Β-lactamase Activity Research Articles

Important Role of a Putative Lytic Transglycosylase Cj0843c in β-Lactam Resistance in Campylobacter jejuni.

Beta-lactam antibiotics are an important class of antibiotics for treating bacterial infections. Despite prevalent β-lactam resistance in Campylobacter jejuni, the leading bacterial cause of human diarrhea in developed countries, molecular mechanism of β-lactam resistance in C. jejuni is still largely unknown. In this study, C. jejuni 81–176 was used for random transposon mutagenesis. Screening of a 2,800-mutant library identified 22 mutants with increased susceptibility to ampicillin. Of these mutants, two mutants contains mutations in Cj0843c (a putative lytic transglycosylase gene) and in its upstream gene Cj0844c, respectively. Complementation experiment demonstrated that the Cj0843 contributes to β-lactam resistance. The Cj0843c insertional mutation was subsequently introduced to diverse C. jejuni clinical strains for MIC test, showing that Cj0843c contributes to both intrinsic and acquired β-lactam resistance of C. jejuni. Consistent with this finding, inactivation of Cj0843c also dramatically reduced β-lactamase activity. Genomic examination and PCR analysis showed Cj0843c is widely distributed in C. jejuni. High purity recombinant Cj0843c was produced for generation of specific antiserum. The Cj0843 was localized in the periplasm, as demonstrated by immunoblotting using specific antibodies. Turbidimetric assay further demonstrated the capability of the purified Cj0843c to hydrolyze cell walls. Inactivation of Cj0843c also significantly reduced C. jejuni colonization in the intestine. Together, this study identifies a mechanism of β-lactam resistance in C. jejuni and provides insights into the role of cell wall metabolism in regulating β-lactamase activity.

Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria.

Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets.

Resistance of Undisturbed Soil Microbiomes to Ceftriaxone Indicates Extended Spectrum β-Lactamase Activity.

Emergence and spread of antibiotic resistance, and specifically resistance to third generation cephalosporins associated with extended spectrum β-lactamase (ESBL) activity, is one of the greatest epidemiological challenges of our time. In this study we addressed the impact of the third generation cephalosporin ceftriaxone on microbial activity and bacterial community composition of two physically and chemically distinct undisturbed soils in highly regulated microcosm experiments. Surprisingly, periodical irrigation of the soils with clinical doses of ceftriaxone did not affect their microbial activity; and only moderately impacted the microbial diversity (α and β) of the two soils. Corresponding slurry experiments demonstrated that the antibiotic capacity of ceftriaxone rapidly diminished in the presence of soil, and ∼70% of this inactivation could be explained by biological activity. The biological nature of ceftriaxone degradation in soil was supported by microcosm experiments that amended model Escherichia coli strains to sterile and non-sterile soils in the presence and absence of ceftriaxone and by the ubiquitous presence of ESBL genes (blaTEM, blaCTX-M, and blaOXA) in soil DNA extracts. Collectively, these results suggest that the resistance of soil microbiomes to ceftriaxone stems from biological activity and even more, from broad-spectrum β-lactamase activity; raising questions regarding the scope and clinical implications of ESBLs in soil microbiomes.

SHV-129: A Gateway to Global Suppressors in the SHV β-Lactamase Family?

Enzymes are continually evolving in response to environmental pressures. In order to increase enzyme fitness, amino acid substitutions can occur leading to a changing function or an increased stability. These evolutionary drivers determine the activity of an enzyme and its success in future generations in response to changing conditions such as environmental stressors or to improve physiological function allowing continual persistence of the enzyme. With recent warning reports on antibiotic resistance and multidrug resistant bacterial infections, understanding the evolution of β-lactamase enzymes, which are a large contributor to antibiotic resistance, is increasingly important. Here, we investigated a variant of the SHV β-lactamase identified from a clinical isolate of Escherichia coli in 2011 (SHV-129, G238S-E240K-R275L-N276D) to identify the first instance of a global suppressor substitution in the SHV β-lactamase family. We have used this enzyme to show that several evolutionary principles are conserved in different class A β-lactamases, such as active site mutations reducing stability and requiring compensating suppressor substitutions in order to ensure evolutionary persistence of a given β-lactamase. However, the pathway taken by a given β-lactamase in order to reach its evolutionary peak under a given set of conditions is likely different. We also provide further evidence for a conserved stabilizing substitution among class A β-lactamases, the back to consensus M182T substitution. In addition to expanding the spectrum of β-lactamase activity to include the hydrolysis of cefepime, the amino acid substitutions found in SHV-129 provide the enzyme with an excess of stability, which expands the evolutionary landscape of this enzyme and may result in further evolution to potentially include resistance to carbapenems or β-lactamase inhibitors.

The challenge of resistance in antimicrobial drug development.

Man has long battled the infectious diseases caused by pathogenic bacteria, fungi, protozoa and viruses. Since the late 1930s, the discovery and development of chemotherapeutic agents has been the central effort toward combating infectious disease. Although many of the major causes of historical pandemics no longer present the threat they once did and we have experienced a multidecade era in which safe and effective antibiotics were plentiful, we are now witnessing the emergence of organisms resistant to the most efficacious agents. The adaptive nature of the pathogens necessitates the discovery of new effective compounds or the re-engineering of existing molecules to combat the resistant organisms. This iterative cycle of discovery and development is a unique hallmark of drug discovery targeting proliferating cells. All major classes of agents with antibacterial, antifungal, antiprotozoal and antiviral activity are subject to lost efficacy owing to resistance through a multitude of pathways. Common mechanisms of resistance and examples of affected classes include point mutations in the target (HIV reverse transcriptase inhibitors), upregulation of efflux pumps (fluoroquinolones), gene amplification or development of bypass pathways (fluconazole) and drug alteration ( β-lactamase activity targeting cephalosporins). As each of these mechanisms is unique to the particular drug:organism system, carefully tailored solutions are necessary to overcome the resistance. Modern medicinal chemistry has confronted numerous resistant organisms with a variety of approaches including the discovery of novel agents without cross-resistance, use of combination therapy, or most frequently, alteration of the chemical structure to circumvent the resistance mechanism. For example, the discovery and development of daptomycin to treat methicillin-resistant Staphylococcus aureus relied on a natural product with a novel mechanism of action. The development of highly active antiretroviral therapy (HAART), a combination of multiple antiviral agents, revolutionized the treatment of HIV. Finally, no class better exemplifies the value of refinement through medicinal chemistry than the β-lactam antibiotics; many successive generations in this class have overcome β-lactamase activity while increasing the spectrum of coverage. In this issue, notable advances in understanding and overcoming resistance in a variety of disease states are presented, including a modern overview of natural product sources for discovery [1], application of combination therapy to prevent resistance [2] and a description of a structure-based approach to antibiotic redesign to evade resistant drug targets [3]. This is followed by more specific approaches to antibiotic resistance in tuberculosis [4], as well as discussion of the critical issue of “The adaptive nature of the pathogens necessitates the discovery of new effective compounds or the re-engineering of existing molecules to combat the resistant organisms.”

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif ‘SXXK’ in addition to a conserve pentapeptide –GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif ‘SXXK’ was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.

Structural and functional studies of a metallo-β-lactamase unveil a new type of structurally encoded nickel-containing heterodinuclear site.

The selection of correct metal ions with high fidelity against competing cellular cations is crucial for the function of many metalloenzymes; however, the understanding of the principles that govern metal selectivity is still incomplete. In this study, the crystal structure of the Tm1162 protein from Thermotoga maritima, a metallo-β-lactamase, is reported. Several crystal structures of wild-type Tm1162 and its mutants were solved. Homologues of Tm1162 are widely distributed in bacteria and archaea, including several human pathogens. The monomer possesses an αβ/βα fold, with the core β-strands having the β-sheet sandwich structure common to the metallo-β-lactamase superfamily. Tm1162 exists as a trimer in the crystal and this trimeric unit is likely to be present in solution. In the trimer, three active sites reside at the interface between subunits, suggesting that the oligomeric assembly is crucial for catalysis. A new type of structurally encoded heterodinuclear site has been identified by confirming the identity of nickel-containing heteronuclear sites in Tm1162 via X-ray absorption spectroscopy and anomalous difference Fourier maps. The second coordination sphere, including His8 and Glu73, maintains the side-chain orientations of histidines and stabilizes the metal-binding site. Nickel coordination was crucial for the oligomerization of Tm1162. The nickel-dependent and manganese-dependent β-lactamase and phosphodiesterase activities of Tm1162 have also been characterized.

A comparative study for the inactivation of multidrug resistance bacteria using dielectric barrier discharge and nano-second pulsed plasma

Bacteria can be inactivated through various physical and chemical means, and these have always been the focus of extensive research. To further improve the methodology for these ends, two types of plasma systems were investigated: nano-second pulsed plasma (NPP) as liquid discharge plasma and an Argon gas-feeding dielectric barrier discharge (Ar-DBD) as a form of surface plasma. To understand the sterilizing action of these two different plasma sources, we performed experiments with Staphylococcus aureus (S. aureus) bacteria (wild type) and multidrug resistant bacteria (Penicillum-resistant, Methicillin-resistant and Gentamicin-resistant). We observed that both plasma sources can inactivate both the wild type and multidrug-resistant bacteria to a good extent. Moreover, we observed a change in the surface morphology, gene expression and β-lactamase activity. Furthermore, we used X-ray photoelectron spectroscopy to investigate the variation in functional groups (C-H/C-C, C-OH and C=O) of the peptidoglycan (PG) resulting from exposure to plasma species. To obtain atomic scale insight in the plasma-cell interactions and support our experimental observations, we have performed molecular dynamics simulations to study the effects of plasma species, such as OH, H2O2, O, O3, as well as O2 and H2O, on the dissociation/formation of above mentioned functional groups in PG.

P05.02 Neisseria gonorrhoeaestrain types and antibiotic susceptibility

Background Molecular surveillance of Neisseria gonorrhoeae will help in understanding the transmission patterns of the infection and the acquisition/development of antibiotic resistant strains. We aimed to determine the antimicrobial resistance (AMR) and the genotypes of the N. gonorrhoeae isolates. Methods Of a total of 287 N. gonorrhoeae isolates the minimal inhibitory concentration (MIC; mg/L) of penicillin, tetracycline, ciprofloxacin, ceftriaxone, azithromycin, spectinomycin and cefixime was determined employing the gold standard agar dilution method. β-lactamase production was detected using nitrocefin solution. The sequence types (STs) of the isolates was obtained using the NG-Multi Antigen Sequence Typing (NG-MAST) method. Results All isolates were susceptible to ceftriaxone, spectinomycin, and cefixime. Resistance to ciprofloxacin, tetracycline, penicillin, and azithromycin was 50.9%, 46.3%, 26.5%, and 2.4% respectively. A total of 10.8% of the strains produced β-lactamase. Overall 74 STs were determined. Five STs made up for 50.3% of all the isolates: ST2992 (19.8%); ST1407 (9.7%); ST2400 (7.6%); ST387 (7.3%); ST2212(5.9%). ST387 was isolated in heterosexuals only and ST2400 and ST2992 in mainly men having sex with men. All ST387 were susceptible to all tested antibiotics with 95.2% of the strains having a MIC of 0.001 mg/L for ceftriaxone. All ST2992 were susceptible to ciprofloxacin and all ST1407, ST2212, and ST2400 were resistant. The median MICs for ceftriaxone were 0.03 µg/ml for ST1407, ST2212, and ST2400 and 0.008 µg/ml for ST2292. None of the five STs showed β-lactamase activity. Conclusions Associations were found between antibiotic susceptibility and sequence type. The most important finding is the absolute susceptibility to the tested antibiotics of ST387, which was identified in heterosexuals only. Our preliminary results are very promising. However, more research is needed to further optimise the NG-MAST method for its use in predicting AMR and in molecular surveillance. Ultimately the method should be applicable directly on biological specimens. Disclosure of interest Nothing to declare.

Interplay among membrane-bound lytic transglycosylase D1, the CreBC two-component regulatory system, the AmpNG-AmpDI-NagZ-AmpR regulatory circuit, and L1/L2 β-lactamase expression in Stenotrophomonas maltophilia.

Lytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in the Stenotrophomonas maltophilia genome, including genes for five membrane-bound LTs (mltA, mltB1, mltB2, mltD1, and mltD2) and a gene for soluble LT (slt). Six LTs of S. maltophilia KJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δslt mutants. Inactivation of mltD1 conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1 mutant-mediated L1/L2 β-lactamase expression involved the creBC two-component regulatory system (TCS) and the ampNG-ampDI-nagZ-ampR regulatory circuit. The inactivation of mltD1 resulted in mltB1 and mltD2 upexpression in a creBC- and ampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via the ampNG-ampDI-nagZ-ampR regulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, the ampNG-ampDI-nagZ-ampR regulatory circuit, and L1/L2 β-lactamase expression in S. maltophilia.

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15.

A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A(77)V, displayed enhanced catalytic activity (kcat/Km) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A(77)V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as β-lactamase substrates

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of β-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of β-lactamases. Kinetic parameters were determined with eight different β-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by β-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

The direct anti-MRSA effect of emodin via damaging cell membrane.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important bacterium for nosocomial infection. Only a few antibiotics can be effective against MRSA. Therefore, searching for new drugs against MRSA is important. Herein, anti-MRSA activities of emodin and its mechanisms were investigated. Firstly, in vitro antimicrobial activity was investigated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-growth curve, and multipassage resistance testing was performed. Secondly, protection of emodin on mice survival and blood bacterial load in mice challenged with lethal or sublethal dose of MRSA were investigated. Subsequently, the influences of emodin on the bacterial morphology, messenger RNA (mRNA) expressions related to cell wall synthesis and lysis, β-lactamase activity, drug accumulation, membrane fluidity, and integrity were performed to investigate its mechanisms. Lastly, in vitro cytotoxicity assay were performed using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. The results showed MICs and MBCs of emodin against MRSA252 and 36 clinical MRSA strains were among 2-8 and 4-32μg/mL, respectively. There was no MIC increase for emodin during 20 passages. In vivo, emodin dose-dependently protected mice challenged with lethal dose of MRSA and decreased bacterial load in mice challenged with sublethal dose of MRSA. Morphology observation showed emodin might disrupt cell wall and membrane of MRSA. Although emodin had no influence on genes related to cell wall synthesis and lysis as well as β-lactamase activity and drug accumulation, emodin reduced membrane fluidity and disrupted membrane integrity. Based on the fact that emodin had no significant cytotoxicity against mammalian cells, it could be further investigated as a membrane-damage bactericide against MRSA in the future.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, β-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, β-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed β-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

Enzymatic functionalization of a nanobody using protein insertion technology.

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays.

Inhibition of the growth of Bacillus subtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome

A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative β-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any β-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies.

A rapid HPLC method for indirect quantification of β-lactamase activity in milk

To circumvent the strictly regulated limits of antibiotics in milk, illegal addition of β-lactamase to lower the antibiotic levels in milk has been reported recently in China. Herein, we describe a fast, sensitive, and robust HPLC-UV method for the determination of β-lactamase activity in milk, based on an indirect quantification strategy. The test milk sample was mixed with a known amount of penicillin G, a specific substrate of β-lactamase. After incubation, an aliquot of the mixture was injected into the HPLC-UV system to quantify the remaining penicillin G in less than 10min. Comparative analysis of the amount of penicillin G before and after incubation was used to indirectly deduce the activity of β-lactamase in the test sample. This method was successfully applied to milk products with different fat percentages, resulting in a detection limit of 0.6 U/mL. Good recoveries, ranging from 94 to 105%, were obtained from blank milk samples spiked with β-lactamase at levels of 2 to 50 U/mL, with relative standard deviations <6%. A good correlation was demonstrated between the HPLC method and the conventional culture-based assay. Using this method, the activity changes in β-lactamase during milk pasteurization, sterilization, and storage were investigated. The advantages of low-cost, accurate quantification and easily accessible instrumentation make the proposed method an ideal alternative for high-throughput routine analysis in the dairy industry.

Clinical management of infections caused by Enterobacteriaceae that express extended-spectrum β-lactamase and AmpC enzymes.

The production of β-lactamase is the principal mechanism by which gram-negative bacteria resist the action of β-lactam antibiotics. In recent decades, there has been an alarming explosion in the diversity, global dissemination, host range, and spectrum of activity of β-lactamases. This has been most clearly reflected by the marked increase in infections caused by bacteria that express extended-spectrum β-lactamases (ESBLs). Some bacterial species possess chromosomally encoded broad-spectrum cephalosporinases (AmpC) that may be expressed at high level by mutational loss of regulatory genes and are intrinsic in some common Enterobacteriaceae, such as Enterobacter spp. Recently, high-level AmpC production has also been seen in new species such as Escherichia coli via plasmid acquisition. ESBL and AmpC producers present challenges to susceptibility testing and the selection of appropriate antimicrobial therapy. This review describes the current global epidemiology of ESBL producers, examines reported risk factors for infections caused by gram-negative bacteria that express ESBL or AmpC enzymes, and discusses the options for antimicrobial therapy, including "re-discovered" older antibiotics and novel agents in development.

Investigation of the in vivo interaction between β-lactamaseand its inhibitor protein

The affinity of β-lactamase inhibitory protein (BLIP) for TEM-1 β-lactamase has raised hopes in the challenge of protein- based inhibitor discovery for β-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the β-lactamase:BLIP complex in vivo in β-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair β-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent β-lactamase inhibitor. To this end, β-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of β-lactamase and BLIP and the formation of the TEM-1 β-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both β-lactamase and its protein inhibitor, complemented with β-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced β-lactamase activity. Although co-expression of β-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind β-lactamase in the bacterial periplasm, regardless of the crowding components.