deficits and identify a potential probiotic therapy for obesity associated with cognitive dysfunction during early life.

The prefrontal cortex (PFC) plays an important role in regulating anxiety-like phenotypes and social behaviors, and impairments in this brain region has been linked to social deficits in mammals. Childhood obesity is associated with an increased risk of neuropsychiatric behavioral abnormalities, including attenuated social preference and increased anxiety-like behaviors in adulthood. However, little data are available on the impact of obesity during adolescence on PFC-dependent behaviors. Herein, we use the mice pups to illuminate whether and how high-fat diet (HFD) feeding in adolescence affects medial prefrontal cortex (mPFC)-dependent behaviors, and what the underlying cellular and molecular mechanism is. We found that juvenile HFD feeding results in the accumulation of senescent astrocytes and microglia in the mPFC of mice. Furthermore, we found a causal link between the accumulation of senescent glial cells and HFD-induced neuropsychiatric behavioral abnormalities. Pharmacological clearance of senescent glial cells in HFD-fed mice enhances neuronal activity and reserves synaptic excitatory/inhibitory balance, thus preserving normal behaviors. Collectively, these results show that senescent glial cells play a significant role in the initiation and progression of juvenile obesity-mediated neuropsychiatric behavioral abnormalities, and suggest that targeting senescent glial cells may provide a therapeutic avenue for the treatment of obesity-related neuropsychiatric disorders in children.

Chronic stress significantly affects neuron morphometry and function in the prefrontal cortex, a brain region controlling cognition and emotion. However, whether and how chronic stress regulates the maturation of NG2-expressing oligodendrocyte precursor cell (NG2(+) cell) and the importance of these changes remained unknown. Here, we report that exposing adult mice to chronic stress results in NG2(+) cell atrophy and myelination arrested in the medial prefrontal cortex (mPFC), and impaired mPFC-dependent functions. These alterations, are phenocopied by overexpression of death receptor 6 (DR6) in NG2(+) cell. Conversely, selectively silencing of DR6 in the NG2(+) cell can partly rescue NG2(+) cell atrophy and cognitive deficiency caused by chronic stress. We further demonstrate that myelination appears necessary for mPFC-dependent cognitive processes, as lysolecithin (LPC)-induced demyelination specifically in the mPFC is sufficient to cause these behavioral and cognitive impairments. Our results indicate that chronic stress impairs cognitive functions, at least in part, through modulation of NG2(+) cell maturation and myelination, and suggest that myelination is require for normal cognitive functions.

Brain structure and functions are significantly affected by enriched environment (EE). Rodent and rhesus monkeys raised in EE will increase myelination in development, and these increase correlate with improved cognitive functions on learning and memory. However, whether and how EE influences remyelination in the adult remained undefined. Here, we used a cuprizone-induced demyelination mouse model demonstrate that EE significantly enhances remyelination. This EE-regulated remyelination is associated with improved motor skills. We found that histone deacetylases 1/2 (HDAC1/2) were drastically increased in EE. EE act mechanistically by inhibition of Wnt signaling pathway during remyelination through promotion of HDAC1/2. Moreover, pharmacological inhibition of HDACs promoted Wnt signaling activation and impaired remyelination in EE. These results suggested that the effect of EE is likely to be mediated, at least in part, by elevating HDAC1/2 expression and inhibiting Wnt signal pathway, which initiates ‘rewiring’ of the neural network and accelerates remyelination. These findings highlighted the potential of EE as a promising noninvasive strategy to accelerate remyelination and to restore motor functions for demyelination related disease.

The immunomodulatory activity of Lactobacillus rhamnosus ZDY114 and donkey milk in BALB/c mice was evaluated by assessing the splenic lymphocyte transformation, haemolytic complement activity, carbon clearance ability and natural killer cell activity. Results showed donkey milk (5 g kg −1 ) in combination with L. rhamnosus ZDY114 (5 × 10 7 cfu mL −1 ) exhibited a significant increase in splenic lymphocyte proliferation, carbon granule engulfing ability and natural killer cell activity when compared with donkey milk or L. rhamnosus ZDY114 alone ( p L. rhamnosus ZDY114. In conclusion, donkey milk (5 g kg −1 ) in combination with L. rhamnosus ZDY114 (5 × 10 7 cfu mL −1 ) was able to enhance specific immune functions.

A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection.

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine.

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes ( Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella . Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10 6 CFU/ml in pure culture or 1 × 10 6 CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10 2 CFU/ml (4.3 × 10 2 CFU/ml for S. Typhimurium, 3.7 × 10 2 CFU/ml for S. Paratyphi B, 7.2 × 10 2 CFU/ml for S. Typhi) in pure culture and 10 3 CFU/g (4.3 × 10 3 CFU/g for S. Typhimurium, 3.7 × 10 3 CFU/g for S. Paratyphi B, 7.2 × 10 3 CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S . Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce.

Beta-lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against beta-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital settings, have reduced activity against newly emerging beta-lactamases. Bacterial production of diverse beta-lactamases including class-A, class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously, several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were shown to be potent and broad-spectrum beta-lactamase inhibitors. As a continuation of our previous work and in order to extend the structure-activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and broad-spectrum inhibitors of the TEM-1 and AmpC beta-lactamases. In combination with piperacillin, their in vitro activities showed enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds such as 12a-c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The 5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA-MurD. Several pulvinones inhibited Mur enzymes with IC(50)'s in the 1-10 microg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives were synthesized, utilizing microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good activity against MurB in vitro and low MIC values against Gram-positive bacteria, particularly penicillin-resistant Streptococcus pneumoniae (PRSP). Derivative 7l demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria. Derivatives 7f and 10a also demonstrated potent nanomolar Kd values in their binding to MurB.

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and PRSP. However, when they were tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128 microg/mL against PRSP. None of the compounds demonstrated activity against gram-negative bacteria at MIC

Metallo-β-lactamases (MBLs) are potent bacterial enzymes that can destroy almost all classes of β-lactams, including carbapenems. Strong hydrolytic activity against carbapenems is observed when these enzymes are produced chromosomally by clinically important pathogens such as Stenotrophomonas maltophilia and Bacteroides fragilis.1, 2, 3 More importantly, many MBLs now appear as plasmid-encoded enzymes that can be transferred among the Enterobacteriaceae as well as the non-fermentors.2, 3 Over 80 unique MBLs have been described in the literature, with at least 23 members in each of the inosine monophosphate and VIM families of plasmid-encoded MBLs.1 Although genes encoding these metalloenzymes may be found in the bacterial chromosome, they are not always expressed in B. fragilis.4, 5 The MBL genes encoded on mobile bacterial plasmids in Pseudomonas aeruginosa,6 Serratia marcescens7 and many other Gram-negative pathogens are also found with genes encoding other types of antibiotic resistance (for example, aminoglycosides),3 resulting in multidrug-resistant pathogens of high clinical importance. Consequently, clinical failures of carbapenems as a result of MBLs continue to be a serious problem. For this reason, an inhibitor of MBLs could provide a distinct advantage when used in combination therapy with susceptible β-lactam antibiotics.

The biochemical properties of tetrahydrofuranyl (THF) carbapenems, carbapenems with THF substituents, were evaluated with respect to enzyme stability, binding to penicillin-binding proteins (PBPs), and penetration into gram-negative organisms. THF carbapenems showed increased stability to hog renal dehydropeptidases (DHPs) compared to that of imipenem or meropenem and were more stable to human DHP than imipenem (k cat value of Staphylococcus aureus PBPs at concentrations that were less than or equal to the MICs. The THF carbapenems bound to PBPs from Escherichia coli and Pseudomonas aeruginosa , with the highest affinities being for PBPs 2 and 4, as noted with imipenem. The affinities for PBPs 1a and 1b in E. coli were reduced for the THF carbapenems compared to that for imipenem, even though the MICs of the THF carbapenems for E. coli strains were lower than those of imipenem. The penetrability of the THF carbapenems into Serratia marcescens S6, which produces the Sme-1 carbapenem-hydrolyzing β-lactamase, was 2.4 to 7.8 times less than that of imipenem. Compounds CL 190,294 and CL 188,624 showed good penetrability, with permeability coefficient values comparable to those of the rapidly penetrating agents cephaloridine, imipenem, meropenem, and biapenem. Decreased penetration into wild-type P. aeruginosa was suggested by the high MICs of the THF carbapenems (MICs, 16 to 32 μg/ml), despite equivalent or better binding to P. aeruginosa PBPs than that of imipenem. However, the MICs of the THF carbapenems for wild-type P. aeruginosa compared to that for an OprD2 mutant generally varied no more than 2-fold, but those of imipenem and other carbapenems differed 16-fold. These data indicated that THF carbapenems do not appear to enter through protein OprD2. In conclusion, the THF carbapenems exhibited stability to hydrolysis by renal DHPs and serine β-lactamases, exhibited strong binding to essential PBPs from E. coli and S. aureus , and penetrated gram-negative enteric bacteria at rates comparable to those for meropenem and biapenem.

At a children’s hospital in Riga, Latvia, isolates identified as Salmonella typhimurium were found to be resistant to expanded-spectrum cephalosporins. Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance. Isoelectric focusing revealed a common β-lactamase with a pI of 8.8. In addition, one of the strains produced a pI 7.6 β-lactamase. A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1. Transformants producing only the pI 8.8 β-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime. A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative k cat value of 95 (benzylpenicillin equal to 100). The enzyme showed lower relative k cat values for ceftazidime (3.3) and aztreonam (9.3). In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively. These results indicated the presence of an unusual extended-spectrum β-lactamase. The gene expressing the pI 8.8 β-lactamase was cloned. Nucleotide sequencing revealed a β-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from S. typhimurium , by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val. These results indicated the presence of a novel extended-spectrum β-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.

Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae (49 and 102 isolates, respectively) were collected from Barnes-Jewish Hospital, St. Louis, Mo., from 1992 to 1996. They were uniformly resistant to ceftazidime, generally resistant to aztreonam, and variably susceptible to cefotaxime. Four representative E. coli strains and 15 Klebsiella strains were examined. From one to four β-lactamases were produced per strain, with three possible enzymes related to ceftazidime resistance: enzymes with pI values of 5.6, 6.1, or 7.6. By pulsed-field gel electrophoresis there were at least 13 different Klebsiella strain types and 3 different E. coli strain types, indicating that the outbreak was not clonal. After cloning and sequencing of the β-lactamase-encoding genes, the enzyme with a pI of 5.6 was identified as TEM-10. The enzyme with a pI of 6.1 was a novel TEM variant (TEM-43) with Lys at 104, His at 164, and Thr at 182. TEM-43 showed broad-spectrum hydrolytic activity against all penicillins, with the highest hydrolysis rate for ceftazidime compared to those for the other expanded-spectrum cephalosporins. Aztreonam was also a good substrate for TEM-43, with hydrolytic activity similar to that of ceftazidime and affinity higher than that of ceftazidime. The TEM-43 β-lactamase was well inhibited by clavulanic acid and tazobactam at concentrations of

Pregnant mothers are susceptible to bacterial infections, which may compromise the health of mothers and offspring. Enterococcus faecalis is a ubiquitous species found in food, restaurants, and hospitals where pregnant woman frequently become exposed to this bacterium. However, the survival, distribution, translocation, and corresponding influence of E. faecalis have not been investigated during the pregnancy period, when the mother and fetus are susceptible to bacterial infection. In this study, a fluorescing E. faecalis strain was used to track the fate of the bacterium in pregnant mice. Orally administered E. faecalis were found to survive and disseminate to all regions of the intestinal tract. It also altered the bacterial community structure by significantly decreasing the diversity of Lactobacillus species, impairing the normal structure and function of the intestinal barrier, which may contribute to the bacterial translocation into the blood, spleen, placenta, and fetus. This may affect fetal and placental growth and development.

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

AsbM1, a carbapenem-hydrolyzing beta-lactamase produced by Aeromonas sobria AER 14M, was purified chromatographically, with anion exchange chromatography performed in the absence of Zn2+. The molecular mass of AsbM1 was approximately 34,000; the isoelectric point was 9.1. AsbM1 had high hydrolytic specificity for carbapenems but low hydrolysis rates for penicillins and cephalosporins. AsbM1 was resistant to the commercially available beta-lactamase inhibitors but was inhibited by pCMB and the chelators EDTA and o-phenanthroline. Zinc, an activator for many metallo-beta-lactamases, inhibited AsbM1 with an IC50 of 8 microM. Analysis of the N-terminal sequence (27 amino acids) showed 26% similarity to the CphA metallo-beta-lactamase. Because of the high specificity for carbapenems and the sensitivity to inhibition by Zn2+, AsbM1 should be included in a new subgroup of metallo-beta-lactamases.

Adverse early-life experience such as depriving the relationship between parents and children induces permanent phenotypic changes, and impairs the cognitive functions associated with the prefrontal cortex (PFC). However, the underlying mechanism remains unclear. In this work, we used rat neonatal maternal separation (NMS) model to illuminate whether and how NMS in early life affects cognitive functions, and what the underlying cellular and molecular mechanism is. We showed that rat pups separated from their dam 3 h daily during the first 3 postnatal weeks alters medial prefrontal cortex (mPFC) myelination and impairs mPFC-dependent behaviors. Myelination appears necessary for mPFC-dependent behaviors, as blockade of oligodendrocytes (OLs) differentiation or lysolecithin-induced demyelination, impairs mPFC functions. We further demonstrate that histone deacetylases 1/2 (HDAC1/2) are drastically reduced in NMS rats. Inhibition of HDAC1/2 promotes Wnt activation, which negatively regulates OLs development. Conversely, selective inhibition of Wnt signaling by XAV939 partly rescue myelination arrestment and behavior deficiency caused by NMS. These findings indicate that NMS impairs mPFC cognitive functions, at least in part, through modulation of oligodendrogenesis and myelination. Understanding the mechanism of NMS on mPFC-dependent behaviors is critical for developing pharmacological and psychological interventions for child neglect and abuse.

Class A-class C mechanism-based beta-lactamase inhibitors were designed on the basis of the intermediacy of an oxycarbenium species capable of cross-linking with amino acids residues in the active site. Penams 24 and 27 were very potent against AmpC in vitro. The MIC values of 24 in combination with piperacillin against class A and class C producing organisms showed improvement over clinically used tazobactam.

The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene.

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10 2 CFU/ml (1.2 × 10 2 CFU/ml for S . Typhimurium, 4.0 × 10 2 CFU/ml for E . coli O157:H7 and 5.4 × 10 2 CFU/ml for L . monocytogenes ) in pure culture and 10 3 CFU/g (5.1 × 10 3 CFU/g for S . Typhimurium, 7.5 × 10 3 CFU/g for E . coli O157:H7 and 8.4 × 10 3 CFU/g for L . monocytogenes ) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S . Typhimurium, E . coli O157:H7 and L . monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use.

Tazobactam was shown to be a potent inhibitor of group 1, 2a, 2b, and 2b' beta-lactamases. Extended kinetic studies with class A and C serine beta-lactamases showed that the PC1, TEM-2, and P99 enzymes all were reversibly inhibited prior to inactivation of the enzymes. The CcrA metallo-beta-lactamase was less well inhibited, with a 50% inhibitory concentration at least 3 orders of magnitude less favorable than those for most serine beta-lactamases. The numbers of hydrolytic turnovers of tazobactam before inactivation were 2 for PC1, 125 for TEM-2, 50 for P99, and 4,000 for the CcrA enzyme. In spectral studies, transient intermediates were formed after reaction of tazobactam with the PC1, TEM-2, and CcrA beta-lactamases, corresponding to enzyme-associated intermediates responsible for hydrolysis of tazobactam. Chromophores absorbing at 270 nm (CcrA) and 288 nm (TEM-2 and PC1) were observed for these reaction intermediates. The P99 cephalosporinase formed a stable complex with a UV maximum at 295 nm. Incubation of tazobactam with all of the enzymes resulted in accumulation of a tazobactam reaction product with a short-wavelength absorbance. This product has characteristics similar to those of the major eucaryotic metabolite of tazobactam. Possible reaction mechanisms are presented to explain the findings. In conclusion, both serine-based and metallo-beta-lactamases were irreversibly inactivated by tazobactam following an initial transient inhibition phase.

Clavulanate and tazobactam (YTR 830) were tested as inhibitors and inducers of the AmpC-type Class I beta-lactamases of Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and the Ic beta-lactamase of Proteus vulgaris. Both clavulanate and tazobactam inhibited the Pr. vulgaris Class Ic beta-lactamase and potentiated ticarcillin and piperacillin against beta-lactamase derepressed variants of this species. Tazobactam, but not clavulanate, also had some ability to inhibit the AmpC Class I enzymes of M. morganii, C. freundii, Ps. aeruginosa, E. cloacae and S. marcescens. The piperacillin + tazobactam combination, unlike ticarcillin + clavulanate, showed some degree of synergy against most derepressed strains of these species. This behaviour partly depended upon the greater inhibitory activity of tazobactam for the enzymes, but also on piperacillin being easier to potentiate than ticarcillin. The synergy between piperacillin and tazobactam was greatest for M. morganii and C. freundii, least for Ps. aeruginosa and E. cloacae. Unfortunately, it is in the last two species that these enzymes pose the greatest resistance threat. Tazobactam caused little or no antagonism of piperacillin against beta-lactamase inducible species, whereas clavulanate antagonized ticarcillin against beta-lactamase inducible strains of E. cloacae and M. morganii (not other species). The antagonism of ticarcillin was attributable to beta-lactamase induction. The lack of antagonism with the tazobactam+piperacillin combination was related to tazobactam being a weaker inducer than clavulanate, not to piperacillin being less susceptible to antagonism than ticarcillin.

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C 55 -MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.

The CcrA beta-lactamase from Bacteroides fragilis TAL3636 was cloned into Escherichia coli and purified from inclusion bodies. This group 3 metalloenzyme hydrolyzed most beta-lactam antibiotics, including cephamycins and carbapenems. Following inhibition by chelators, enzyme activity was recovered with the cations Zn2+ and Co2+. Clavulanate and sulbactam were activators; tazobactam at 10 microM inactivated the enzyme.

Beta-Lactamases of Ambler's Class A are the most commonly encountered mechanism of bacterial resistance to beta-lactam antibiotics. In the face of selective pressure arising from use of either newer cephalosporins or beta-lactam/beta-lactamase inhibitor combinations, mutations arose among Class A beta-lactamase genes, leading to resistance. Clavulanic acid, a naturally occurring clavam, and the penicillanic acid sulfones sulbactam and tazobactam are the inhibitors in clinical use. This review focuses on the mechanism of inhibition by these currently marketed beta-lactamase inhibitors and on the mechanism by which specific amino acid substitutions might lead to resistance. The key amino acid positions important for inhibitor-resistance include Met69, Ser130, Arg244, Arg275, and Asn276. Ser130 is vital to the chemical mechanism of inhibition. Arg244 appears to be coordinated to Arg275 and Asp276 by hydrogen bonds. Arg244 is involved in positioning beta-lactams, especially penicillins and beta-lactamase inhibitors, via their carboxyl groups. Site-directed mutagenesis studies confirm the role of Arg244 and its coordinating partners in beta-lactam turnover and in the reactions leading to enzyme inactivation. This mechanism is dependent on the donation of a proton via a water coordinated to Arg244 and Val216 to clavulanic acid to allow formation of a favorable leaving group. This proton donation is probably not required for formation of a favorable leaving group for the sulfone inhibitors sulbactam and tazobactam. Therefore, some amino acid substitutions have differing effects on inhibition by clavulanic acid compared with the penicillanic acid sulfones. Met69 may play a more structural role in beta-lactam positioning within the oxyanion hole.

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.

Biological activities of biapenem, imipenem, and meropenem were compared with respect to permeability into Gram-negative bacteria, binding to penicillin-binding proteins (PBPs), and hydrolysis by beta-lactamases. Permeability for the three carbapenems was similar when measured in Serratia marcescens S6 producing a carbapenem-hydrolyzing beta-lactamase. Penetration of the carbapenems was comparable with cephaloridine and faster than piperacillin or the extended spectrum cephalosporin cefotaxime. All the carbapenems bound most strongly to PBP 2 of Escherichia coli and Pseudomonas aeruginosa, and to PBP 1 of Staphylococcus aureus. In addition, biapenem showed strong affinity with PBP 1a of E. coli and PBP 1b of P. aeruginosa. Selected serine beta-lactamases, including the extended spectrum plasmid-mediated beta-lactamases, hydrolyzed these carbapenems at rates < 0.1% that of cephaloridine. Metallo-beta-lactamases hydrolysed the carbapenems at measurable rates, with enzymes from Bacteroides fragilis and Xanthomonas maltophilia hydrolyzing biapenem at lower Vmax values than meropenem or imipenem. In conclusion, all the carbapenems exhibited good rates of penetration, bound strongly to PBPs in both Gram-negative and Gram-positive bacteria, and were stable to most Group 1 and Group 2 serine beta-lactamases, but were hydrolyzed by metallo-beta-lactamases.

Reexamination of Serratia marcescens isolates obtained in 1982 revealed two organisms that were resistant to the penem FCE 22101 (MIC, 512 micrograms/ml) and imipenem (MIC, 16 micrograms/ml) and that had slightly reduced susceptibilities to meropenem (MIC, 0.12 micrograms/ml). MICs of these agents for typical S. marcescens isolates were 1 to 8, 0.25 to 0.5, and 0.03 micrograms/ml, respectively. The two isolates were fully susceptible to broad-spectrum cephalosporins, and only one was highly resistant to ampicillin and carbenicillin (MICs, greater than 1,024 micrograms/ml). Both isolates had beta-lactamases that focused at pIs 8.2 and 9.7. The penicillin-resistant isolate additionally produced the TEM-1 enzyme. The enzymes with pIs of 8.2 and 9.7 were separated by cation-exchange chromatography. The pI 8.2 beta-lactamase was a class I enzyme of the type found in most S. marcescens isolates. It was almost inactive against carbapenems and penems, as was the class I enzyme from another S. marcescens strain. The pI 9.7 enzyme hydrolyzed penems and carbapenems rapidly: kcat (turnover number) values for FCE 22101, imipenem, and meropenem were 3.4, 26, and 1% of the kcat value for cephaloridine, respectively; kcat/Km values were 140, 915, and 150% of the kcat/Km value for cephaloridine, respectively. Otherwise, the pI 9.7 enzyme had predominantly penicillinase activity. It was inhibited more readily by clavulanate than by tazobactam and was inactivated by the chelating agents EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Expression of the pI 9.7 enzyme was not associated with any plasmid, and production was not transferred to Escherichia coli K-12 recipients, even after the mobilizing plasmid pUZ8 was inserted into the S. marcecens donor strains.

Tigemonam was compared in vitro with other orally active beta-lactams, aztreonam and ciprofloxacin against a selection of Gram-negative clinical isolates and laboratory-derived beta-lactamase-producing variants. Of the orally active beta-lactams, tigemonam was the most potent, with a spectrum of activity similar to that of aztreonam. This included stability to plasmid-mediated beta-lactamases and poor induction of chromosomal beta-lactamases. The susceptibility to Class I enzymes was only clinically significant for derepressed Enterobacter cloacae mutants. Tigemonam may have a valuable role in the management of infection caused by enterobacteria, particularly if bioavailability following oral administration is confirmed in human subjects.

Hyperproduction of chromosomal Class I beta-lactamase causes resistance to newer cephalosporins in Enterobacter cloacae, Citrobacter freundii, Serratia spp. and indole-positive Proteeae. We examined the interactions of FCE 22101 with these enzymes, measuring (i) antibiotic lability to pure enzyme, (ii) inducing power and (iii) activity against beta-lactamase-inducible strains and their -depressed and -basal mutants. Turnover numbers of FCE 22101 were between 0.7 and 1.5 molecules hydrolysed/min/enzyme molecule, compared with ranges of 0.5-40 for cefotaxime (950 for the Proteus vulgaris cephalosporinase) and 4400-22,000 for cephaloridine. FCE 22101 induced enzyme synthesis strongly below the MIC and antagonized labile weak inducers such as cefotaxime and mezlocillin. Like imipenem, but unlike newer cephalosporins, FCE 22101 had equal activity against inducible strains and derepressed mutants, and did not significantly select derepressed mutants from inducible populations. beta-Lactamase basal mutants of E. cloacae and C. freundii were four- to eight-fold more sensitive than inducible and derepressed organisms whereas beta-lactamase-basal, -inducible and -derepressed Proteeae and Serratia spp. were equally sensitive. This difference reflected the larger amounts of enzyme produced by the E. cloacae and C. freundii strains, rather than kinetic differences in enzyme activity.

LXA-1, a novel plasmid-mediated β-lactamase, was observed in clinical isolates of Klebsiella oxytoca, Klebsiella pneumoniae, Citrobacter freundii and Enterobacter cloacae. All the strains additionally produced TEM-1 β-lactamase. LXA-1 had an Mr of 24 000 and a pI of 6.7. It hydrolysed benzyl-penicillin, ampicillin, carbenicillin and first generation cephalosporins, but not methicillin, oxacillin or cefotaxime. Clavulanate and cloxacillin were inhibitors. Studies of one of the E. cloacae isolates showed that LXA-1 was encoded by a 41-MDa IncFII plasmid distinct from that encoding TEM-1 enzyme in the strain. Transconjugants which acquired LXA-1 production, but not TEM-1, exhibited only low-level resistance to substrate β-lactams.

The interactions of imipenem, carbenicillin, cefotaxime, ceftriaxone, and azlocillin with the chromosomal beta-lactamase of Pseudomonas aeruginosa were compared. Imipenem was hydrolyzed very slowly (kcat, 1/min) and induced beta-lactamase synthesis strongly. Its minimal inhibitory concentrations (MICs) reflected this behavior, being equal (1-2 micrograms/ml) for enzyme-inducible strains and their stably derepressed mutants. Mutants that had basal (i.e., minimal and uninducible) enzyme production were eight- to 16-fold more susceptible to imipenem than were inducible or stably derepressed strains. Carbenicillin was stable to hydrolysis (kcat, less than 0.1/min) and induced weakly at low concentrations. Consequently its MICs were equal for beta-lactamase-inducible strains and for their basal mutants. Stable beta-lactamase derepression generally did not increase resistance to carbenicillin significantly. Azlocillin, cefotaxime, and ceftriaxone were labile to hydrolysis (kcat, 12-297/min) but induced poorly. Consequently their MICs for enzyme-inducible strains equaled those for basal mutants but were elevated for derepressed mutants.

Concern has been expressed that clavulanate can antagonize ticarcillin against enterobacteria and pseudomonads that have inducible expression of chromosomal 'Class I' beta-lactamases. It is suggested that clavulanate-induced enzyme inactivates ticarcillin, which itself is a feeble inducer. We confirmed that this mechanism applied, showing that antagonism was abolished in beta-lactamase-basal mutants of inducible strains. Antagonism has been reported in double disc tests with strains of Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, Serratia spp. and indole-positive Proteeae. Only with some strains of Ent. cloacae and Morganella morganii, however, did the presence of 1-32 mg/l clavulanate elevate the MIC of ticarcillin by more than one or two dilutions in chequerboard studies. Clavulanate was synergistic with ticarcillin against Proteus vulgaris strains, being a potent inhibitor of the unusual Class I enzyme of this species. Induction-determined antagonism was not reduced in Ent. cloacae transconjugants that produced the plasmid-mediated TEM-1 beta-lactamase, despite the ability of this enzyme to bind clavulanate. Our results suggest that Ent. cloacae and M. morganii strains should be confirmed not to be more sensitive to ticarcillin alone than to ticarcillin/clavulanate, before the latter combination is used clinically. Otherwise, it appears that beta-lactamase induction is unlikely to cause significant antagonism. It is emphasized that induction is reversible, causing, at worst, a transient resistance. It should not be confused with the selection of stably-derepressed mutants that can occur, for example, in the clinical use of newer cephalosporins.

Indole-positive members of the Proteeae usually have inducible expression of chromosomal beta-lactamases. Mutants with stably derepressed beta-lactamase expression occur in inducible populations at frequencies in the range of 10(-6) to 10(-8). The contribution of these beta-lactamases to drug resistance was examined in Morganella morganii and Proteus vulgaris. The M. morganii enzyme was a high-molecular-weight (49,000) class I cephalosporinase with low Vmax rates for ampicillin, carbenicillin, and and broad-spectrum cephalosporins. The P. vulgaris enzyme had a lower molecular weight (32,000) and high Vmax rates for ampicillin, cephaloridine, cefotaxime, and ceftriaxone. Imipenem and cefoxitin inactivated the P. vulgaris enzyme but were low-Vmax, low-Km substrates for that of M. morganii. Despite these differences, the two beta-lactamases caused similar resistance profiles. Ampicillin and cephaloridine were strong inducers for both species, and beta-lactamase-inducible strains and their stably derepressed mutants were resistant, whereas basal mutants (those with low-level uninducible beta-lactamase) were susceptible to these two compounds. Mezlocillin, cefotaxime, ceftriaxone, and (usually) carbenicillin were almost equally active against beta-lactamase-inducible organisms and their basal mutants, but were less active against stably derepressed mutants. This behavior reflected the beta-lactamase lability of these drugs, coupled with their weak inducer activity below the MIC. Carbenicillin was a labile strong inducer for a single P. vulgaris strain, and inducible enzyme was protective against the drug in this atypical organism. Cefoxitin and imipenem, both strong inducers below the MIC, were almost equally active against beta-lactamase-inducible organisms and their basal and stably derepressed mutants.

Most newer penicillins and cephalosporins are labile to the Class I beta-lactamases that are inducible in Pseudomonas aeruginosa. Enterobacter spp., Citrobacter spp., Serratia spp. and indole-positive Proteeae, but fail to induce enzyme synthesis below the MIC. They remain active against the inducible strains but not against mutants (termed 'stably depressed') that manufacture beta-lactamase copiously without induction. Such mutants are segregated at high frequency and may be selected during therapy, sometimes causing clinical failure. Meropenem induced Class I enzymes weakly below the MIC in Ps. aeruginosa, Ent. cloacae, C. freundii, Ser. marcescens, Morganella morganii and Pr. vulgaris. Nonetheless it remained equally active against inducible strains and their derepressed mutants, and was no more active against laboratory-derived beta-lactamase basal mutants. It did not select derepressed mutants from beta-lactamase-inducible populations. These data suggested great stability to Class I beta-lactamases, and this deduction was confirmed by direct assays with purified beta-lactamases. Initial hydrolysis rates ranged from 0.9-10.0 drug molecules hydrolysed/enzyme molecule/minute but these values declined by 80-90% before 5% of the antibiotic had been hydrolysed, indicating that the carbapenem could deactivate the enzymes. Deactivation was reversible, full activity being restored once the meropenem was removed. These results suggest that meropenem, like imipenem and various experimental penems, may overcome the resistance problems presented by Class I beta-lactamases.